低极性化合物的电喷雾质谱检测方法及几种中藏药材的活性成分研究

本学位论文首先报道了为解决低极性化合物的电喷雾质谱（ESI-MS）分析难题而建立的一种衍生化分析方法；然后从色谱-质谱联用分析、分离纯化和结构鉴定等方面分别报道了几种中藏药材的活性成分研究。论文由下述六章组成： 第一章报道了盐酸羟胺衍生化方法在电喷雾质谱 (ESI-MS) 分析中的应用。该方法利用盐酸羟胺和羰基成肟的快速反应，建立了针对三萜酮等含酮或醛羰基低极性化合物的ESI-MS 信号增强技术。此方法不仅可应用于增强羰基化合物的ESI-MS 质谱信号，还可检测化合物中羰基的个数以及辨别涉及羰基官能团的同分异构体。此外，通过简单的氧化反应，还可将该方法拓展到三萜醇、甾醇等含羟基的低极性化合物，增强它们的ESI-MS 信号。对比已报道的相关ESI-MS 增强质谱信号的衍生化方法，此方法有经济、实用、快速和简便的显著特点。 第二章是关于野生羌活及其栽培品种化学成分的色谱-质谱联用分析。对不同产地野生羌活生长过程中活性成分的动态变化、野生羌活不同形态部位和人工栽培羌活中的活性成分含量进行了HPLC 定量分析。结果表明主要活性成分羌活醇和异欧前胡素都随生长期存在规律性变化，羌活不同形态部位中的活性成分含量也有明显不同。这些实验结果有些较好地印证了传统中医的用药理论，有些也对羌活的传统使用方法提出了新的建议。 第三章介绍了几种传统中藏药材的色谱-质谱联用及串联质谱分析。通过GC-MS 方法，从藏药材长花党参挥发油中共分离鉴定出45 个化合物；利用HPLC方法测定了该藏药材中的主要化学成分——木犀草素的含量（0.7%）；利用串联质谱技术，对西番莲和射干中的主要成分进行了快速鉴定，从西番莲中鉴定了4个黄酮碳苷；从不同产地的射干和川射干中鉴定了8 个主要异黄酮成分，其中包括一个未见报道的化合物。 第四章的内容为藏药材石莲叶点地梅的活性成分研究。从植物石莲叶点地梅(Androsace integra (Maxim.) Hand.-Mazz.) 乙醇提取物的正丁醇萃取部分共分离和鉴定了6 个化合物，利用MS 和NMR 等现代波谱学技术阐明了它们的结构：其中包括4 个三萜类化合物：分别是androsacin (1)、 ardisiacrispin A (2) 、saxifragifolin A (3) 和20(29)-lupen-3-one (4)；一个神经酰胺：4-羟基-Δ8,9(Z)-鞘氨醇-2'-羟基正二十四碳酸酰胺(5)；一个甾体类化合物：胡萝卜苷(6)。化合物1为新的13,28-epoxy-oleanane 型三萜皂苷，在其结构表征的过程中，采用LC-MS 进行糖分析，获得了值得推广的好结果。通过活性筛选发现化合物1~3 对HepG2肝癌细胞表现出不同程度的抑制活性，其中化合物2 活性最好，其IG50 为1.65μg/mL。 第五章是关于一些传统中藏药材的农药活性筛选。利用Syngenta 公司的活性筛选平台对68 种传统中藏药材醇提物进行了抗菌和除草的生物源农药活性筛选。结果表明所筛选的68 种植物提取物中，共有14 种样品表现出明显的除草/杀虫活性，其中水母雪莲花、松萝和茯神木等植物提取物还具有多种生物活性。活性成分还有待进一步追踪分离、纯化和结构鉴定。 第六章为文献综述，概述了羌活药材的研究进展。对羌活属及药用羌活植物从分类学、本草学、品质评价、人工栽培、化学成分及药理作用等方面进行了文献归纳和总结。 In this dissertation, an electrospray ionization mass spectrometry (ESI-MS) signal enhancement method, as well as the work of bioactive components study, HPLC-MS/MS application, bioassay screening, chromatograph separation and structure identification of the metabolites in several medicinal herbs have been reported. First chapter expounded a rapid, simple ESI-MS sensitivity enhancement method for detecting carbonyl groups in natural products has been developed by using hydroxylamine hydrochloride (NH2OH·HCl) as a derivatization reagent. We use the oxime formed during the derivatization reactions and its Beckmann rearrangement intermediates as a means of detecting the carbonyl groups originally present in these triterpenoids. In comparison with other derivatization methods in the literature, this method is simple, specific and can be used to detect carbonyl groups in triterpenoids which have low polarity and are poorly or non-ionizable. Moreover, it can also be used to detect hydroxyl groups by using the Dess-Martin periodinane (DMP) to convert primary and secondary hydroxyls into carbonyl groups. Chapter 2 reported an HPLC-MS method for analyzing the main bioactive compounds in both wild and cultured Notopterygium incisum. The results indicated that the main bioactive compounds varied through different seasons regularly, and in different commercial parts of this herb the content of these compounds also differed from each other. The quantitative analysis results showed that in the traditional commercial parts, the content of main chemical constitutes in Silkworm Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are higher than that in Striped Notopterygium. This result is tally with the traditionally concept that the quality of Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are better than that of Striped Notopterygium, which means that the quality of rhizomes is better than main roots. The chemical constituents of cultured N. incisum is reported for the first time in this dissertation and the analysis results showed some growth curves of chemical constituents in this plant, but still left some questions unanswered. Chapter 3 discussed the GC/LC-MS analysis of the traditional Chinese medicines Codonopsis thalictrifolis, Passiflora incarnate, Belamcanda chinensis and Passiflora incarnate. The main constituent, luteolin was isolated and identified from the traditional Tibet medicine of C. thalictrifolis. The quantitative analysis by HPLC has revealed that the content of luteolin in this herb is 0.7%. GC-MS was employed to analyzed chemical constituents of the essential oil from the flower of C. thalictrifolis. More than 60 peaks were detected and 45 of them were identified by comparing their spectra with that of the standards in the database and literatures. ESI-MS/MS was used to analyze the n-butanol extract of Passiflora incarnate. Based on the information of pseudo molecular ions and fragment ions of the glycosides, four major flavone-C-glycosides have been detected and identified as 7-methoxyluteolin-6-C-β-D-glucopyranoside, vitexin, swertisin and orientin. The isoflavone compounds in theextracts of three samples of B. chinensis collected in Gansu, Sichuan and Hunan, and the extract of Iris tectorum collected in Sichuan were analyzed by using TOF-HRMS and IT-MS. From the extracts of these herbs, a new isoflavone, identified as 5’,5,6,7-tetrahydroxy-3’4’-dimethoxyl isoflavon, and 7 known ones have been identified by analyzing the fragmentation patterns and their molecular formulas given by HRMS and the tandem mass spectrometry acquired by IT-MS. Chapter 4 elucidated the isolation and identification of a new triterpene saponin, androsacin (1), along with five known compounds (2-6) were isolated from the whole plants of Androsace integra (Maxim.) Hand.-Mazz., an herb used in traditional Chinese and Tibetan medicine. The chemical structure of the new compound was established as 3β-O-{β-D-glucopyranosyl-(1→4)-O-β-D-xylopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-[O-β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl}-16α-hydroxy-13β,28-epoxy-olean-30-al by analyzing its MS, 1D- and 2D-NMR spectra. Compound 2 was cytotoxic toward HepG2 cancer cell with the GI50 value of 1.65 μg/mL. Chapter 5 described the biogenic pesticide activity screening of 68 traditional Chinese and Tibetan medicine extractions. The intention of this study is to explore bioactive natural compounds from these traditional medicinal herbs for biogenic insecticides use. Based on Syngenta’s bioassay, 14 extractions of these traditional medicines showed pesticide activities, and some of them had multi-activities on antibacterial and insecticidal. Chapter 6 is a review on the chemical and bioactivity research progress of Notopterygium incisum and N. forbesii.

环糊精衍生物的设计、合成及其对环辛烯光异构化研究

环糊精(Cyclodextrins, CDs)经化学修饰后可以得到各种类型的衍生物，不仅可以扩展其原有的键合能力，而且还可以改变其选择性，是当代超分子化学的一个研究热点。环糊精第二面的仲羟基比第一面的伯羟基有着更好的催化性能，第二面的选择性修饰将产生更多有价值的衍生物，可用于催化、酶模拟、手性识别等方面。 取代苯甲酰基修饰环糊精对顺式环辛烯(cis-cyclooctene)光异构化反应有非常重要的影响作用，苯环上取代基的性质和取代位置与产物的%ee值和对映体构型之间存在某种内在联系。有目的地选择适宜取代基，设计、合成新型环糊精光增感剂, 有可能按预定目的得到更高%ee值的反式环辛烯；同时，取代苯甲酰基修饰环糊精对cis-cyclooctene光异构化的增感机理有待于进一步阐明。 本论文工作对环糊精的化学修饰以及超分子体系对cis-cyclooctene不对称光异构化反应方面的进展进行了调研。合成了一系列单-6-位取代苯甲酰基修饰环糊精，用于cis-cyclooctene光异构化增感反应，并用圆二色光谱滴定法研究这些环糊精衍生物与cis-cyclooctene的相互作用，以探索光增感反应机理。在此基础上，探讨了环糊精第二面的选择性修饰方法。内容主要包括： 1. 简要介绍了超分子化学的概况，并对环糊精的选择性修饰方法和超分子体系对cis-cyclooctene不对称光异构化反应的主要成果和最新进展进行了评述。 2. 合成了12种单-6-O-(取代苯甲酰基)-β-环糊精，其中10种为新化合物。采用紫外光谱、红外光谱、核磁共振波谱以及质谱等手段对化合物的结构进行了表征。 3. 探索了直接选择性修饰环糊精第二面的便捷新方法。用取代苯甲酰咪唑酯为酰化试剂，0.2M碳酸盐缓冲溶液(pH=9.9)作催化剂，能够有效地活化2-位仲羟基，对环糊精第二面进行选择性修饰，此方法既简便又经济；同时，发现取代苯甲酰基能够在β-CD第二面的2-位、3-位羟基间相互迁移。 4. 用单-6-O-(取代苯甲酰基)-β-环糊精作光增感剂，对cis-cyclooctene光异构化反应进行研究。实验结果证明：取代苯甲酰基上的取代基性质、位置、长度对反应的对映选择性有很大影响；此外，反应体系溶剂极性对产物的%ee值和对映体构型也有重大影响。用单-6-O-(3-甲氧苯甲酰基)-β-CD作增感剂，cis-cyclooctene光异构化反应产物(R)-trans-cyclooctene的对映选择性为45.8%ee，是到目前为止取得的最好对映选择性。 5. 采用圆二色光谱滴定法研究环糊精衍生物与cis-cyclooctene的相互作用，计算包结物的平衡常数，研究包结物的相对稳定性，为探索光增感反应机理提供基础。我们猜测：电子效应对cis-cyclooctene光异构化反应的影响，可能比取代基位置对反应的影响更大，借助电子效应有希望获得更高的%ee值。 Cyclodextrins can be subjected to diverse modifications to give a wide variety of cyclodextrin derivatives, which could not only extend their original molecular binding ability, but also alter their molecular selectivity. Therefore, cyclodextrin chemistry is currently a significant topic in supramolecular chemistry. The more open secondary hydroxyl side of CDs is stated to be catalytically very important, modifications of this face are believed to produce valuable derivatives for catalysis, enzyme mimic, chiral discrimination, etc. Mono-6-O-(substituted benzoyl)-β-CDs as novel supramolecular photosensitizing hosts have recently excited considerable attention in photochirogenesis. The supramolecular photosenstization of cis-cyclooctene mediated by them gave chiral trans-cyclooctene, enantiomeric excess of which was critically affected by the substituent introduced to the sensitizer moiety. In order to enhance the photoenantiodifferentiating ability, and elucidate the origin mechanisms of substituent-dependent enantioselectivity, in this work a series of mono-6-O-(substituted benzoyl)-β-CDs have been synthesized, and applied for enantiodifferentiating photoisomerization of cis-cyclooctene. The major contents are as follows: 1. The general aspects of supramolecular chemistry were descibed briefly. The new progress and important achievements on methods of selective modification of cyclodextrin and supramolecular enantiodifferentiating photoisomerization of cis-cyclooctene were reviewed. 2. Twelve mono-6-O-(substituted benzoyl)-β-CDs including ten novel compounds have been synthesized. Their structures have been characterized by using UV-vis, IR, NMR and MS methods. 3. A new convenient strategy for direct acylation of β-cyclodextrin on the secondary hydroxyl face was achieved by using the combination of N-benzoylimidazole and carbonate buffer in DMF, and the acyl migration between the C-2 and C-3 hydroxyl groups of β-cyclodextrin was found. 4. Experiments using mono-6-O-(substituted benzoyl)-β-CDs as chiral sensitizing hosts for mediating the enantiodifferentiating photoisomerization of cis-cyclooctene, were carried out. The results indicate that enantiomeric excess was critically affected, or even switched in sign, by the substituent introduced to the sensitizer moiety, and polarity of solvent. Using mono-6-O-(3-methoxybenzoyl)-β-CD as chiral sensitizing host, (R)-trans-cyclooctene was obtained in up to 45.8% enantiomeric excess, which is the highest value ever reported for supramolecular photochirogenesis with analogous hosts. 5. The conformational variation of these modified CDs and their complexation behaviors with cis-cyclooctene were examined by circular dichroism spectroscopy in water-methanol mixed solvents, which reveal that the orientation of chromophore was highly sensitive to the type, position and length of the introduced substituents. In the end, the complex stability constants(Ks) were calculated, and the mechanisms of reaction were discussed. Maybe, electronic effects are more important than positions of substituents for mediating the enantiodifferentiating photoisomerization of cis-cyclooctene.

三种藏药材的化学成分研究

本学位论文报道了西藏产三种藏族传统植物药材的化学成分研究。论文由四章组成，前三章是实验部分，分别报道了尼泊尔黄堇(Corydalis hendersonii Hemsl.)、藏波罗花(Incarvillea younghusbandii Sprague)和全缘叶绿绒蒿(Meconopsis interifolia Franch.)的化学成分研究结果。从这三种青藏高原药用植物中共分离鉴定出33 个化合物，其中1 个是新化合物。第四章概述了罂粟科紫堇属植物的化学和药理研究进展。 第一章为尼泊尔黄堇的化学成分研究。通过正、反相硅胶柱色谱等分离方法从药用植物尼泊尔黄堇的地上部分共分离纯化得到12 个化合物。运用MS、1H-NMR、13C-NMR、DEPT、HMBC、NOESY 等现代波谱学方法将它们的结构鉴定为：刺罂粟碱(1) ， 普托品(2) ， 新那亭(3) ， 斯可任(4) ， tetrahydrothalifendine (5) ，9-methyl-decumbenine C (6)，tetrahydroberberrubine (7)，隐品碱(8)，α-别隐品碱(9)，6,7-methylenedioxy-1(2H)-oxoisoquinolinone (10)，6-丙酮基-5,6 -二氢血根碱(11)和β-谷甾醇(12)。其中化合物6 为新化合物，为首次发现的分子骨架上C-9 位连有甲基的苯肽异喹啉类型生物碱。另外，除化合物1 和2 外，其它9 个生物碱（3～11）均为首次从该种植物中分离得到。同时，我们还对对尼泊尔黄堇中的总生物碱进行了串联质谱分析。 第二章为藏波罗花的化学成分研究。从该药用植物的地上部分共分离得到16个化合物，通过理化常数和波谱数据鉴定为：异佛手柑内酯(1)，6-甲氧基当归素(2)，欧前胡素(3)，花椒毒内酯(4)，珊瑚菜素(5)，水合氧化前胡素(6)，rivulobirin A (7)，齐墩果酸甲酯(8)，咖啡酸甲酯(9)，银桦酸(10)，(D)-boschniakinic acid (11)，对羟基苯甲酸(12) ， tert-O-β-D-glucopyranosyl-(R)-heraclenol (13) ， 5-methoxy-8-O-β-D-glucopyranosyloxypsoralen (14)，前胡苷V(15)和苯乙醇-O-β-D-吡喃葡萄糖-(1→2)-O-β-D-吡喃葡萄糖苷(16)。所有以上化合物均为首次从该种植物中分离得到。另外我们还首次对藏波罗花挥发油的化学成分进行了气相色谱-质谱(GC-MS)联用分析，共鉴定出39 个挥发性成分。 第三章为全缘叶绿绒蒿化学成分的分离鉴定。从传统藏药材全缘叶绿绒蒿地上部分共分离纯化出8 个化合物。通过理化常数和波谱数据将他们的结构分别鉴定为：去甲血根碱(1)，β-谷甾醇(2)，3-羟基-齐墩果烷-12(13)-烯-30-酸(3)，6-丙酮基-5,6-二氢血根碱(4)，木犀草素(5)，胡萝卜苷(6)，quercetin 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (7)和普托品(8)。其中化合物1，4 和7 为首次从该种药用植物中分离得到。 第四章为综述，总结和归纳了近年来罂粟科紫堇属植物的化学和药理研究进展。 This dissertation consists of four parts. The first, second and third parts report the studies on the chemical constituents from the medicinal plants of Corydalis hendersonii, Incarvillea younghusbandii and Meconopsis interifolia. The forth part reviews the progress of the studies on Corydalis species. The first chapter is about the isolations and identifications of alkalids from the aerial parts of C. hendersonii which is a traditional Tibetan medicine to treat febrifuge, high blood pressure and hepatitis. A new isoquinoline alkaloid, 9-methyl-decumbenine C (6), together with ten known alkaloids, stylopine (1), protopine (2), canadine (3), scoulerine (4), tetrahydrothalifendine (5), tetrahydroberberrubine (7), cryptopine (8), α-allocryptopine (9), 6,7-methylenedioxy-1(2H)-isoquinolinone (10) and 6-acetonyl-5,6-dihydrosanguinarine (11), and β-sitosterol (12) were isolated. Their structures were elucidated by spectroscopic methods. Furthermore, the total alkaloids were analyzed by ESI-MSn. The second chapter is about the isolations and identifications of chemical constituents from the aerial parts of I. younghusbandii. Sixteen compounds were isolated and purified by normal and reversed phase silica gel column chromatography. By spectral analysis, there structures were identified as isobergapten (1), sphondin (2), imperatorin (3), xanthotoxin (4), phellopterin (5), heraclenol (6), rivulobirin A (7), methyl oleanolate (8), methyl caffeate (9), grevillic acid (10), (D)-boschniakinic acid (11), 4-hydroxybenzoic acid (12), tert-O-β-D-glucopyranosyl-(R)-heraclenol (13), 5-methoxy-8-O-β-D-glucopyranosyloxypsoralen (14), decuroside Ⅴ(15), and phenylethyl-O-β-Dglucopyranosyl-(1→2)-β-D-glucopyranoside (16). All of these compounds were isolated from this plant for the first time．By the way, the chemical components of the essential oil from I. younghusbandii were analyzed by GC-MS for the first time. The third chapter is about the the isolations and identifications of the chemical constituents of M. interifolia. Eight compounds were isolated and identified as norsanguinarine (1), β-sitosterol (2), 3-hydroxyolean-12(13)-en-30-oic acid (3), 6-acetonyl-5,6-dihydrosanguinarine (4), luteolin (5), daucosterol (6), quercetin 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (7) and protopine (8). The compounds 1, 4 and 7 were isolated from this herb for the first time. The last chapter is a review of the research progress of the studies on Corydalis species, which includes the chemical constituents in this genus and their pharmacology.

不同放牧强度下高山草甸生态系统碳氮分布格局和循环过程研究

本研究针对川西北高山草甸缺乏科学管理，过度放牧导致草场退化，并由此引发的一系列生态环境问题，选取红原县瓦切乡1996 年草地承包后形成的四个放牧强度草场，即不放牧、轻度（1.2 头牦牛hm-1）、中度（2.0 头牦牛hm-1）和重度放牧（2.9 头牦牛hm-1），作为研究对象，研究了不同放牧强度对草地植物-土壤系统中碳、氮这两个最基本物质的分布格局和循环过程的影响，并探讨了放牧干扰下高山草甸生态系统的管理。 1．放牧对草地植物群落物种组成，尤其是优势种，产生了明显的影响。不放牧、轻度、中度和重度放牧草地群落物种数分别为22，23，26，20 种，群落盖度分别是不放牧96.2%＞中度93.6%＞轻度89.7%＞重度73.6%。随放牧强度的增加， 原植物群落中的优势种垂穗鹅冠草（ Roegneria nutans ）、发草（Deschampsia caespitosa）和垂穗披碱草（Elymus nutans）等禾草逐渐被莎草科的川嵩草（Kobresia setchwanensis）和高山嵩草（Kobresia pygmaea）所取代成为优势种。同时，随放牧强度的增加，高原毛茛（Ranunculus brotherusii）、狼毒（Stellera chamaejasme）、鹅绒委陵菜（Potentilla anserina）和车前（Plantagodepressa）等杂类草的数量也随之增加。 2．生长季6～9 月份，草地植物地上和地下生物量（0～30cm）都是从6 月份开始增长，8 月份达到最高值，9 月份开始下降。每个月份，通常地上生物量以不放牧为最高，重度放牧总是显著小于不放牧；地下生物量随放牧强度的增加表现为增加的趋势，通常重度和中度放牧显著高于不放牧和轻度放牧草地。不放牧、轻度、中度和重度放牧草地6～9 月份4 个月的植物总生物量平均值分别是1543、1622、2295 和2449 g m-2，但随放牧强度的增加越来越来多的生物量被分配到了地下部分，地下生物量占总生物量比例的大小顺序分别是重度88%＞中度82%＞轻度76%＞不放牧69%。生物量这种变化主要是由于放牧使得群落优势种发生改变而引起的，其分配比例的变化体现了草地植物对放牧干扰的适应策略。 3．植物碳氮贮量的季节变化类似与生物量的变化。每个月份，不同放牧强度间植物地上碳氮的贮量有所不同，一般重度放牧会显著减少植物地上碳氮贮量。植物根系(0～30cm)碳氮贮量随放牧强度的增加表现为增加的趋势，通常重度和中度放牧显著高于不放牧和轻度放牧草地。不放牧、轻度、中度和重度放牧草地6～9 月份4 个月的植物总碳平均值分别是547、586、847 和909 g m-2，根系碳贮量占植物总碳的比例大小顺序分别是重度88%＞中度82%＞轻度76%＞不放牧69%；放牧、轻度、中度和重度放牧草地6～9 月份4 个月的植物总氮平均值分别是17、17、23 和26 g m-2，根系氮贮量占植物总氮的比例大小顺序分别是重度79%＞轻度71%＞中度70%＞不放牧65%。 4. 土壤有机碳贮量（0～30cm）的季节变化表现为7 月份略有下降，8 月开始增加，9 月份达到的最大值。土壤氮贮量的季节变化表现为随季节的推移逐渐增加的趋势。增加的放牧强度不同程度的增加土壤有机碳氮的贮量。不放牧、轻度、中度和重度放牧6～9 月份4 个月的土壤有机碳贮量的平均值分别是9.72、10.36、10.62 和11.74 kg m-2，土壤氮贮量分别为1.45、1.56、1.66 和1.83 kg m-2。土壤中有机碳（氮）的贮量都占到了植物-土壤系统有机碳（氮）的90%以上，但不同放牧强度之间的差异不明显。 5. 土壤氮的总硝化和反硝化，温室气体N2O 和CO2 的释放率的季节变化表现为从6 月份开始增加，7 月份达到最大值，8 月份开始下降，9 月份降为最小值。增加的放牧强度趋向于增加土壤氮的总硝化和反硝化作用，温室气体N2O和CO2 的释放率，通常情况下，中度放牧和重度放牧显著地加强了这些过程。 6．垂穗鹅冠草（Roegneria nutans）和川嵩草（Kobresia setchwanensis）凋落物在不同放牧强度下经过1 年的分解，两种凋落物的失重率及其碳氮的损失率3都随放牧增加表现为增加的趋势。在同一放牧强度下，川嵩草凋落物的失重率和碳氮的损失率都高于垂穗鹅冠草凋落物。 7. 尽管重度放牧显著增加了土壤碳氮的贮量，但同时也显著降低了植被群落盖度，降低了植物地上生物量，因此，久而久之会减少植物向土壤中的碳氮归还率；与不放牧和轻度放牧相比，重度放牧又显著增加了土壤CO2 和NO2 的排放量，这是草地生态系统碳氮损失的重要途径。由此可见，对于这些地处青藏高原的非常脆弱的高山草甸生态系统，长期重度放牧不仅导致植物生产力降低，而且将导致草地生态系统退化，甚至造成土壤中碳氮含量减少。 Long-term overgrazing has resulted in considerable deterioration in alpine meadowof the northwest Sichan Province. In order to explore management strategies for thesustainability of these alpine meadows, we selected four grasslands with differentgrazing intensity (no grazing-NG: 0, light grazing-LG: 1.2, moderate grazing-MG: 2.0,and heavy grazing-HG: 2.9 yaks ha-1) to evaluate carbon, nitrogen pools and cyclingprocesses within the plant-soil system in Waqie Village, Hongyuan County, Sichuan Province. 1. Grazing obviously changed the plant species composition, especially ondominant plant species. Total number of species is 22, 23, 26, and 20 for NG, LG, MGand HG, respectively. Vegetation coverage under different grazing intensity ranked inthe order of 96.2% for HG＞93.6% for MG＞89.7% for LG＞73.6% for NG. Thedominator of HG community shifted from grasses-Roegneria nutans andDeschampsia caespitosa dominated in the NG and LG sites into sedges-Kobresiapygmaea and K. setchwanensis. At the same time, with the increase of grazingintensity, the numbers of forbs, such as Ranunculus brotherusii, Stellera chamaejasme,Potentilla anserine and Plantago depressa, increased with grazing intensity. 2. Over the growing season, aboveground and belowground biomass showed a 5single peak pattern with the highest biomass in August. For each month, abovegroundbiomass usually was the highest in the NG site and lowest in the HG site.Belowground biomass showed a trend of increase as grazing intensity increased and itwas significantly higher in the HG and MG site than in the NG and LG sites. Totalplant biomass averaged over the growing season is 1543, 1622, 2295 and 2449 g m-2for NG, LG, MG and HG, respectively. The proportion of biomass to total plantbiomass for NG, LG, MG and HG is 88%, 82%, 76% and 69%, respectively. Higherallocation ratio for is an adaptive response of plant to grazing. 3. Carbon and nitrogen storage in plant components followed the similar seasonalpatterns as their biomass under different grazing intensities. Generally, heavy grazingsignificantly decreases aboveground biomass carbon and nitrogen compared to nograzing. Carbon and nitrogen storage in root tended to increase as grazing increasedand they are significantly higher in the HG and MG sites compared to the LG and NGsite. Total Carbon storage in plant system averaged over the growing season is 547,586, 847 and 909 g m-2 for NG, LG, MG and HG, respectively, while 17, 17, 23 and 26g m-2 for nitrogen. The proportion of carbon storage in root to total plant carbon forNG, LG, MG and HG is 88%, 82%, 76%, 69%, respectively, while 65%, 71%, 70%and 79% for nitrogen. 4. Carbon storage in soil (0-30cm) decreased slightly in July, then increased inAugust and peaked in September. Nitrogen storage in soil tended to increase withseason and grazing intensity. Total Carbon storage in soil averaged over the growingseason is 9.72, 10.36, 10.62 and11.74 kg m-2 for NG, LG, MG and HG, respectively,while 1.45, 1.56, 1.66 and 1.83 for nitrogen. The proportion of carbon (nitrogen)storage in soil to plant-soil system carbon (nitrogen) storage for NG, LG, MG and HGis more than 90%, which is not markedly different among different grazing intensities. 5. Gross nitrification, denitrification, CO2 and N2O flux rates in soil increasedfrom June to July and then declined until September, all of which tended to increasewith the increase of grazing intensity. Generally, heavy and moderate grazing intensitysignificantly enhanced these process compared to no and light grazing intensity. 6. After decomposing in situ for a year, relative weight, carbon and nitrogen loss in the litter of Roegneria nutans and Kobresia setchwanensis tended to increase asgrazing intensity increased. Under the same grazing intensity, relative weight, carbonand nitrogen loss in the litter of Kobresia setchwanensis were higher than these in thelitter of Roegneria nutans. 7. Although heavy grazing intensity resulted in higher levels of carbon andnitrogen in plant and soil, it decreased vegetation coverage and aboveground biomass,which are undesirable for livestock production and sustainable grassland development.What is more, heavy grazing could also introduce potential carbon and nitrogen lossvia increasing CO2 and N2O emission into the atmosphere. Grazing at moderateintensity resulted in a plant community dominated by forage grasses with highaboveground biomass productivity and N content. The alpine meadow ecosystems inTibetan Plateau are very fragile and evolve under increasing grazing intensity by largeherbivores; therefore, deterioration of the plant-soil system, and possible declines insoil C and N, are potential without proper management in the future.

生姜化感作用机理及其栽培模式研究

近年来，随着对作物重茬（连年种植）障碍原因的深入研究，植物的化感作用越来越受到国内外众多学者的重视。而作为重要调料和药用植物的生姜，其连作障碍也备受关注，系统地研究生姜化感作用将有助于理解和最终解决生姜连作障碍问题。本文通过研究生姜不同部位、不同浓度的水浸液对与其间作的两个物种（大豆和四季葱）种子的萌发及幼苗生长的影响，从而证明生姜化感作用的存在；并通过温室盆栽实验研究了生姜的自毒作用（即研究生姜不同部位、不同浓度的水浸液对其幼苗的形态、生理生化、光合作用、土壤酶、土壤微生物多样性及土壤养分的影响），从而揭示生姜退化和衰老的机制，并为生姜筛选出合适的间作物种提供科学依据，对生姜连作障碍提出科学的解决方法。主要研究结果如下： 1. 与对照相比，生姜所有部位（根茎、茎、叶）、所有浓度（10、20、40、 80 g l-1）的水浸液均抑制了大豆种子和葱籽的萌发率、幼苗生长、水分吸收和脂肪酶活性，并且其抑制程度随着水浸液浓度的增加而增强，其生姜各部位水浸液抑制效应的强弱顺序为茎＞叶＞根茎。这一结果表明生姜根茎、茎、叶含有能够抑制大豆种子和葱籽种子萌发和幼苗生长的水溶性化感物质。根茎是生姜的主要收获部位，而生姜的残株（主要是茎和叶）应该从大田中处理掉以减轻其抑制效应。生姜水浸液中主要化感成分包括：根茎水浸液中主要是丁香酸和伞花内脂；茎水浸液中主要是阿魏酸，且其含量最高为73.4 ug/g；叶水浸液中除了阿魏酸，其他六种物质均检测出来，但含量较高的主要有丁香酸、伞花内脂和香豆酸。 2. 生姜茎和叶不同浓度的水浸液均显著抑制了生姜幼苗的株高、每株叶片数和叶面积，其抑制程度随着水浸液浓度的增加而有所增强，而生姜幼苗每株分枝数差异不显著；同时生姜水浸液也极大程度地影响了生姜幼苗的生物量（包括地下生物量、地上生物量和总生物量，均为鲜重）。在同一浓度下，茎水浸液对生姜幼苗形态指标及生物量指标均显示出最强的抑制作用，叶水浸液次之，根茎水浸液最弱。与对照相比，低浓度的生姜根茎水浸液提高了生姜幼苗叶片内四种抗氧化酶（SOD、POD、CAT、APX）活性，高浓度的根茎水浸液抑制了四种抗氧化酶活性，而茎和叶水浸液均随着浓度的增加而抑制了四种抗氧化酶活性，三种水浸液均随着浓度的增加降低了生姜幼苗叶片内叶绿素的含量，而增加了生姜幼苗叶片的相对电导率和丙二醛含量。同时，三种水浸液均随着浓度的增加降低了生姜幼苗的光合参数（包括胞间CO2浓度、气孔导度、蒸腾速率及净光合速率）。 3. 三种生姜水浸液对所测六种土壤酶活性均产生了不同程度的影响，其中影响最大的是酸性磷酸酶和蔗糖酶，在10 g l-1 时就达到了显著水平，并且所有酶均有随着水浸液浓度增加而增大的趋势；相同部位的水浸液随着浓度的增加，细菌和真菌的数量呈增加趋势，而放线菌的数量呈减少趋势；三种生姜水浸液均随着浓度的增加降低了土壤中有机质的含量，加剧了土壤中硝态氮含量的积累，根茎水浸液对土壤有效磷、速效钾和铵态氮均显示出低浓度提高其含量而高浓度降低其含量的趋势，而茎和叶水浸液则随着浓度的增加均降低了其含量。 4. 与生姜单作相比，所有间作系统均在旺盛生长期和收获期不同程度地提高了土壤酶活性，同时也增加了土壤细菌数量及土壤微生物总数但不显著；所有间作系统在旺盛生长期和收获期均不同程度地影响了土壤真菌及放线菌数量（增加或减少），所有间作系统间的多样性指数差异不显著，除了旺盛生长期四种作物（生姜-大豆-四季葱-大蒜）的间作模式显著降低了多样性指数，其值仅为生姜单作的33.18%；生姜与大豆间作不仅提高了19.6%的生姜产量而且获得了较好的经济效益，并且，所有间作系统均显著抑制了生姜姜瘟病的发生。 5. 不同栽培模式不同程度地影响了收获期生姜的株高、分枝数、根茎产量及内在品质。其中处理2显著地促进了生姜的分枝（10.5%），同时处理2、3和4也促进了生姜的生长（株高分别增加了15.0%、11.4%和14.0%），并且这三个处理提高了生姜的产量；处理2和3能有效提高生姜块茎中维生素C（分别较单作生姜显著提高了3.29%和4.05%）、处理3显著提高了可溶性糖（8.2%）、姜辣素（4.6%）和蛋白质等有益物质的含量，降低硝酸盐有害物质的含量（处理2显著降低了14.0%），改善了姜块的外观和内在品质。并且，生姜与大豆间作具有最高的纯收入和产投比，分别较生姜单作提高了24.80%和8.8%。Recently, allelopathy has been more and more paid attentions by national and foreign scholars with profound research on reasons of crop replanted (continuous planted) obstacle. Ginger rhizome is valuable all over the world either as a spice or herbal medicine and ginger replanted obstacle is also paid attentions. Systematic research on ginger allelopathy will contribute to understanding and ultimate solving problem of ginger replanted obstacle. The effects of ginger aqueous extracts with different parts and concentrations on seed germination and early seedling growth of soybean and chive were studied in this article to testify that ginger existed allelopathy. Furthermore, ginger autotoxicity was also studied by pot experiment in greenhouse (namely research on effects of ginger aqueous extracts with different parts and concentrations on morphological indexes, physiological and biochemical indexes, photosynthesis, soil enzymes, soil microbial diversity and soil nutrients) to reveal mechanism of ginger degeneration and senescence, provide scientific basis for selecting appropriate intercropping species and put forward scientific resolvent for ginger replanted obstacle. The main results were as follows: 1. All aqueous extracts at all concentrations inhibited seed germination, seedling growth, water uptake and lipase activity of soybean and chive compared with the control, and the degree of inhibition increased with the incremental extracts concentration. The degree of toxicity of different ginger plant parts can be classified in order of decreasing inhibition as stem＞leaf＞rhizome. The results of this study suggested that rhizome, stem and leaf of ginger contained water soluble allelochemicals which could inhibit seed germination and seedling growth of soybean and chive. The rhizome is the main harvested part of ginger. The residue (mainly stems and leaves) of the ginger plant should be removed from the field so as to diminish its inhibitory effect. The main allelopathic components of three kind of aqueous extracts were as follows: Rhizome extract chiefly contained syringic acid and vmbelliferone and stem extract mainly contained frulic acid whose content was the highest (73.4 ug/g). The other six substances were detected except of frulic acid, but only contents of syringic acid, vmbelliferone and p-coumaric acid were higher. 2. Stem and leaf aqueous extracts of ginger with different concentrations significantly inhibited plant height, leaf numbers per plant and leaf area, and the degree of inhibition increased with the incremental extracts concentration. However, tiller number per plant of ginger seedling showed no significant difference. At the same time, ginger aqueous extracts also influenced biomass including under-ground biomass, above-ground biomass and total biomass (fresh weight) to a large extent. Under the same concentration, stem aqueous extract showed the mostly inhibitory effect on morphological indexes and biomass indexes of ginger seedling. Rhizome aqueous extract showed the leastly inhibitory effect and leaf aqueous extract was intervenient. Enhanced concentration of ginger aqueous extracts significantly reduced total chlorophyll content, accompanying with increases in memberane permeability (REL) and lipid peroxidation (MDA). Compared with the control, rhizome ginger aqueous extract of lower concentration (10 g l-1) increased the activities of major antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT; ascorbate peroxidase, APX) of ginger leaf tissue and higher concentration inhibited the activities of four antioxidant enzymes. However, stem and leaf aqueous extract inhibited the activities of four antioxidant enzymes with increase in concentration. Meanwhile, enhanced concentration of ginger aqueous extracts significantly reduced photo-parameters of ginger seedling (including CO2 concentration, stoma conductivity, net photosynthesis rate and transpiration rate). 3. Rhizome, stem and leaf ginger aqueous extract showed different effect on six soil enzyme activities, and acid phosphatase and invertase showed significant effect when aqueous extract concentration got 10 g l-1. Furthermore, six soil enzyme activities increased with increase in aqueous extract concentration. Bcterial and fungi number tended to increase while antinomyces tented to decrease with the increase in aqueous extract concentration of identical part. Ginger aqueous extracts reduced soil organic matter content with increased concentration, accompanying with NO3-—N accumulation in soil. Rhizome aqueous extract showed the same tendency for available P, available K and NH4+—N, namely lower concentration increased their contents in soil and higher concentration reduced their contents. While stem and leaf aqueous extracts reduced their contents with the increamental concentration. 4. All intercropping systems increased soil enzyme activities to different extent both at VGS and at HS compared to solo ginger. All intercropping systems increased the colony numbers of soil bacteria and total of soil microbe but not significantly either at VGS or at HS. All intercropping systems increased the colony numbers of soil fungi and actinomytes to a different extent (increase or decrease) both at VGS and at HS. For DI, difference between all cultivation patterns and S-G was not significant either at VGS or at HS except that G-S-C-G whose value was only 33.18% of S-G at VGS significantly decreased. G-S not only increased ginger yield by 19.6% but also obtained better economic benefit. Furthermore, all intercropping systems significantly inhibited occurrence of bacterial wilt of ginger. 5. Different cultivated pattern influenced plant height, tiller numbers, rhizome yields and intrinsic quality of ginger. Treatment 2 significantly facilitated tiller occurring (10.5%). Treatment 2, 3 and 4 promoted ginger growth (plant height respectively increased 15.0%、11.4% and 14.0%) and enhanced rhizome yields. Treatment 2 and 3 effectively increased vitamin C content (significantly increased 3.29% and 4.05% compared to solo ginger). Treatment 3 significantly increased contents of beneficial substances such as soluble sugar (8.2%), gingerols (4.6%) and protein. Treatment 2 significantly decreased contents of deleterious substance namely nitrate (14.0%) and improved appearance and intrinsic quality of ginger rhizome. Furthermore, treatment 2 (ginger/soybean intercropping) could obtain better economic benefit and showed the highest net income and ratio of benefit and cost whose values respectively increased by 24.80% and 8.8% compared to solo ginger.

同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

青藏高原栽培青稞遗传多样性研究

青稞，是我国藏区居民对裸大麦的称谓，它不仅是藏民的主要食粮、燃料和牲畜饲料，而且也是啤酒、医药和保健品生产的原料；青稞不仅为藏区人民的健康和经济发展做出了很大的贡献，而且对人类健康和社会经济的可持续发展都有重要的意义。青藏高原是我国及世界上青稞分布和种植面积最大的地区，资源极其丰富。虽然从经典遗传直到分子标记对我国大麦遗传多样性都有研究，但研究手段、数量仍然不够深入，对我国大麦资源遗传多样性研究的信息非常有限，不能很好地满足大麦遗传研究和育种应用的需要，尤其是对西藏栽培大麦的遗传多样性的研究还只是刚刚开始，关于栽培青稞多态性的研究报道很少。本研究采用SSR标记和蛋白质电泳两类技术，从SSR标记位点、单体醇溶蛋白、B组醇溶蛋白和淀粉粒结合蛋白（SGP）等四个方面对我国青藏高原栽培青稞的遗传多样性进行了综合评价。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究采用SSR标记分析了64份青藏高原栽培青稞的遗传多样性，同时评估SSR标记在我国大麦育种和品种鉴定中的应用潜力。选择了30个已知作图位点SSR标记，其中25个标记与重要性状的控制位点连锁紧密。选择的30个SSR标记，5个未得到很好的扩增产物，3个无多态性。22个多态性SSR标记位点中，每位点检测出等位基因2~15个，共检测出等位基因132个，平均每位点6.0 个。各多态位点检测出基因型为2~11种，位点HVM33的基因型最多。各多态位点的多态信息指数为0.16~0.91, 平均为0.65。根据PIC值选择了13个SSR标记用于我国青藏高原栽培青稞基因型鉴定，这些标记的PIC值为0.6以上。结合PIC值和基因型差异，选择了8个多态信息含量高的SSR标记，构建了高效指纹图谱，此图谱能把64份材料完全区分。 贮藏蛋白电泳分析是研究相关编码蛋白基因多态性的非常有效的方法。大麦单体蛋白与小麦醇溶蛋白相对应，具有丰富的多态性，可用于大麦遗传多样性、品种鉴定和群体进化等研究。本研究通过A-PAGE电泳技术研究了84份青藏高原栽培青稞的单体醇溶蛋白多态性。大麦单体醇溶蛋白图谱与小麦醇溶蛋白电泳图谱类似，所分离的蛋白清晰地分为ω－，γ－，β－和α－四个部分。青藏高原栽培青稞单体醇溶蛋白具有丰富的多态性，84份青稞材料中存在43条不同的蛋白带，75种组合带谱；其中67种为单一材料所独有，另8种则分别包含了2-3份材料。每份材料中拥有醇溶蛋白带为6－16条，含有6-10条单体醇溶蛋白带材料较多。西藏和四川材料群体单体醇溶蛋白多态性不同，具有区域特异性。西藏材料中发现了40条不同蛋白带，3条特异带，46 种蛋白组合；四川材料中出现了40种不同蛋白带，26种条带组合, 3条特异带。基于单体蛋白多态性的聚类与材料的来源有一定的相关性。A-PAGE单体蛋白具有丰富的多态性，可作为遗传研究和品种鉴定的标记。 大麦醇溶蛋白（hordein）是大麦籽粒的主要贮藏蛋白，与大麦的营养品质和加工品质密切相关，而且具有丰富的多态性，广泛用于品种鉴定、种质筛选、遗传多样性和亲缘关系研究。B组醇溶蛋白是主要的醇溶蛋白组份，约占总醇溶蛋白的80%，而且具有丰富的多态性。本研究采用SDS-PAGE分析了72份青藏高原栽培青稞B组醇溶蛋白的遗传多样性。青藏高原栽培青稞B组醇溶蛋白具有丰富的多态性，72份青稞材料中存在15种蛋白带，30种组合带谱，其中15种为单一材料所独有，另15种则分别包含了2-10份材料。每份材料中B组醇溶蛋白条带数为4-8条，含5、6条的材料较常见。不同来源的群体材料间B组醇溶蛋白组成存在差异，西藏青稞含有26种蛋白组合带谱，其中有19种特异带谱；四川群体中共发现11种蛋白组合带型，其中有4种特有带谱。两群体中都存在稀有条带。聚类分析将材料分成三组，材料聚类与材料来源地没有明显的相关性。 淀粉粒蛋白（Starch granule proteins, SGPs）是一类与淀粉粒结合的微量蛋白，一些淀粉粒蛋白具有淀粉生化合成中主要的酶蛋白功能，其变异会影响淀粉含量和特性，从而影响淀粉的应用。关于我国大麦淀粉粒组成研究还未见报道。本实验首次开创了我国大麦淀粉粒结合蛋白的研究工作。采用SDS-PAGE电泳技术研究了青藏高原栽培青稞的SGP组成，并分析了不同SGP组合间淀粉含量的差异，初步探索了所分离的SGP蛋白与淀粉合成的关系。66份青稞材料中分离了10种主要的SGP，其表观分子量为40-100KD，低于60KD的SGP带有7条，共有16种组合带谱；各SGP蛋白和组合带谱出现的频率存在差异，青藏高原青稞的SGP组成存在多态性。西藏青稞和四川青稞的SGP组成有很大差异，SGP组成具有地域差异性，西藏青稞含有12种蛋白组合带谱，其中有9种特异带谱；四川群体中共发现7种蛋白组合带型，其中有4种特有带谱；两群体中仅有3种共同的蛋白组合带谱。SGP蛋白特性将66份青稞分为三组, 即Ⅰ、Ⅱ、Ⅲ，材料聚类与材料来源具有一定的相关性。不同组合带谱材料间淀粉含量差异显著性检验结果显示，不同带谱间材料的总淀粉含量、直链淀粉含量和支链淀粉含量有差异，带谱2（SGP1+3+7+9+10）和8（SGP1+2+4+6+8）的总淀粉含量及支链淀粉含量显著大于组合带谱3（SGP1+3+7+10）的总淀粉含量。组合带谱7（SGP1+2+6+8）的直链淀粉含量显著低于带谱11（SGP1+5+8）的直链淀粉。带谱SGP2、3、4、5、6、7、8、9、10可能参与淀粉合成，SGP9可能与高支链淀粉的合成相关。 SSR标记位点、单体醇溶蛋白、B组醇溶蛋白、淀粉结合蛋白等四个方面的研究结果表明青藏高原SSR标记多态性、单体醇溶蛋白多态性、B组醇溶蛋白多态性和SGP多态性都非常丰富，与青藏高原是栽培青稞的多样性分布中心的观点一致。 青藏高原栽培青稞的SSR标记、单体醇溶蛋白、B组醇溶蛋白和SGP多态性表现出很大差异。SSR标记覆盖了整个基因组，多态性非常高。单体蛋白、B组醇溶蛋白、SGP蛋白是育种中非常关注的性状，他们只是代表基因组中的某一区域或位点，多态性相对较低。但单体蛋白多态性很高，84份材料中检测出43条不同蛋白带，75种不同的组合带谱。SSR标记技术和单体蛋白技术都是遗传多样性研究的有力工具，但单体蛋白技术不仅多态性高，而且经济、操作简便，是种质鉴定的理想方法。 对不同标记的多态性材料数据进行聚类，聚类图能为我们提供各材料间的遗传相似信息，为材料选择提供参考。但材料聚类与材料来源的地理区域的相关性表现不一致。SSR聚类和B组醇溶蛋白聚类与材料的来源地无相关性，而单体醇溶蛋白和SGP聚类与材料来源地有一定相关性，即西藏群体和四川群体分别有集中类群，这可能是人为选择的附加效应。 不同来源的群体材料的遗传多样性不同，具有区域特异稀有基因，加强不同地区间资源的交换和配合使用，有利于增加群体遗传多样性和新品种培育。 青藏高原栽培青稞的麦芽浸提性状、淀粉性状、病虫及裸粒等重要农艺性状控制位点存在丰富的变异，遗传基础宽广，可能蕴藏着多种不同的等位基因，是研究重要性状遗传特性、基因资源挖掘和遗传育种的宝贵资源库。 Hulless barley, due to its favorable attributes such as high feed value, good human nutrition，rich dietary fiber and ease processing, attracts people,s attention . Hulless barley plays a very important role in Tibetan life, used as essential food crop, main animal feed and important fuel. In addition to tsampa (roasted barley flour), a main food for Tibetan, hulless barley is also made into cake, soup, porridge, recent naked barley liquor and cornmeal. Qinghai-Tibet Plateau is one of a few areas which plant naked barley widely in the world and also has a long growing history. Genetic diversity of the cultivated hulless barley in this region , however, has not been documented. The study of genetic diversity existing within this population is of particular interest in germplasm identification, preservation, and new cultivar development. This study analyzed the genetic diversity of the cultivated naked barley from Qinghai-Tibet plateau through the study of SSR marker loci and monomeric prolamins, B-horden and starch granule proteins. SSRs are present abundantly in genomes of higher organisms and have become a popular marker system in plant studies. SSRs offer a number of advantages, such as the high level of polymorphisms, locus specificity, co-dominance, reproducibility, ease of use through PCRand random distribution throughout the genome. In barley, several hundred SSRs have been developed and genetically mapped and can therefore be selected from specific genomic regions. The genetic diversity of 64 cultivated naked barley from Tibet and Sichuan was studied with 30 SSRs of known map location.Among the selected SSR markers, PCR products of 5 SSR markers were not obtained and 3 SSR marker loci were monomeric. A total of 132 alleles were identified at 22 polyomeric SSR loci. The number of alleles per locus ranged from 2 to 15, with an average of 6.0. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94, with an average of 0.65. 13 SSR markers with the PIC value >0.6 have been selected for discrimination of Qinghai-Tibet naked barley genotypews. A finger Print map was developed through 7 SSR markers with the high PIC value. It could be used as an efficient tool for gene discovery and identification of gernplasm. Hordeins, the main storage proteins of the barley seed, are composed of momomeric and polymeric prolamins and divided into -A, B, C and D groups in order of decreasing electrophoretic mobility. Hordeins show high inter-genotypic variation and have been extensively used as markers for cultivar identification and analyzing the genetic diversity. This study analyzed the genetic diversity of B-hordein in 72 naked barley from Qinqhai-Tibet Plateau. Extensive diversity was observed. A total of 15 different bands and 30 distinct patterns were found. Jaccard's coefficient of similarity was calculated, and the accessions were divided into three　main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions based on the polymorphism of B-hordein was investigated. Monomeric prolamins show high inter-genotypic variation and have been used as molecular markers for cultivar identification, analyzing the genetic diversity in collections and investigating the evolution processes and structure of populations However, the cultivated hulless accessions from Qinghai-Tibet Pateau in China have never been examined with respect to monomeric prolamins. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-four cultivated hulless barley from Qinqhai-Tibet Plateau in China. Extensive diversity was observed. A total of 43 different bands were found, of which 21 different bands were in the region of ω group, 8 in the region of γ, 8 in the region of β, and 6 in the region of α group. Among the 86 accessions, 75 distinct patterns were identified. The number of bands ranged from 6 to 16, depending on the variety. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A dendrogram was obtained from the analysis of the groups and five main clusters were identified. No relationship between the distribution in the dendrogram and growth habits and origins of the cultivars could be detected. Starch is the major constituent of the cereal endosperm, comprising approximately 65% of the dry weight of the mature wheat grain. The starch formed in all organs of plants is packaged into starch granules, which vary widely between species and cultivars in size and shape. Wheat endosperm starch granules contain about corresponding to the main biosynthase of starch. This report firstly dealed with intraspecific variation of the major SGPs in cultivated naked barley from Qinghai-Tibet plateau. A total of 10 major SGPs were observed in the range of 40KD-100KD and 16 types of patterns were found. Based on the variation of SGPs, accessions studied were classified into 3 groups. A geographical cline of electrophoregram was observed. In addition, significance test of the difference of starch content among groups and types of patterns were done, and the results indicated those SGPs could be related to the content of starch. Diagram obtained through cluster analysis exhibited a structuration of diversity and genetic relationship among cultivated hulless accessions. In breeding program, parents with genetically distant relationship for hybridization will increase genetic diversity of progenies. In conclusion, cultivated naked barley from Qinghai-Tibet Plateau in China presents a high variability with respect to monomeric prolamins，SSR markers , B- hordeins and SGPs. The result of this study supports Qinghai-Tibet Plateau is the center of cultivated hulless barley and the cultivated naked barley is considered to be a gene pool with large diversity and could be applied to breeding for cereal.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.

亚硝化—厌氧氨氧化处理猪场废水研究

猪场废水COD浓度高、氨氮浓度高、悬浮物浓度高，已成为农村面源污染的主要来源，并严重威胁到农村饮用水安全。猪场废水氨氮浓度高、处理难度大，如何采用经济高效的方法，去除氨氮使其达到排放标准，一直是猪场废水处理中面临的重要难题。 厌氧氨氧化是近年受到国内外水处理研究者广泛关注的新型生物脱氮技术，具有不需要外加有机碳源、节省供氧量、降低能耗等优点。虽然国内外研究者对厌氧氨氧化过程的脱氮机理、厌氧氨氧化菌的生理生化特性等进行了多方面的研究，但已有的报道大多以模拟废水为研究对象，以猪场废水为研究对象的报道，在国内外文献中极少有报导。 本论文以猪场废水为主要研究对象，考察了猪场废水的亚硝化过程、厌氧氨氧化的启动过程，并对亚硝化和厌氧氨氧化联合用于猪场废水脱氮进行了探索。 1．论文首先研究了猪场废水的亚硝化过程，考察了废水水质和主要运行条件对亚硝化过程的影响。实验表明：（1）亚硝化阶段反应时间为8到10h时，出水中氨氮和亚硝酸盐浓度比可达到1：1～1：1.23，满足厌氧氨氧化反应对二者比例的要求；达到前述要求时，氨氮去除率达到58.3～65.6 %，亚硝化率在整个过程均保持在97 %以上，COD去除率在59.2～68.6 %；（2）曝气量（溶解氧）对亚硝化过程影响显著，随着曝气量增大，达到厌氧氨氧化要求的氨氮与亚硝酸盐氮浓度比例所需水力停留时间τ越短，pH出现明显下降的时间越短；（3）τ对应的pH在7.8～8.1之间，无需进行pH调节即可满足厌氧氨氧化反应对pH的要求；（4）氨氮和COD降解过程遵循一级反应动力学，氨氮和COD降解的速率常数分别为0.0656～0.0724 1/h和0.0491～0.0664 1/h。 2．在进行亚硝化过程研究的同时，以模拟废水为试验对象，进行厌氧氨氧化启动研究。以反硝化污泥和养殖厂储水池厌氧底泥的混合污泥作为接种污泥，历时大约100天，培育出具有厌氧氨氧化活性的污泥，氨氮和亚硝酸盐氮最高进水浓度分别为223.8 mg/L和171.4 mg/L，去除率最高分别达48%和41.5%，此时二者消耗比例为1.33：1。 3．在猪场废水的亚硝化研究完成和厌氧氨氧化过程初步启动成功后，在模拟废水中逐步加入猪场废水的亚硝化处理出水，逐步实现亚硝化和厌氧氨氧化的组合。亚硝化出水添加到厌氧反应器后，厌氧氨氧化反应仍可继续进行，且去除效率逐步提高。研究发现添加的亚硝化出水中携带的亚硝化细菌在厌氧氨氧化菌膜外层生长并累积，增加了厌氧氨氧化反应基质的传质阻力，妨碍了厌氧氨氧化效率的提高。 4．亚硝化－厌氧氨氧化实际工程应用探索中，生物接触氧化池可在有效去除废水中的有机物的同时实现亚硝化，出水中氨氮和亚硝酸盐比例平均为1.10，可满足后续厌氧氨氧化的要求；在适宜的进水浓度和温度下，ABR池出现了厌氧氨氧化启动的迹象；研究同时发现，水质的波动和气温的变化是工程中影响厌氧氨氧化菌活性的重要因素。 论文的主要创新点在于：（1）以猪场废水为研究对象，以实现厌氧氨氧化为目标，对亚硝化过程进行了比较详细的考察，获得了亚硝化出水满足厌氧氨氧化要求的工艺条件，通过对其COD和氨氮降解过程的考察，得出亚硝化阶段COD降解和氨氮去除的动力学模型；（2）对亚硝化－厌氧氨氧化处理猪场废水进行了探索，确立了影响其污染物去除率稳定的重要因素。 论文的上述研究成果，为厌氧氨氧化技术的实用性研究提供理论依据。 Piggery wastewater, which is characterized by high concentration of COD、ammonium and suspend substance, has become a most important source of non-point source pollution and also severely threats drinking water security in rural area. How to discharge piggery wastewater with the ammonium concentration meeting standard by economical and effective method? This is the most urgent problem in piggery wastewater treatment. As a new biological nitrogen removal technology, Anammox process has been paid more and more attention by researchers all over the world. Anammox has advantages of no need of organic carbon addition, low oxygen consumption and energy consumption. Plenty of investigations have been carried out to the mechanism, physiological and biochemical characteristic of bacteria about Anammox. Most of researches focused on synthetic wastewater, there is rare report about its application in piggery wastewater. In this paper，experimental studies were performed to investigate Sharon process in treatment of piggery wastewater，the start up process of Annammox using synthetic wastewater were studied, the feasibility of applying Sharon-Anammox process in the nitrogen removal of piggery wastewater was evaluated. 1. Sharon process of piggery wastewater was firstly investigated to analyze the effects of water quality and main running parameters, which meet the NH4+-N to NO2--N ratio requirement of successive Anammox. Results showed: (1)During Sharon Process，after 8～10 hours’ reaction the NH4+-N to NO2--N ratio in effluent reached 1:1.0～1:1.23, when the removal percentage of NH4+-N was 58.3～65.6 %, a semi-nitration rate of above 97 % was achieved during the process; meanwhile 59.2～68.6 % of the COD was also removed. (2)The aeration rate（oxygen） had obvious effect on the hydraulic retention time(τ) which met the NH4+-N to NO2--N ratio requirement of Anammox. As aeration rate increased, the hydraulic retention time(τ) was shortened. (3) The pH corresponding to τ was between 7.8 and 8.1, thus it needed no artificial adjustment. (4) The reduction of ammonia and COD followed the first-order reaction kinetics. The velocity constants of ammonia and COD were 0.0656～0.0724 1/h and 0.0491～0.0664 1/h, respectively. 2. The startup of Anammox process using the artificial wastewater was performed simultaneously with Sharon. The aim was to investigate the running parameters of Anammox and make foundation for the combination stage. By using the mixture of denitrifying sludge and anaerobic sludge in tank of the breeding factory, sludge of Anammox activity was cultivated in UASB after 100 days. The removal percentage of NH4+-N and NO2-N were up to 48% and 41.5%, respectively, when the NH4+-N and NO2-N influent concentration were 223.8 mg/L and 171.4 mg/L, respectively, the NH4+-N and NO2-N removal rate was 1.33:1. 3. After investigation of Sharon and startup of Anammox, effluent of Sharon process was added into the synthetic wastewater to combine Sharon and Anammox step by step. It took some time after the addition of Sharon effluent that Anammox reaction continued and the removal rate kept increasing. It indicated that nitrifying bacteria were carried by the Sharon effluent cumulated in the outer layer of Anammox. This enhanced transfer resistance of Anammox reaction and the increasing removal rate was restrained. 4. In the bio-contact oxidation pond of practical project, Sharon process were carried out successfully and organic compounds were removed effectively. An average NO2-N/ NH4+-N rate of 1:1.0 was achieved in the effluent, which met the requirement of successive Anammox. Under condition of suitable influent concentration and temperature, there was evidence that Anammox could start up in ABR. The variety of wastewater and temperature had great affects on Anammox activity in practical engineering. Innovation of this paper: (1) The Sharon process for treating piggery wastewater was discussed in details. Technological parameters that met requirement of Anammox were obtained. The dynamic models of COD and ammonium removal in the process were educed. (2) Sharon-Ananmmox for treatment of piggery wastewater was investigated, and the primary influencing factors was studied. This paper could be a theoretical consult for research of Anammox utility.

影响沼气甲基产孢弧菌生长及其甲烷单加氧酶活性的若干因素研究

本文叙述了影响甲烷氧化细菌沼气甲基产孢弧菌81Z菌株生长和甲烷单加氧酶(MMO)活性的若干因素。沼气甲基产孢弧菌81Z菌株细胞生长被高浓度PO43-(>8mM),NH4+([NH4cl]>500mg/l)抑制；[CuSO4·5H2O]在0～4mg/l范围内。生长随[Cu2+]升高而加强，低[Cu2+](0.1mg/l CuSO4·5H2O)培养基中，添加Cocl2·6H2O(0.238mg/l)；促进菌体细胞生长。发酵罐分批培养过程中，生长延迟期过后，沼气甲基产孢弧菌81Z菌株细胞MMO比活很快达到最高，并稳定至对数生长中后期，随即急剧下降至初始水平。发现沼气甲基产孢弧菌81Z细胞中存在一种MMO活性，它不同于已报道过的两种MMO,MMOL最适PH6.2～6.4，4℃相对稳定，其产生不受培养基中[Cu2+]调控能与甲醇-甲醇脱氢酶系统相偶联，在无细胞抽提液中其活性被400μM[Cu2+]抑制。在低[Cu2+]发酵罐培养条件下，沼气甲基产孢弧菌81Z菌株产生可溶性MMC，其最适PH7.0，4℃不稳定，可被DE-52分离为三组分：A、B、C。为了获得沼气甲基产孢弧菌81Z细胞MMO的最佳催化活性，①采用高[Cu2+]培养基进行发酵罐培养，收集对数生长中期的细胞；②选择反应缓冲液PH6.3；③反应体系中添加5mM甲醇或甲酸是有效的方法。在本研究所采取过的最佳条件下，测得MMO活性为15.9nmol/min·mg干细胞，是以前报道的该菌株活性0.97nmol/min·mg干细胞的十六倍。Some factors which influence growth and MMO activity of Methylosporovibrio methanica 81Z were described. The growth of Methylosporovibrio methanica 81Z is inhibited by high concentration of PO43-(8mM)or NH4+(500mg/lNH4cl). The growth of Methylosporovibrio methanica 81Z increased with rising of copper concentration up to 4mg/l CuSO4·5H2O. At low copper concentration(0.1mg/lCuSO4·5H2O),adding Cocl2·6H2O(0.238mg/l)could enhance the growth of Methylosporovibrio methanica 81Z.With batch culture of Methylosporovibrio methanica 81Z in a fermentor, after lag phase, the activity of MMO reached the highest level rapidly and steady until later log phase, then falled to initial level.MMOL activity differenct from that of two types of MMO reported before was found from Methylosporovibrio methanica 81Z with optimum PH value from 6.2 to 6.4 and relative stabilty at 4℃. Synthsis of the MMOL was not regulated by copper concentaration in medium. Its activity could couple with methane-l-methanoldehydrogenase system, and in cell-free extract, were inhibited by 400μm copper ion. At low copper concentration(0.1mg/lCuSO4·5H2O) and in a fermentor, Methylosporovibrio methanica 81Z could syntheis soluble MMO similar to solble MMO reported before by Palton and Patel. Its optimum PH value was 7.0. It was unstable at 4℃. It could be resoluted into three components: A, B, and C. It was effentive for obtaining the maxtmum MMO with Methylosporovibrio methanica 81Z that (1) to keep high copper concentration(4mg/lCuSO4·5H2O) in a fermentor and harvest cell at middlel lag phase;(2) to choose 6.3 as the PH value of reaction buffer;(3)and to add 5mM methanol or formate into reaction system. In this dy, the MMO activity of cells of Methylosporovibrio methanica 81Z was reached 15.9 nmol/min.mg, dry weight, sixteen times as high as the value(0.97nmol/min.mg, dry weight) reported with the same strain.

泥生绿菌的分离鉴定及其在净化废水中作用的初步探讨

本文介绍了从厌氧间歇膨胀光合反应器内的活性污泥中分离并鉴定的泥生绿菌(Chlorobium limicola Nadson)S1,它属严格厌氧光能自养型细菌，在有硫化物和少量碳酸氢盐存在下，有广泛利用有机物的能力，它的最适生长温度为28-30℃,最适生长PH为6.5-7.0，且含有氢化酶。因此，它能与甲烷发酵菌共存而共同作用，达到废水净化之目的。通过光照(2#反应器)和黑暗(1#反应器)对比实验，表明了在光照条件下即有泥生绿菌S1存在下，反应系统能更好地降低CODcr、BOD5 和提高CH4 含量，在四个负荷段的运行中，2#反应器在后三个负荷段的甲烷含量能稳定在91.6%而1#反应器为87%，2#反应器的二氧化碳含量为4.5%而1#反应器为8.8%，于28.35g/l.d的负荷下，2#反应器CODcr去除率达83.4%，BOD去除率达74.53%，分别较1#反应器高10.8%,6.4%。COD去除率提高了14%，BOD去除率提高了9.3%。本试验的试验条件为：白天自然光照，晚上电源光照，光照强度为1000-2500lux，通过连续动态运转，并以恒定的流速将废液注入反应器中，进水PH控制在6.5-7.2，反应器厌氧，恒温室温度控制在30±1℃。为使整个试验中同一水质条件下进行，进水采用化学合成培养基。This paper reports a Chlorobium Liwicola S1's isolation and identification. It is a strictly anaerobic and photosynthetic autotrophic bacterium. Along with sulfidedepondent CO2 assiwilaton,a few simple organic compounds can be photoassimilated. Acetate is most effectively used. Its best conditons of growth are 28-30℃,PH 6.5-7.0, and it contains hydrogenase. So it can live with methanefermentative bacteria in order to treat wastewater. At the same time, the treatment of wastwater using Chlorobium Limicola S1 with methane-fermontative bacteria under dark anaerobic and light anaerobic conditions is studied. In contrast with 1# reactor-darken, 2# reactor-illuminated can lossen wastewater's CODcr, BOD5 and on hance CH4 content better. In the test, 2# reactor's CH4 content is stable at 91.6%, but 1# reactor's is 87%. The CO2 content of 2# reactor is 4.5%, but 1# reactor's is 8.8%. When the load of teatment is 28.35g/l.d, the COD removal effficiency is 83.4% and the BOD removal efficiency is 74.53% in 2# reactor. They are separately 10.8%, 6.4% higher than 1# reactor's.

超声与磁粉强化含单宁废水生物处理研究

单宁是一种典型的有毒难降解污染物，在制革、造纸、制药、印染等行业废水中广泛存在，对水环境造成污染并且影响废水生物处理效果。本研究针对含单宁废水生物处理效率低、较高浓度时微生物受抑制且污泥容易膨胀等问题，采用超声和磁粉来强化含单宁废水生物处理，研究超声和磁粉对微生物活性、污染物去除及污泥沉降性能的影响，并对其作用机理进行了分析和探讨。 研究结果表明，活性污泥系统中单宁酸容积负荷可以达到1.8kgCOD/(m3·d)，单宁酸和COD去除率分别达到85.2%和79.6%，但如果负荷进一步增大则微生物活性迅速降低。系统在pH 5~8、温度20~35℃、DO>1 mg/L的条件下具有较好的单宁酸降解效果和处理稳定性。单宁降解动力学参数为：μmax =0.208h-1；Ks=226mg/L；Ki=522mg/L；kd=0.0092h-1；Y =0.594。 磁粉对系统处理效果和污泥沉降性能有一定的促进作用，且效果要优于外磁场。适宜的磁粉粒径和投加量分别为0.05~0.15mm和1.0g/L，COD去除率比对照系统提高6.4%，SVI降低28.6%，污泥絮体结构紧密。磁粉强化主要是通过其对污泥菌胶团的凝聚、吸附作用以及对微生物活性的强化作用实现。 在适当强度（0.4W/cm2）和辐照时间（20min）的超声作用下污泥絮体和细胞膜通透性增大，酶分泌也增多，系统的COD去除率比对照提高了8.8%，单宁酶酶活提高了11%。但超声也使污泥絮体结构松散，沉降性能下降，SVI比对照系统升高9.3%。 由于污泥流失加剧导致污泥浓度相对较低，声磁联合强化系统相对于磁粉强化系统其处理效果并没有提高。但相对于单纯活性污泥系统，声磁联合作用下系统处理效果、污泥沉降性能以及系统运行稳定性都得到明显改善。本研究为难降解废水的生物处理提供了一个新的思路。 Tannins are typical refractory and toxic pollutants that commonly exist in wastewater from dye, medicine, paper and leather industries and cause many problems associated with environmental pollution and biological treatment of wastewater. Biological treatment efficiency of tannin-containing wastewater is usually low owing to its biological toxicity and low biodegradability, microbes are usually inhibited under high tannin concentration and sludge bulking frequently occurs. In this study, ultrasound and magnetic powder were used to improve the biological treatment performance of simulated tannic acid-containing wastewater. The effects of ultrasonic irradiation and magnetic powder on microbial activity, tannic acid degradation rate and sludge sedimentation were investigated. The augmentation mechanisms were analyzed and discussed. The experimental results showed that the microbes were prominently inhibited under high tannic acid concentration, but moderate degradation efficiency can be maintained under a tannic acid load of up to 1.8kgCOD/(m3·d), with the tannic acid degradation and COD removal percentage of 85.2% and 79.6% respectively. The highest degradation rates and treatment stability were achieved at pH range of 5~8, temperature range of 20~35℃ and DO concentration of above 1mg/L. The kinetic parameters were estimated, including: μmax =0.208h-1；Ks=226mg/L；Ki=522mg/L；kd=0.0092h-1；Y =0.594. The microbial activity, tannic acid degradation rate and sludge sedimentation were improved by adding Fe3O4 magnetic powder, and the augmentation performance was better than external magnetic field. The appropriate particle size and dosage of magnetic powder were found to be 0.05~0.15mm and 1.0g/L, respectively, under which the COD removal percentage was improved by 6.4% and SVI value decreased by 28.6%, and compact floc structure was observed. This was mainly caused by the flocculation and adsorption effects of magnetic powder against sludge floc and the stimulation of microbial activity under appropriate magnetic field. Under appropriate ultrasonic irradiation (ultrasonic intensity 0.4W/cm2, ultrasonic irradiation time 20min), the permeability of floc and cell membrane are improved, transfer of substrate and oxygen were reinforced; meanwhile, more enzyme were produced by microbes under the slight damage caused by ultrasound. However, the floc structure became loose under ultrasonic irradiation, leading to relatively poor sedimentation, with the SVI value 9.3% higher than the control system. Although the magnetic powder-ultrasonic irradiation combined augmentation system showed no improvement in treatment performance compared with sole magnetic augmentation system owing to its relatively low sludge concentration, it guaranteed the stable operation of system, meanwhile the tannic acid degradation and sludge sedimentation were significantly improved compared with sole activated sludge system. This study gives a new idea for biological treatment of refractory wastewater.

Gamma decay of the lowly excited states of 189Re

189W activities were produced via the 192Os(n, α) reaction using irradiation of isotopically enriched 192Os metallic powder of ～100 mg/cm2 with 14 MeV neutrons. The X-γ and γ-γ coincidence measurements were made so as to obtain γ rays from 189W decay and its coincidence relations. A new simple decay scheme of 189W including three γ rays of 210.2, 229.6 and 260.2 keV is proposed. Two new levels of 189Re at 470.4 and 489.8 keV are assigned.

腹腔温热灌洗联合术后静脉微泵及置泵区域性动脉灌注化疗综合治疗胃肠道恶性肿瘤

目的:探讨胃肠道癌术后预防腹腔转移及肝转移的有效方法。方法:对157例胃肠道癌切除术后病人,随机分成术中腹腔温热灌洗及术后静脉微泵和置泵持续动脉灌注化疗组72例(简称治疗组),单纯静脉化疗组85例(简称对照组),并对其腹腔转移率、肝转移率及3年生存率进行对照研究。结果:治疗组腹腔转移率21.5%、肝脏转移率13.4%、3年生存率71.6%,对照组腹腔转移率41.2%、肝脏转移率22.6%、3年生存率47.5%。两组之间比较有显著性差异(P<0.05)。结论:术中腹腔温热灌洗及术后静脉微泵和置泵持续动脉灌注化疗,对胃肠道癌病人术后腹腔转移及肝转移有良好的防治作用。

~(89)Ru的β缓发质子衰变性质

藉助氦喷嘴带传输系统 ,采用“p γ”符合的鉴别原理 ,成功地合成与鉴别了5 8Ni(36 Ar,2p3n)反应产生的核素89Ru ,并研究了它的 β缓发质子衰变性质 .实验给出89Ru的衰变半衰期为 (1 .2± 0 .2 )s,测定了它的β缓发质子能谱 ,提取出了89Ru经缓发质子衰变布居到子核88Mo的 2 + 和 4+ 态的相对终态分支比 (1 0 0∶6 ) .通过与统计模型计算结果进行对比 ,初步指定了89Ru基态的自旋 宇称 (5 / 2 + 或 7/ 2 ±)和质量过剩 (- 5 9.5MeV) .