谷子体细胞无性系变异和抗除草剂基因转化研究

谷子是我国北方具有区域重要性的禾谷类粮食作物。谷子的体细胞无性系变异和外源基因转化相对其它作物研究较少。本研究利用谷子生产的育种中应用的多个优良品种为材料，分析了谷子体细无性系变异发生的影响因素、变异性和频率、DNA分子水平变异和育种应用等问题;采用基因枪法和花粉管通道法，进行了谷子抗除草剂基因转化研究。 主要结果包括： 1. 通过几种外植体的愈伤组织诱导分析,找到了小花分化期前后的谷子幼穗是愈伤组织诱导的最好外植体.提出谷子愈伤组织可按生长状态和结构划分为质密型、松软型和松散型三种基本类型的观点。前两种为胚性愈伤组织并可相互转换，松软型愈伤组织生长状态稳定，可塑性好，是继代培养和因基化的首选类型。供试品种中豫谷1号、高39、郑407和矮宁黄为易培养品种，矮88、豫谷2号、系238、冀14号、C445和青丰谷为相对难培养品种。这些结果为谷子组织培养和生物技术操作提供了基础资料。 2. 首次对多个谷子品种较大群体的无性系变异进行了分析。结果表明，以R_2株系为单位的谷子体细胞无性系表现型变异频率平均为13.0%，不同基因型的变幅为4.3～32.9%;变异涉及株高、抽穗期、穗粒重、出谷率、叶鞘色、育性、抗病性、米色、穗型等多个性状，多数变异为株高和抽穗期等数量性状，少数为矮秆等质量性状：变异的性状多数能在R_3稳定并遗传给后代。从谷子体细胞无性系变异中选出了一批农艺性状得到改良的新品系，其中系103已进入省区域试验，并提供给多家育种单位作为亲本应用。 3. 将RAPD分析技术引入谷子体细胞无性系变异研究。豫谷2号无性系的RAPD多态性变化既有亲本带的缺失，也有新带的产生。用RAPD多态性变化的SMC值度量无性系同亲本相比DNA水平变异的大小，表现型发生变异的无性系,其SMC值分别为0.905～1.0;表现型未发生变异的无性系，RAPD多态性也可能发生变化，其SMC值分布为0.953～1.0。 4. 通过Gus基因瞬时表达单位数的比较，优化了JQ－700基因枪转化谷子松型愈伤组织的操作参数：质粒DNA用量3μg/mg钨粉，CaCl_2农度1.5M，亚精胺浓度40mM，样品室高度7cm，粗弹头为微弹载体，每皿愈伤组织用量1～2g，每次轰击钨粉用量50μg，轰击前4小时和轰击后16～20小时，用含蔗糖150g/l的高渗培养基处理。利用该技术体系，以bar基因为目的基因转化豫谷2号愈伤组织。经选择培养和植株再生，首次获得了抗0.1% bialaphos的正常可育植株，经PCR和Southern blot分析，bar基因已整合到转化体的基因组中，为创造抗除草剂的谷子新种质提供了材料和技术基础。 5. 研究了花粉管通道法转化和花粉介导的基因枪转化谷子的可行性。花粉管通道法转化后代中，获得了抗0.1% bialaphos的抗性植株，经X-gluc组织化学检测，抗性植株叶片的Gus反应为阳性。初步说明该植株可能为转化体，在谷子上为花粉管通道法转化的可行性提供了佐证。花粉介导的基因枪转化未获得转化体。 本文对体细胞无性系变异形成的原因和应用、无性系变异的分子生物学分析、以及谷子的外源基因转化方法等问题进行了讨论。

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in surface sediment and bivalve from the Changjiang Estuary, China

Surface sediments and bivalves were collected from the Changjiang Estuary in December 2003 and November 2004, respectively. Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in these samples were measured with high-resolution chromatography (HRGC)/High Resolution Mass Spectrometer (HRMS). The concentrations of total PCDD/Fs and toxic equivalent (TEQ) were 169.83 +/- 119.63 and 0.81 +/- 0.36 pg/g dry weight (dw) in sediments, and 580.33 +/- 240.17 and 7.24 +/- 3.65 pg/g dw in bivalves. The homolog compositions of PCDD/Fs were similar among samples, the most abundant congener was octa-chlorinated dibenzo-p-dioxin (OCDD) and then octa-chlorinated dibenzofuran (OCDF) and 1,2,3,4,6,7,8-hepta-chlorinated dibenzo-p-dioxin (HOCDD). The herbicide pentachlorophenol (PCP) and sodium pentachlorophenol (Na-PCP) were proved the main source of PCDD/Fs in this area.

Xenobiotics removal from polluted water by a multifunctional constructed wetland

Removal efficiencies on xenobiotics from polluted water in a twin-shaped constructed wetland consisting of a vertical flow chamber with the crop plant Colocasia esculenta L. Schott and a reverse vertical flow one with Ischaemum aristatum var. glaucum Honda, were assessed by chemical analysis and bioassays. After a four-month period of application, removal efficiencies of the applied pesticides parathion and omethoate were 100%, with no detectable parathion and omethoate in the effluent. For the applied herbicides, the decontamination was less efficient with removal efficiencies of 36% and 0% for 4-chloro-2-methyl-phenoxyacetic acid and dicamba, respectively. As shown by toxicity assay with duckweed Lemna minor L., growth retardation may occur if the water treated for herbicide removal is used in irrigation of sensitive cultivars in agriculture or horticulture. In contrast to I. aristatum var. glaucum Honda, the crop C esculenta L. Schott has a high yield in biomass production as a valuable source of renewable energy. (C) 2002 Elsevier Science Ltd. All rights reserved.

A column method for determination of soil organic partition coefficients of eight pesticides

A column method was developed to conveniently and reliably determine the soil organic partition coefficients (K-oc) of three insecticides (methiocarb, azinphos-methyl, fenthion), four fungicides (triadimenol, fuberidazole, tebuconazole, pencycuron), and one herbicide (atrazine), in which real soil acted as a stationary phase and the water solution of pesticide as an eluent. The processes of sorption equilibrium were directly shown through a breakthrough curve(BTC). The log K-oc values are 1.69, 1.95, 2.25, 2.55, 2.69, 2.67, 3.10, and 3.33 for atrazine, triadimenol, methiocarb, fuberidazole, azinphos-methyl, tebuconazole, fenthion and pencycuron, respectively.

施用农用化学品及生防制剂对土壤线虫群落动态影响的研究

Dynamics of soil nematode communities amended with agrochemicals and bio control preparations were investigated in a soybean field. The results showed that the frequency of plant non parasitic nematodes were obviously higher in soil amended with bio control preparations (Doufeng 1) than with urea and herbicide, however, that of plant parasitic nematodes exhibited an inverse trend.

Establishment of a micro-particle bombardment transformation system for Dunaliella saliva

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. saliva with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 mu g/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Simple, mild and convenient synthesis of two novel N-dichloroacetyl oxazolidines and their biological activities

Two N-dichloroacetyl oxazolidines were synthesized with a simple, mild and convenient method. All the compounds were characterized by IR, (HNMR)-H-1 and elemental analysis. The preliminary biological test showed that the compounds protected maize against injury by some herbicides to some extent.

A column method for determination of soil organic partition coefficients of eight pesticides

A column method was developed to conveniently and reliably determine the soil organic partition coefficients (K-oc) of three insecticides (methiocarb, azinphos-methyl, fenthion), four fungicides (triadimenol, fuberidazole, tebuconazole, pencycuron), and one herbicide (atrazine), in which real soil acted as a stationary phase and the water solution of pesticide as an eluent. The processes of sorption equilibrium were directly shown through a breakthrough curve(BTC). The log K-oc values are 1.69, 1.95, 2.25, 2.55, 2.69, 2.67, 3.10, and 3.33 for atrazine, triadimenol, methiocarb, fuberidazole, azinphos-methyl, tebuconazole, fenthion and pencycuron, respectively.

Soil column chromatography for correlation between capacity factors and soil organic partition coefficients for eight pesticides

A soil column chromatographic method was developed to measure the capacity factors (k') of pesticides, in which soil acted as a stationary phase and methanol-water mixture as an eluent. The k' values of eight pesticides, including three insecticides (methiocarb, azinphos-methyl, fenthion), four fungicides (triadimenol, fuberidazole, tebuconazole, pencycuron), and one herbicide (atrazine), were found to be well fitted to a retention equation, ln k'=ln k(w)'-S-phi. Due to similar interactions of solutes with soil and solvent in both sorption determination and retention experiment, log k' has a good linear correlation with log K-oc for the eight pesticides from different classes, in contrast with poor correlation between log k' from C-18 column and log K-oc. So the method provides a tool for rapid estimation of K-oc from experimental k'. (C) 1999 Elsevier Science Ltd. All rights reserved.