甲基对硫磷水解酶结构和功能的研究

本研究以本实验室分离的有机磷农药高效降解菌Pseudomonas sp．WBC-3为材料，通过X-射线晶体学研究确定了甲基对硫磷水解酶（MPH）的三维结构，并在结构基础上探讨了甲基对硫磷水解酶的结构与功能的关系。利用生物信息学手段对Pseudomonas sP.WBC-3中的甲基对硫磷水解酶基因进行分析，推测论H的结构基因的编码序列为398-1393 bp，蛋白大小为331个氨基酸，且具有一个由35个氨基酸组成的信号肤。同时发现，MPH是一个不同于具有相似生物学功能的有机磷水解酶（OPH）的蛋白质，而且在PDB库中尚无与MPH同源性较高的蛋白结构。晶体的生长依赖于高纯度的蛋白质的获得。在本研究中，我们采用阳离子交换树脂和凝胶过滤两步纯化获得了纯度达95％以上的MPH溶液，并采用等离子体质谱仪测定MPH为含锌的金属酶。采用悬滴蒸汽扩散法获得了PI和P43212两种晶型的晶体以及硒标记的MPH晶体。在获得单晶以后，最终利用尸43212晶型的晶体通过多波长反常散射法（MAD）解析了MPH的三维结构。MPH的晶体结构为同源二聚体，具有与OPH类似的二价金属离子组成的活性中心：其中一个单体含有两个锌离子，另一个单体含有一个锌离子和一个锅离子；每个单体的两个金属离子通过AsP 151、His 152、His 302、His 147、His 149、His 234和A印255与蛋白质相连，一个H2O分子桥连于两个金属离子，还有一个H2O分子只与一个金属离子配位。在MPH三维结构知识的基础上，利用分子生物学手段对MPH活性中心附近可能的底物结合氨基酸位点进行了研究。通过定点突变获得了针对Trp179，Phe 196和Phe 119三个位点的六个突变体W179F、W179A、F196W、F196A、FllgW和Fll9A，系统比较了突变体与野生酶的催化动力学特点。结果表明Trp 179，Phel％是MPH活性中心的底物结合部位的关键氨基酸。而Phe 119在与底物结合中的作用不明显。

黄河上游青海沙蜥种组的系统地理学研究

沙蜥属（Phrynocephalus）的卵胎生类群主要分布在我国青藏高原，包括南疆沙蜥（P. forsythii）、西藏沙蜥（P. theobaldi）、红尾沙蜥（P. erythrurus）、贵德沙蜥（P. putjatia）和青海沙蜥（P. vlangalii）。其卵胎生生殖方式适应了高寒生境，与青藏高原隆升有关。纵观前人的研究，上述几种卵胎生沙蜥的分类、系统发育关系以及生物地理都还存在疑问。本文研究了分布在若尔盖湿地的青海沙蜥红原亚种（P. v hongyuanensis）以及分布在黄河上游其它地区青海沙蜥种组的地理分布格局，并探讨了其形成机制。 青海沙蜥在黄河上游主要分布于若尔盖湿地以及青海湖周边地区。若尔盖湿地青海沙蜥红原亚种的生境由于沼泽的形成被切割成不连续的斑块，通过遗传分析可以推测这种特殊生境对它们遗传结构的影响。其次，贵德沙蜥、青海沙蜥的青海湖周边各居群以及若尔盖湿地居群之间的系统地理格局还未见报道。因此本文以居群为单位，将它们作为一个复合体，通过系统地理研究，可以了解其种群遗传结构，据此分析相关的地质历史事件对其分布的影响。主要结果如下： 1. 若尔盖湿地青海沙蜥红原亚种的种群遗传结构： 共研究了三个地理单元（红原（HY）、辖曼（XM）、玛曲（MQ））的7个采集点的72个个体。所有ND4-tRNALeu序列比对得到785 bp的片断，定义了9种单倍型。结果显示总的核苷酸多样性较低，单倍型多样性较高。分子变异分析（AMOVA）显示3个单元间差异显著（P<0.01），遗传变异主要存在于地理单元间，占62.61%。除MQ单元，XM各居群与HY居群混杂在一起，单倍型网络图没有显示出单倍型和地理位置的对应关系。XM单元单倍型的不配对分布（Mismatch distribution）为明显左移的单峰，且Fu’s Fs test为负值，表明XM单元可能经历了近期种群扩张，有足够的时间积累单倍型的多态性，还不足以大幅提高核苷酸多样性，这是其单倍型多样性较高和核苷酸多样性较低的原因。MQ单元遗传多样性低而与其他单元显著分化，推测这与3万年前黄河在若尔盖玛曲之间贯通有关。近期沼泽的形成对XMb居群的隔离时间短，使得其遗传多样性低但还不足以形成大的遗传差异。无论黄河的贯通还是沼泽的形成其隔离形成的时间都不长，其作用改变了单倍型出现的频率，也出现了一些特有单倍型，但共享单倍型还广泛存在，还不足以使得不同居群之间形成较大的遗传距离。 2. 黄河上游青海沙蜥种组的分布格局与地史过程的关系： 黄河上游青海沙蜥种组包括贵德沙蜥、青海沙蜥指名亚种的青海湖周边各居群、青海沙蜥红原亚种若尔盖湿地居群、以及青海湖以西的部分居群（序列由Genbank下载获得），总计22个居群189个样品。所有ND4-tRNALeu序列比对得到703个位点，定义了39种单倍型。以南疆沙蜥为外群构建的贝叶斯树以及MP法构建的无根树，都分为A、B两大组。其中A包括若尔盖湿地居群以及玛多居群（A1）、青海湖以西的居群和兴海居群（A2）、西藏沙蜥；B包括青海湖以南的居群和天祝居群（B1）、青海湖以东北的居群（B2）。单倍型网络图分别对应了系统发育树上的各支。按照系统发育结果分组进行分子变异分析，得到组间变异占88.63％，各组间差异显著（P=0.000）。种群遗传结构分析得到，A1和B2可能经历了近期的种群扩张，前者扩张时间约为0.105－0.189 Ma B.P.（million years before present），后者为0.057－0.102 Ma B.P.，可能与末次间冰期的气候变暖有关。A2和B1对应的两个地理单元都具有较强的种群遗传结构，较为稳定。 青海沙蜥种组A、B两大支之间遗传距离大，分化明显，分化大约发生在4.29－2.38 Ma B.P.，推测青藏运动的A幕运动后复杂的地形变化可能是它们产生分化的原因。B1和B2分化大约发生在1.73－0.96 Ma B.P.，这与湟水流域构造运动发生的时间相符。在早、中更新世时期，B1支内部各居群可能有交流，中更新世末共和盆地出现的抬升以及河流溯源改道等事件可能是引起这支内部多个单倍型丢失的原因。A1、A2支的分化可能与倒数第三次冰期降临之后气候变冷、阿尼玛卿山的大冰帽有关。 The viviparous group of genus Phrynocephalus is mainly distributed in the Qinghai –Tibetan Plateau, including P. forsythii、P. theobaldi、P. erythrurus、P. putjatia and P. vlangalii. These species are adapted well to the cold clime there, and the origin of this group was the result of a vicariance event associated with the uplifting of the Qinghai -Tibetan Plateau. Although many works have been done, there are still several questions about classification、phylogenetic relationships and the biogeography of this group. The phylogeographic pattern of the P. vlangalii complex on the upper reaches of the Yellow River and the P. v. hongyuanensis in Zoige Wetland were studied in this thesis. On the upper reaches of the Yellow River, P. vlangalii complex are distributed in Zoige Wetland and the southeast and northeast region of Kuku-noor Lake. Because of the forming of the wetland in Zoige, the habitats for sand lizards are divided into many discontinuous ones, and it is necessary to analyze genetic structure in these unique habitats. The phylogeographic patter among P. putjatia、populations of P. vlangalii in the southeast region of Kuku-noor Lake and populations of P. vlangalii in Zoige Wetland hasn’t been studied yet, and the complicated geological events of the Plateau may play an important role in the populations’ diversity and species forming there. So these populations were gathered as a complex, and phylogeographic analysis were used to clarify these doubts. According to the two topics above, this thesis has two parts of results as follows: 1. Three geographic units of P. vlangalii hongyuanensis in Zoige Wetland were defined, and they were Xiaman （XM）、Hongyuan （HY） and Maqu （MQ）. 785bp fragments of the mtDNA ND4-tRNAleu were determined from 72 samples and nine haplotypes were identified. As a whole, the nucleotide diversity was low，but the haplotype diversity was high. Analysis of molecular variance （AMOVA） showed that the three units were distinctly different（P<0.01），and 62.61% of the total genetic diversity was attributable to variation among units. There were 3 haplotypes shared among XM and HY，and no geographic clustering was observed except MQ from the TCS network. The results from the mismatch distribution analysis and Fu’s Fs test implied that there might be a recent population expansion in the XM unit, and this may be the reason why XM had a high haplotype diversity but a low nucleotide diversity. We estimate that the MQ and XMb have lower diversities because of some very recent geographic events, such as the formation of the Yellow river’s upriver and the Zoige Wetland. Although they are distinctly different, not enough time has passed for them to have diverged a great genetic distance. 2. 189 samples in 22 populations of P. vlangalii complex were collected, including P. putjatia、populations of P. vlangalii in the southeast and northeast region of Kuku-noor Lake、 populations of P. vlangalii in Zoige Wetland and the data from Genbank. 703bp ND4-tRNALeu sequences identified 39 haplotypes. P. forsythii was selected as outgroup, and both the Bayesian tree and the MP unrooted tree were divided into two groups（A、B）. A included populations in Zoige Wetland and Xinghai（A1）、populations in the west of Kuku-noor Lake（A2）、P. theobaldi, and B included populations in the southeast of Kuku-noor Lake and Tianzhu（B1）、populations in the northeast of Kuku-noor Lake（B2）. The haplotype network agreed with these groups. AMOVA showed that these five groups were distinctly different（P<0.01）, and 88.63% of the total genetic diversity was attributable to variation among groups. There might be recent population expansion in A1 and A2, which corresponded to the dry climate of the last interglacial period. The expansion times were 0.189-0.105 Ma B.P. and 0.102-0.057 Ma B.P., respectively. A2 and B1 had strong genetic structure. The large genetic distance between A and B showed that they had been separated from each other for a long time（about 4.29-2.38 Ma B.P.）, and it corresponded to the A phase of Qingzang Movement. The diversity between B1 and B2 at 1.73-0.96 Ma B.P. may be caused by the geological event in Huangshui valley. In early Pleistocene, populations in B1 may have gene flow because of geographic linkage, and later the uplift of the Plateau and the change of river route there made a few haplotypes lost. A1 and A2 were divided into two parts by A’nyemaqen Mountains at 0.66-0.37 Ma B.P., which maybe corresponded to glaciations at about 0.7 Ma B.P.

小麦抗白粉病新基因PmCD1和抗条锈病基因YrCD的鉴定及分子标记筛选

本研究对自育小麦白粉病抗源“07鉴126”和条锈病抗源CD1437、CD0534-5进行抗性遗传分析和微卫星引物的筛选，建立了与PmCD1和YrCD抗病基因连锁的SSR分子标记，主要研究结果如下： 1.小麦白粉抗源“07鉴126”抗白粉病基因的鉴定和分子标记的建立 品系“07鉴126”对我国目前白粉菌强优势生理小种E09、E11和其它多种小种表现免疫或高度抵抗。Pm-sus是07鉴126的自然突变感病株。利用“07鉴126”和Pm-sus的F2抗病性分离群体进行抗条锈病性遗传分析和分子标记定位，结果表明，“07鉴126”的白粉抗性为显性单基因控制的全生育期抗性，暂命名为PmCD1；并筛选到了与PmCD1共分离的显性SSR分子标记Xbarc183。系谱分析和分子标记分析表明PmCD1来源于荆州黑麦。抗谱分析表明PmCD1不同于已知的黑麦抗白粉基因，是一个新的抗白粉病基因。Xbarc183这一分子标记的建立为PmCD1的分子标记辅助选择和抗病基因累加提供了方便。 2.小麦条锈抗源CD1437抗条锈病基因的鉴定和分子标记的建立 利用对优势条锈菌小种条中32免疫的小麦品系CD1437及其自然突变感病株Yr-sus杂交构建F2、F3抗病性分离群体。抗条锈病性遗传分析结果显示，1437的抗条锈性为显性单基因控制的全生育期抗性，该基因暂命名为YrCD。SSR分析发现，位于1B染色体上的7个SSR标记Xcfd65、Xgwm11、Xgwm18、Xbarc187、Xwmc406、 Xwmc419、Xwmc216依次分布在YrCD的一侧，与YrCD的遗传距离在1.7 cM至9.2 cM。YrCD和YrCH42的等位性分析显示二者可能为等位基因。YrCD和Yr24、Yr26的抗谱相似。系谱分析和分子标记分析表明贵农20是YrCD的供体。本研究推测YrCD、Yr24、Yr26和YrCH42可能是等位基因，并推测Yr-sus是缺失突变体。 3. 小麦条锈抗源CD0534-5抗条锈病基因的鉴定 利用对条中32免疫的小麦抗条锈病品系CD0534-5及其感病重组自交系CD0534-4建立F2抗病性分离群体。抗条锈病性遗传分析表明，CD0534-5的条锈抗性由两对独立的显性主效基因控制。用BSK法分析，发现其中一对基因与SSR分子标记Xgwm11、Xgwm18、Xwmc128、Xwmc419连锁，该基因是来源于贵农20的YrCD。另一抗性基因来源贵农19，是极有利用价值的未知抗性基因。 This study focused on the investigation and identification of a novel powdery mildew resistant gene PmCD1 in wheat lines 07jian126 and stripe rust resistant gene YrCD in wheat lines CD1482 and CD0534-5, and screened SSR markers tightly linked to them. The main results were as follows: 1.Identification and SSR markers screening of a novel powdery mildew PmCD1 in wheat line 07jian126. Using a Pm resistant wheat line 07jian126 and its Pm susceptible mutant, a F2 population was constructed. Pedigree and genetic analyses indicated that the Pm resistance in 07jian126 was tranderred from rye (Secale cereale L.) cv. Jinzhou and was controlled by a single dominant gene. Differential test using 21 Bgt isolates revealed that the Pm resistant gene in 07jian126 is novel and was temporarily designated as PmCD1. A dominant SSR marker Xbarc183/130 bp was found co-segregated with PmCD1 in the F2 population. The diagnostic band of Xbarc183/130 bp co-segregating with PmCD1 could be used as an ideal marker in marker-assisted-selection during wheat breeding program. 2. Identification and SSR markers mapping of yellow rust resistant gene YrCD in wheat line CD1437. Wheat line CD1437 was highly resistant to predominant Chinese stripe rust race CYR32 at both seedling and adult stages. A F2 population was developed from the cross of CD1437 and its Yr susceptible mutant Yr-sus. Genetic analysis indicated line CD1437 contains a single dominant gene, temporarily designated YrCD. Seven SSR markers on the chromosome 1BS including Xcfd65, Xgwm11, Xgwm18, Xbarc187, wmc406, Xwmc419and Xwmc216 were close linked to YrCD with a genetic dsitance 1.7 cM to 9.2 cM. YrCD came from wheat cultivar Guinong 20. Allelic test of CD1437 and Chinese cultivar Chuanmai 42 indicated that YrCD and YrCH42 were allelic. Reaction patterns of YrCD and Yr24, Yr26 to 21 PST isolates were the same. These results suggested that YrCD and Yr24, Yr26, YrCH42 might be allelic. 3.Detection and identification of yellow rust resistance genes in wheat line CD0534-5 Wheat line CD0534-5 was highly resistant to predominant Chinese stripe rust race CYR32, while its recombinant inbred line CD0534-4 was susceptible. Genetic analysis with a F2 population developed from the cross of CD0534-5 and CD0534-4 indicated line CD0534-5 contains two independent dominant genes. Four SSR markers on the chromosome 1BS including Xgwm11, Xgwm18, Xwmc128, Xwmc419 were found to linked with one gene in CD0534-5. According the locations of makers and pedigree, this gene in CD0534-5 was YrCD, from cultivar Guinong 20. Another resistant gene was from cultivar Guinong 19, different with those genes on 1B such as Yr10, Yr15, Yr5 etc, was a valuable resistant gene in wheat breeding.

青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.

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.

RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

基于ITS 序列和matK 基因序列的中国药用石斛及其混伪品的分子鉴定

为了从分子水平对中国药用石斛及其混伪品进行鉴定，本文选取了核rDNA ITS 序列和叶绿体DNA 的matK 基因序列进行研究。采用改良的CTAB 法提取石斛的基因组DNA，PCR 产物直接测序法对17 种（共32 份）药用石斛的核糖体内转录间隔区ITS 全序列进行测定，克隆测序法对12 种（共22 份）药用石斛的叶绿体的matK 基因序列进行测定，运用BioEd it，MEGA4.0 等生物软件分析了石斛属植物的rDNA ITS 序列及叶绿体的matK 基因序列的特征，比较了石斛属间、种间、种内不同居群（品种）间的序列碱基差异及遗传距离，应用邻接法构建分子系统树。主要研究结果如下： （1）建立了17 种（共32 份）药用石斛rDNA ITS 区碱基全序列数据库，其中，ITS1 的长度为228~234 bp，GC 含量为45.7%~53.0%，变异位点167 个，占总位点67.34%，信息位点106 个，占总位点42.74%，ITS2 长度为241~247 bp，GC含量为44.8%~55.7%，变异位点165 个，占总位点66.27%，信息位点115 个，占总位点46.18%。 （2）建立了12 种（共22 份）药用石斛的叶绿体matK 基因全序列数据库，叶绿体matK 基因长1410 bp，变异位点51 个，信息位点11 个。除了存在碱基替换的遗传变异外，还存在碱基的插入和缺失。 （3）通过ITS 序列比较分析了各材料间的遗传距离和碱基差异，属间的遗传距离为0.295，石斛种间的平均遗传距离为0.142，碱基相差2~156 个，种内各居群间的平均遗传距离为0.002，碱基相差1~2 个。属间的遗传距离大于种间的遗传距离，种间的遗传距离大于种内不同居群（品种）间的遗传距离。 （4）根据分析石斛叶绿体的matK 基因序列得到，外类群（密花石豆兰）与石斛属间最小遗传距离为0.027，石斛种间的平均遗传距离为0.008，种间最大的遗传距离0.014， 最小的遗传距离为0.003，碱基相差8~20 个。种内不同居群（品种）遗传距离为0.001，相差1~5 个碱基。 （5）利用17 种石斛的全序列数据库及遗传分析软件，通过对待检种rDNA I TS区进行序列测定，成功地对10 个待检种进行了鉴定，并且在原植物开花后得到了验证。 （6）运用12 种石斛的matK 基因全序列数据库及遗传分析软件，成功地对4个待检种进行了鉴定，同样在原植物开花后得到了验证。 （7）本文利用石斛的核糖体内转录间隔区ITS 序列和叶绿体的matK 基因序列数据库分别构建了NJ 树，外类群与石斛属间石斛种间以及种内不同居群（品种）间均能在NJ 树中明显分化开来，二者构建的分子系统树一致，为石斛的分子鉴定提供了依据。 In order to identify Chinese Herba Dendrobii and its adulterant species on molecular level, we studied the sequences of rDNA ITS and chloroplast matK gene. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materia ls) were purified and then sequenced. The PCR products of chloroplast matK gene of Dendrobium (22 materia ls) were purified, cloned and then sequenced. The characteristic of the sequences and the genetic dista nce were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies, and different populations. Phylogenetic trees were constructed using the NJ method by the biology softwares including BioEd it, MEGA4.0 etc. The ma in results as follows: (1) It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materia ls). The ITS1 was 228~234 bp, the GC content accounting for 45.7%~53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241~247 bp, the GC accounting for 44.8%~55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. (2) The database of the chloroplast matK gene sequences was built up, which contained 12 species of Herba Dendrobii (22 materia ls). The matK gene sequences were about 1410bp in length. There were 51 variable sites and 11 Parsim-Informative sites. And there were nucleotides insertions and deletions in some species , in addition to the nucleotides substitutions. (3) The rDNA ITS sequences were compared and analyzed by the biology softwares. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was 0.295. The avera ge genetic dista nce was 0.142 between Dendrobium species, and there were 2~156 variable nucleotides. The avera ge genetic dista nce between different populations was 0.002, and there were 2~156 variable nucleotides. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was greater tha n that of Denrobium interspecies. Meanwhile, the genetic dista nce between Denrobium species was also greater tha n that of different populations (variaties). (4) The characteristics of the chloroplast matK gene sequences were obtained after analyzing by the biology softwares. The minima l genetic dista nce was 0.027 between Bulbophyllum odoratissimum and Dendrobium . The ma xima l genetic dista nce was 0.014 between Dendrobium species, and there were 20 variable nucleotides. The minima l genetic dista nce between populations was 0.003, and there were 8 variable nucleotides.The genetic dista nce between populations was 0.001, and there were 1~5 variable nucleotides. (5) The molecular Phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then we authenticated 10 materia ls on molecular level. What’s more, they had been proved when these pla nts flowered. (6) The molecular Phylogeny tree was built up on the database of chloroplast matK gene sequences of 12 species of Herba Dendrobii with the biology softwares.Then 4 materia ls were authenticated on molecular level. Moreover, they had also been proved when these pla nts were in flower. (7) The Phylogenetic trees were separately constructed on the sequences of rDNA ITS and chloroplast matK gene B. odoratissimum and Dendrobium all could be distinguished on the Phylogenetic trees. Meanwhile, the Phylogenetic trees based on two groups of sequences were coincident. rDNA ITS and matK gene sequence could be used as molecular markers for authentication of Herba Dendrobii.

红发夫酵母发酵虾青素的研究

红发夫酵母分离于北美西部高山地区和日本一些岛屿上落叶树的渗出液中，因其所产主要色素为在水产养殖、食品和医药工业有广阔应用前景的虾青素而成为研究的热点。本论文对红发夫酵母Phaffia rhodozyma 的生长特性、培养参数与培养基组分对生长和虾青素积累的影响及其优化、虾青素合成的调节控制、虾青素的提取测定及红发夫酵母耐高温菌种的诱变进行了系统的研究。 虾青素是红发夫酵母的胞内色素，要对其进行分析首先要对红发夫酵母进行破壁处理，实验发现二甲亚砜是最有效的破壁溶剂，用氯仿和丙酮可以有效地把类胡萝卜素从二甲亚砜破壁后的红发夫酵母细胞中提取出来。 在固定摇床转速为200 rpm，温度为20 ℃的条件下，当种龄为36 h，以10％的接种量接入装液量为30 mL的250 mL三角瓶，初始pH为5.5时最有利于红发夫酵母的生长及类胡萝卜素的合成。 本实验中红发夫酵母最佳利用碳、氮源分别为蔗糖和蛋白胨，但蛋白胨价格昂贵，不适宜作单一氮源，因此使用硫酸铵和酵母膏作为复合氮源。 本论文采用了BP神经网络结合遗传算法的方法来优化红发夫酵母的发酵培养基，得到红发夫酵母发酵培养基的最佳配比为：蔗糖45.10 g/L、硫酸铵3.00 g/L、硫酸镁0.80 g/L、磷酸二氢钾1.40 g/L、酵母膏3.00 g/L、氯化钙0.50 g/L，使用优化后的培养基发酵类胡萝卜素产量达到8.20 mg/L，干重达到9.47 g/L，类胡萝卜素的产量比起始培养基提高了95.90%，干重提高了89.40%。 从代谢途径出发对红发夫酵母合成虾青素调控调控，选择谷氨酸、乙醇、VB1作为添加剂，通过正交试验设计得出三者添加水平分别为0.2 g/L，0.1% (V/V)，10 mg/L时，类胡萝卜素产量提高了25.73%，达到了10.31mg/L。 通过上述优化培养，本论文中红发夫酵母的虾青素产量从1.33 mg/L提高到9.12 mg/L，产量提高了6.86倍；总类胡萝卜素产量从4.23 mg/L提高到10.31 mg/L，产量提高了2.44倍；细胞干重从5.00 g/L提高到11.35 g/L，提高了2.27倍，总体提高效果显著。 红发夫酵母属于中低温菌，本论文采用紫外复合诱变的方式，通过高温筛选，得到一株能在35 ℃下能生长的突变株，但所产类胡萝卜素中虾青素所占比例很小，可能是诱变改变了红发夫酵母的代谢途径，阻断了虾青素的合成。 Phaffia rhodozyma is a heterobasidiomyceteous yeast that was originally isolated from the slime fluxes of brich tree wounds in mountain regions of northern Japan and southern Alaska. Phaffia rhodozyma produces astaxanthin as its principal carotenoid pigment, which has potential applications in acquaculture, food and pharmaceutical industry. This paper researched ways to break cell, analysis of astaxanthin, characteristics of growth, culture parameters and the effects of components of medium on growth and astaxanthin formation , optimization of culture medium, control of astaxanthin synthesis and mutagenesis of Phaffia rhodozyma. It is necessary to disrupt the yeast cell for extracting astaxanthin considering the yeast accumulating carotenoids in cell. Dimethyisulphoxide was the most effective solvent for breaking the yeast cell; acetone and chloroform were effective to extract carotenoids out of the disrupted cell. The optimum pH for growth and carotenoids synthesis is 5.5, the optimum medium volume is 30 mL (in 250 mL flask), the optimum culture time of inoculum is 36 h, the optimum inoculum concentration is 10%. The research on culture medium showed: sucrose is the best one of 6 carbon sources for growth and astaxanthin synthesis. Peptone is the best nitrogen source for growth and astaxanthin synthesis. Uniform Design was used for trial design of the formula medium components, then back-propagation neural network was established to modeling the relationships between the carotenoid yield and the concentration of medium components. Genetic algorithm (GA) was used for global optimization of the model. The optimum combination of the medium was obtained: sucrose 45.10 g/L, ammonium sulfate 3.00 g/L, magnesium sulfate 0.80 g/L, potassium dihydrogen phosphate 1.40 g/L, yeast extract 3.00 g/L, calcium chloride 0.50 g/L. The yield of carotenoid reached 8.20 mg/L, which was 95.90% higher than that of the original medium. Glu, VB1 and ethanol were selected as fermentation addictives, after Orthogonal Test, the carotenoid contents increased by 25.73% when adding 0.16 g/L Glu, VB1 10 mg/L and ethanol 0.1% (V/V). After the above optimization, the astaxanthin content increased 6.86 folds, which is 9.12 mg/L. The carotenoids content increased 2.44 folds, which is 10.31 mg/L. The biomass increased 2.27 folds, which is 11.35 g/L. Phaffia rhodozyma grows in the mild temperature range of 0 to 27 ℃, in this work, a thermotolerant mutant was selected through UV-irradiation. It can grows at 35 ℃, and showed increased carotenoid content. The optimal growth temperature for this mutant is 30 ℃. But the mutant can only produce carotenoids with little astaxanthin accumulation.

苯并(a)芘及其代谢产物的连续降解研究

在以驯化过的芽孢杆菌(BA-07)降解BaP的过程中,鉴定出2个BaP的未开环代谢产物顺式-4,5-二氢-4,5-二醇-BaP(cis-BP4,5-dihydrodiol)和顺式-7,8-二氢-7,8-二醇-BaP(cis-BP7,8-dihydrodiol).由于该产物对微生物有一定毒性,所以难于进一步降解.为提高BaP降解的同时,降低cis-BP4,5-dihydrodiol和cis-BP7,8-dihydrodiol的累积,对2种降解方法(即单纯用BA07降解和运用高锰酸钾与BA-07耦合的方法降解)进行了比较,并且优化了连续降解的参数.结果表明,①对BaP及其代谢产物的连续降解,化学氧化与微生物耦合(高锰酸钾与BA-07)的降解效果明显好于单纯利用微生物(细菌BA-07)的降解;②在同一时间取样,cis-BP4,5-dihydrodiol的残留率均高于cis-BP7,8-dihydrodiol;③当BaP的浓度为40μg/mL,培养基的最佳pH为7.0,以琥珀酸钠为共代谢底物,可以显著提高BaP降解率,降低cis-BP 4,5-dihydrodiol和cis-BP7,8-dihydrodiol的累积.同时提出了化学氧化与微生物协同的方法可以有效促进环境中持久有机污染物的连续降解.

Performance improvement in direct methanol fuel cell cathode using high mesoporous area catalyst support

Black Pearls 2000 (designated as BP- 2000) and Vulcan XC-72 (designated as XC-72) carbon blacks were chosen as supports to prepare 40 wt % (the targeted value) Pt/C catalysts by a modified polyol process. The carbon blacks were characterized by N-2 adsorption and Fourier tranform infrared spectroscopy. The prepared catalysts were characterized by inductively coupled plasma atomic emission spectroscopy, transmission electron microscopy, scanning electron microscopy (SEM), in situ cyclic voltammetry, and current-voltage curves. On BP- 2000, Pt nanoparticles were larger in size and more unevenly distributed than on XC-72. It was observed by SEM that the corresponding catalyst layer on BP- 2000 was thicker than that of XC-72 based catalyst at almost the identical catalyst loading. And the BP- 2000 supported catalyst gave a better single cell performance at high current densities. These results suggest that the performance improvement is due to the enhanced oxygen diffusion and water removal capability when BP- 2000 is used as cathode catalyst support. (C) 2004 The Electrochemical Society.

Heat capacities and thermodynamic functions of p-chlorobenzoic acid

The heat capacities of p-chlorobenzoic acid were measured in the temperature range from 80 to 580 K by means of an automatic adiabatic calorimeter equipped with a small sample cell of internal volume of 7.4cm(3). The construction and procedures of the calorimetric system were described in detail. The performance of this calorimetric apparatus was evaluated by heat capacity measurements on alpha-Al2O3. The deviations of experimental heat capacities from the corresponding smoothed values lie within +/-0.28 per cent, while the inaccuracy is within +/-0.40 per cent, compared with the recommended reference data in the whole experimental temperature range. A fusion transition at T = 512.280 K was found from the C-p-T curve of p-chlorobenzoic acid. The enthalpy and entropy of the phase transition, as well as the thermodynamic functions {G((T)) - G((298.15))}, {H-(T) - H-(298.15)} and {S-(T) - S-298.15}, were derived from the heat capacity data. The mass fraction purity of p-chlorobenzoic acid sample used in the present calorimetric study was determined to be 0.99935 by fraction melting approach. (C) 2002 Elsevier Science Ltd. All rights reserved.