条锈菌诱导的小麦抗病与感病近等基因系SSH文库构建及分析

为分析条锈菌诱导下的小麦抗病与感病近等基因系之间差异表达的基因,以接种小麦条锈菌CY26小种的抗病近等基因系Yr4/6×Taichung 29幼苗叶片cDNA作为实验方,接种CY26的感病亲本Taichung 29幼苗叶片cDNA为驱动方,利用抑制消减杂交(SSH)技术构建了一个包含1 300余克隆的消减文库。对文库中600个克隆进行了反向Northern点杂交筛选,对获得的阳性克隆进一步进行了Northern杂交验证,获得显著差异的克隆12个。经测序和BlastX分析,其中6个差异表达序列的推测产物分别为亮氨酸重复序列蛋白、过氧化氢酶、硫氧还蛋白、RNA结合蛋白、抗坏血酸过氧化物酶和热激蛋白。除亮氨酸重复序列为信号传导类蛋白外、其他几个均为抗病防御类蛋白。     

中华鳖孵化一周胚胎的肾脏/尿生殖嵴混合组织的cDNA文库构建与特征

为克隆到与胚胎发育有关的新基因,以孵化一周的中华鳖(Trionyxsinensis)胚胎的肾脏及尿生殖嵴混合组织为原始材料,采用SMART和长距离PCR技术,构建了一个中华鳖cDNA表达文库。分析结果表明,该未扩增的cDNA文库大约含有4.134×105个克隆。任意挑取了192个克隆,提取质粒后用SfiⅠ酶切鉴定表明没有插入片段的克隆为9个,插入了cDNA片段的克隆为183个,插入的cDNA片段大小范围为0.4-3.8kb。其中,插入片段在0.4-1.0kb之间的克隆为19个,在1.1-2.0kb之间的克隆为53个,在2.1-3.0kb之间的克隆为92个,大于3.1kb的克隆为19个。另外,任意挑选45个克隆,提取质粒后,从5′端进行测序,Blast分析表明,除了21个序列在GenBank中找不到同源序列外,其余24个序列在GenBank中均被证实有各自相应的同源序列,这些序列代表6种类型的基因核糖体蛋白基因、代谢酶基因、组织特异性表达的基因、转录因子基因、受体蛋白基因及其它基因。该发育阶段特异性cDNA文库可以进一步用于中华鳖胚胎发育过程中相关基因的鉴定分离、结构功能分析、以及表达调控机制等研究。     

草豆蔻cDNA文库的构建及鉴定

以草豆蔻花序原基为材料,构建了cDNA文库。原始文库滴度为0.8×106pfu/mL,扩增后滴度为4.23×1011pfu/mL。插入片段大小在500bp～1.5kb之间,重组率为95.3%。以水稻RAP1A基因中包含MADS-box保守区段的序列为探针对该文库进行筛选,获得的阳性克隆经测序及序列比对分析,确认其中共有10个含MADS-box的阳性克隆。     

鹅源新城疫病毒ZJI株基因组cDNA克隆的序列修饰

将鹅源新城疫病毒ZJI株全基因组cDNA克隆通过酶切切下包含T7启动子区域和转录载体的片段,将其自身环化后获得约6．5kb的质粒。设计引物,利用基因定点突变技术,在此质粒上T7启动子与NDV Leader序列之间突变插入额外的3个G碱基,将此突变最终引入到原基因组cDNA克隆中。应用RT—PCR技术从尿囊液中扩增NDV基因组F／HN基因区域部分片段,利用限制性内切酶BsmBI将扩增片段连接,最终将原cDNA克隆中相应片段替换下。测序结果表明,原基因组cDNA克隆中特定位置碱基插入突变成功,F／HN基因区域碱基突变均得以纠正。以上cDNA克隆的修饰与替换为该毒株的反向遗传研究打下了基础。     

陆地棉GhSUMO基因的克隆与黄萎病菌诱导表达分析

SUMO化修饰与植物抗病防御、信号转导和耐旱等有着直接的关系.本文以抑制差减杂交(suppression subtractive hybridization,SSH)技术获得SUMO的EST为信息探针,对棉花EST数据库进行同源搜索和电子克隆,获得了全长为396 bp的SUMO基因编码区cDNA全长,我们将该基因命名为GhSUMO,推测该基因编码95个氨基酸.分别以抗黄萎病陆地棉品种豫棉21号的cDNA和DNA为模板,对该基因进行了PCR扩增验证,测序结果表明,GhSUMO基因序列与电子克隆序列一致,且没有内含子.蛋白序列分析表明,该蛋白具有保守泛素结构域和C端双Gly的断裂/连接位点,以及保守的疏水表面和Ulp1-Smt3互作位点.系统进化分析表明,该蛋白与蓖麻的同源序列表现了最高的相似性,与其它双子叶植物同源序列次之,而与单子叶植物的相似性较低.棉苗接菌后实时定量PCR结果显示,该基因表达量在接黄萎病菌的量48 h后明显上调,96 h达到未接菌对照的5倍以上.GhSUMO基因可受黄萎病菌诱导表达,表明该基因在陆地棉抗黄萎病的机制中可能发挥重要作用.     

结球甘蓝抗TuMV相关基因的克隆

以结球甘蓝高抗TuMV自交不亲和系84075为材料,构建了cDNA文库。根据抗病基因保守序列（NBS－LRR）设计一对简并引物,以84075的基因组DNA和cDNA为模板,进行PCR扩增,分别扩增出一条513bp片段,扩增片段进行克隆测序。选取两个与抗病基因同源性较高的克隆片段作探针（命名Borl,Bor2）,对构建的cDNA文库进行筛选,得到一个阳性克隆（命名TuR2）,测序及序列分析表明,该基因总长为762bp,编码226个氨基酸、包含681bp的开放阅读框。与已克隆的抗病基因有不同程度的同源性。利用TuR2作探针,进行了Southern杂交、Northern杂交以及抗病性的共分离检测分析。结果表明,TuR2可能吧单拷贝形式存在,其表达是组成成型的,且无组织特异性;初步确定是一个与结球甘蓝抗TuMV相关的基因。     

利用抑制差减杂交技术分离马铃薯晚疫病抗性相关基因

以晚疫病病原菌混合小种接种处理48h的马铃薯水平抗性材料(R-gene-free)叶片为目的材料,以未处理材料作为对照,用抑制差减杂交技术构建了一个富集晚疫病抗性相关基因的差减文库。应用反向Northern技术对840个克隆进行斑点杂交筛选,筛选出150个病原诱导后信号明显增强的克隆。26个片段测序结果表明：部分片段基因功能与抗病性明显相关。7个差异表达片段与GenBank EST数据库中已有晚疫病原诱导马铃薯叶片得到的EST有很高同源性(达95％～100％);部分片段核苷酸或氨基酸序列分别与番茄、烟草、拟南芥等的EST序列或氨基酸序列有较高同源性;另有4个基因片段在GenBank EST数据库中未找到明显的同源序列,可能为新发现的基因片段。     

DADS诱导HL-60 G2/M期阻滞消减文库的构建与初步筛选

目的:构建DADS诱导HL60-细胞G2/M期阻滞的差异表达文库,初步筛选相关基因.方法:分别提取无DADS和有DADS处理HL-60细胞的总RNA和mRNA,构建消减cDNA文库.随即挑选正向SSH的阳性克隆,PCR检测插入片段,将含插入片段的克隆测序.Blastn分析差异cDNA片段的同源性.结果:构建了DADS诱导人白血病HL-60细胞G2/M期阻滞差异表达文库,其中包含120个正向SSH的克隆和100个反向SSH的克隆.50个随机正向SSH的克隆测序、比较同源性,发现5个新EST片段,已经在GenBank中登录.结论:所构建的DADS诱导人白血病HL-60细胞G2/M期阻滞消减文库为进一步筛选白血病HL-60细胞G2/M期阻滞相关基因奠定了基础.     

拟南芥psy基因cDNA的克隆及其植物表达载体的构建

为了获得胚乳组织特异性表达八氢番茄红素的转基因小麦,以拟南芥幼叶RNA为模板,由特异型引物通过RT-PCR一步法得到大小约为1.3kb的基因片段,将此片段连接在克隆载体pMD18-T进行测序,结果表明,该基因片段为八氢番茄红素合成酶基因(psy)cDNA片段。将psy基因片段正向插入植物表达载体pLRPT中高分子量麦谷蛋白亚基基因1Dx5启动子与nos终止子之间,pLRPT载体无1Dx5基因开放阅读框,运用菌落PCR对重组子进行筛选与鉴定,说明拟南芥psy基因已正确插入pL-RPT,成功构建了植物表达载体pLRPTPSY。     

鹅源新城疫病毒ZJI株基因组cDNA克隆的序列修饰*

将鹅源新城疫病毒ZJI株全基因组cDNA克隆通过酶切切下包含T7启动子区域和转录载体的片段,将其自身环化后获得约6.5kb的质粒。设计引物,利用基因定点突变技术,在此质粒上T7启动子与NDV Leader序列之间突变插入额外的3个G碱基,将此突变最终引入到原基因组cDNA克隆中。应用RT-PCR技术从尿囊液中扩增NDV基因组F/HN基因区域部分片段,利用限制性内切酶BsmB I将扩增片段连接,最终将原cDNA克隆中相应片段替换下。测序结果表明,原基因组cDNA克隆中特定位置碱基     

Identification of Alternaria brassicicola genes expressed in planta during pathogenesis of Arabidopsis thaliana

Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on cruciferous plants including economically important Brassica species. The purpose of this study was to identify fungal genes expressed during infection of Arabidopsis. In order to identify candidate genes involved in pathogenicity, we employed suppression subtractive hybridization (SSH) between RNA isolated from A. brassicicola spores incubated in water and on the leaf surface of the Arabidopsis ecotype Landsberg. Two populations of cDNA were created from total RNA extracted after 24h when approximately 80% of the spores had germinated either on the leaf surface or in water. Following SSH, expression of clones was examined using dot-blot macro-arrays and virtual Northern blots. 47 cDNA clones differentially expressed between Alternaria infected Arabidopsis leaves and spore germination in water were selected for sequencing. Seventy-seven percent (36) of the cDNAs had significant homology to fungal sequences from databases examined, including available fungal genomes, while 13% (11) had no homology to sequences in the databases. All 36 genes had significant matches with genes of fungal origin, while 11 genes did not have significant hits in the databases examined. Five sequences were expressed on the plant leaf surface but not during spore germination in water according to virtual Northern blots. These five cDNAs were predicted to encode a cyanide hydratase, arsenic ATPase, formate dehydrogenase, major Alternaria allergen, and one unknown. RT-PCR was used to examine the expression of these five genes during infection of Brassica oleraceae var. capitata (cabbage), in vitro growth in nutrient rich media, and infection of Arabidopsis thaliana. Four of these genes are expressed in the nutrient rich medium, while the unknown gene P3F2 was only expressed during plant infection. The results of this study provide the first insight into genes expressed during A. brassicicola infection of Brassica species that may be involved in fungal pathogenesis.     

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells weresuccessfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase Ⅱ and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.     

Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.

YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.     

用抑制差减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段

以Hoagland溶液培养的梭梭幼苗(H)为对照群体,甘露醇处理的梭梭幼苗(M)为目标群体,进行抑制差减杂交.用经过H cDNA差减的M cDNA构建了一个含有大约400个独立克隆的差减文库;采用差减前的H cDNA和M cDNA以及正向/反向差减杂交后的cDNA为模板标记探针,对随机挑取的100个重组质粒进行差示筛选,获得了21个阳性候选克隆.从这些阳性候选克隆中随机挑取了8个进行Northern blot分析,证实其中3个候选克隆代表了在M中特异表达或表达增强的基因,序列分析和同源性比较表明它们与逆境胁迫有关;而另外5个候选克隆无Northem杂交信号,推测它们为低丰度转录本.     

Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones

As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined. The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA. The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively. These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation. Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A. thaliana mitochondria. The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells.