盐胁迫下胡杨cDNA文库的构建及其nhx基因的克隆

采用CTAB法提取经600 mmol·L^-1 NaCl胁迫处理后的胡杨总RNA,利用SMART技术构建载体为λTriplEx2的盐胁迫条件下胡杨的表达型cDNA文库（SSL）。文库的滴度为1.2×10⁶ pfu·ml^-1,重组率约为90.6%,扩增后的滴度为3×10⁹ pfu·ml^-1,容量约为2.4×10¹¹。插入片段长度均大于400 bp,平均长度约为790 bp。从文库中随机挑选32个阳性克隆进行3′端测序,并将测序结果通过NCBI (National Center for Biotechnology Information) 数据库进行比较,发现有23个EST序列与已知序列有明显的同源性,而其余的与NCBI中的已知序列相似性较低（e值>10^-4）,可能是未知基因。其中序列SSL061、SSL179与脱水素序列有较高的序列一致性。同时合成引物,通过PCR扩增文库的方法,从中筛选获得存在于植物细胞膜上并在植物受盐胁迫时起重要作用的nhx基因,进一步验证了文库的质量。     

小麦抗病基因表达谱中的文库构建与筛选方法研究

以抗白粉病品系“百农 32 17×Mardler”BC5F4为材料 ,构建了白粉病菌诱导的普通cDNA文库和抑制消减杂交(SSH)cDNA文库。分别对两文库进行了一定规模的测序 ,获得普通cDNA文库不重复ESTs 387条和SSHcDNA文库ESTs 76 0条。将获得的ESTs与GenBank序列进行了BLASTn、BLASTx同源性分析。结果表明 :在普通文库中 ,一些参与光合作用与核糖体构成等的基因出现频率较高 ,而获得的抗病相关基因则较少。消减文库在构建方法、抗病相关基因的富集等方面具有明显的优越性 ,是目前抗病基因表达谱研究中的较好方法。利用高密度点阵膜杂交技术对两文库的筛选结果表明 ,该方法具有相对简便易操作、杂交膜可反复使用等优点 ;但也存在mRNA及同位素用量大等问题。经筛选 ,消减文库中有 5 4 1%的功能已知ESTs为抗病相关基因 ,被证明参与了小麦抗白粉病反应     

The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.     

47个早期人胚胎低丰度表达基因ESTs筛选及结果分析

构建高质量ｃＤＮＡ文库在基因克隆、ｍＲＮＡ差异展示、表达序列标签测序和基因定位等研究中具有十分重要的作用。为了从早期胚胎中分离人类新基因,构建了受精后３周龄的人ｃＤＮＡ文库,用标记的一链ｃＤＮＡ探针对该文库的６５０８个克隆子进行菌落原位杂交,得到１６７７个无任何杂交信号的低丰度表达克隆子,从中随机挑选了４７个进行５′端部分测序,将测序结果与三大基因库进行序列同源性比较,发现１８个克隆（３８．３％）     

Classification of sequences expressed during the primordial and basidiome stages of the cultivated mushroom Agaricus bisporus

Two complementary DNA (cDNA) libraries were constructed from tissues isolated from primordia and basidiomes of Agaricus bisporus to characterize genes involved in mushroom development. Using single-pass sequencing of 869 cDNA clones, we found 477 expressed sequence tags (ESTs), including 466 not previously described in the databases for A. bisporus. A BLASTX search revealed that 374 ESTs had similarities with protein sequences available from databases; 193 of these ESTs were categorized according to their putative function. Most ESTs were assigned to one of four roles: metabolism (23%), cell structure (15%), cell growth and division (12%), and protein destination and storage (10%). The remaining ESTs with putative homologues were classified in 10 additional categories. Many ESTs could not be functionally assigned. Based on redundancy levels, at least 4 ESTs were preferentially expressed in each tissue type. Sequence analysis also suggested the presence of paralog tyrosinase genes in the A. bisporus genome.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.Communicated by R. Hagemann     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。