玉米混合组织的EST文库的构建和部分EST序列的分析

Construction of EST library of mixed tissues from maize and analysis of sequences of partial ESTs

以CLONTEECH的pDNR-LIB质粒为载体,构建了干旱和盐碱共胁迫48h和相应的无胁迫正常生长条件下耐旱玉米(ZeamaysL.)YQ7-96品系的雌雄分化期(12片叶龄)的混合组织(叶、茎和花器)EST文库。该文库容量>2×105个克隆,覆盖了预计的玉米基因组5万个基因的4倍以上。随机对3168个克隆中插入的cDNA进行测序,获得有效序列2098条,其中,2002个克隆中的cDNA进行了5'端测序,96个克隆中的cDNA的进行了5'和3'两端测序。结果表明,3'端测序的96个克隆的cDNA都含有polyA尾巴;进一步对这2098条组装分析,形成168个重叠群(Contig)和1693个单拷贝EST(Singlet),代表了共计1861条独立基因(Unigene);根据GenBank中的玉米EST数据库和BLASTn分析软件进行核苷酸同源性分析,1731条(93.01%)EST的score值大于或等于100,130条(6.99%)的EST的score值小于100,说明这130条属于新的表达基因的序列,有21条可以定位在玉米的不同染色体上;BLASTx分析表明,2098条EST中,1007条(48%)有功能注释信息,1091条(52%)无功能注释信息。该文库中的EST将会对公共数据库中现存的玉米EST数据起到补充作用。

With the CLONTEECH vector pDNR-LIB,an EST library of the drought-tolerant maize (Zea mays L.) line YQ7-96 was made from the mixed tissues containing leaves,stems and floral organs at the stage( i.e. up to 12 leaves) of differentiation of the sex organs under normal cultivation without stresses and in the presence of co-stress of water deprivation,salt and alkali for 48 h.This library consisted of over 2×10~5 EST clones,which covered over 4 times of about 50000 genes predicated in the maize genome. The insertion cDNA fragments of 3168 clones selected randomly from the library were sequenced. The total 2098 valid sequences were finally obtained,in which 2002 ESTs were sequenced from 5' end of the cDNAs,96 ESTs were sequenced from 5' and 3' end of the cDNAs. Sequencing results showed that 3' ends of cDNA fragments of all 96 clones sequenced from 3' end had poly A tails. The 2098 valid sequences formed 168 contigs and 1693 singlet,which represented the total 1861 unigenes; Based upon the maize EST database and BLASTn analysis in the GenBank,1731 (93.01%) ESTs had score of > or = 100,130 (6.99%) ESTs had score of <100 . This implied that the latter 130 ESTs belonged to sequences of novel genes expressed. 21 ESTs were localized on different chromosomes of maize; The BLASTx analysis showed that in ESTs of 2098 unigenes 1007(48%) ESTs could be functionally annotated,and 1091 (52%)ESTs had no functional information. The above results implied that ESTs in our library would be complementary to the existing maize EST database in the public database.