虾夷扇贝外套膜和肾脏组织cDNA文库构建以及EST的初步分析

Construction of cDNA libraries from mantle and kidney of Japanese scallop (Mizuhopecten yessoensis) and ESTs analysis

利用SMARTcDNA文库构建试剂盒首次构建了健康虾夷扇贝(Mizuhopecten yessoensis)外套膜和肾脏2种组织的cDNA文库。2个文库容量分别为2.30×106CFU/mL和1.20×106CFU/mL,重组率均超过98%,平均插入片段长度均大于1kb,文库质量符合标准要求。将随机选取的4009个克隆进行5′端测序,得到3884个有效序列(外套膜923个,肾脏2961个),测序成功率96.9%。经过质量控制和拼接,共得到2007条单基因簇(Unigenes),包含363个序列重叠群(Contigs)和1644条单一序列(Singletons)。通过BLASTx和BLASTn搜索比对,共有659个Unigenes(外套膜157个;肾脏502个)获得了基因注释(E<e-5),占Unigene总数的32.84%。对Unigene的功能和分类进行了初步分析,发现多种与免疫相关的基因,如凝集素、超氧化物歧化酶、溶菌酶、大防卫素等。利用SSRIT在线工具对2007条Unigene查找微卫星,发现有69个EST中含有微卫星序列,其中40个可以用于引物设计。虾夷扇贝cDNA文库的成功构建,为进一步的EST分析、功能基因的表达和克隆、多态性EST-SSR的筛选以及虾夷扇贝分子遗传学研究、种质资源评价和分子选育等奠定了基础。

The cDNA libraries of mantle and kidney of Japanese scallop（Mizuhopecten yessoensis）were constructed with SMART cDNA library construction kit.The mantle and kidney libraries have high library titers of 2.30×106 and 1.20×106 CFU/mL,respectively.The recombined efficiency of each cDNA library exceeds 98%,with an average insert size of 1 kb.Total 4 009 clones from two libraries were randomly picked out and sequenced from the 5′end using a T7 universal primer,and 3 884 raw sequences of the ESTs were obtained and then assembled into 2 007 unigenes including 363 contigs and 1 644 singletons.These genes exhibited homology with those having known functions.Many genes were related to immune functions,such as lipase,C-type lectin,superoxide dismutase,goose- type lysozyme,serine protease inhibitor and big defensin.Sixty-nine microsatellite-containing ESTs were identified from the 2 007 unigenes by using the online bioinformatics software SSRIT.Based on the flanking sequences of the microsatellite loci,40 pairs of microsatellite primers were designed.The successful construction of cDNA libraries from Japanese scallop made the bases of further analysis of ESTs functional gene discovery,and cloning and screening polymorphic EST-SSR primers.