副溶血弧菌(Vibrio parahaemolyticus)诱导拟穴青蟹(Scylla paramamosain)血淋巴cDNA文库构建及表达序列标签初步分析  

CONSTRUCTION OF HAEMOLYMPH cDNA LIBRARY OF SCYLLA PARAMAMOSAIN INDUCED BY VIBRIO PARAHAEMOLYTICUS AND ANALYSIS OF EXPRESSED SEQUENCE TAGs

作　　者：蔡小辉[1] 彭银辉[1] 赵鹏[1] 宋忠魁[1] 张琴[1] 黄国强[1] 

机构地区：[1]广西海洋研究所广西海洋生物技术重点实验室,北海536000

出　　处：《海洋与湖沼》2013年第3期684-690,共7页Oceanologia Et Limnologia Sinica

基　　金：广西自然科学基金资助项目;2010GXNSFA013079号;2012GXNSFAA053065号;广西科技攻关资助项目;1114012-11号;南海水产经济动物增养殖广东普通高校重点实验室开放课题;2011003号;国家海洋公益资助项目;201205028-4号

摘　　要：利用SMART技术构建副溶血弧菌(Vibrio parahaemolyticus)诱导的拟穴青蟹(Scylla paramamosain)血淋巴cDNA文库。结果表明,该文库的滴度为1.04×107CFU/mL,文库总容量达1.144×107CFU,克隆子插入片段大小为500—2000bp,重组率达98%。将随机挑选的300个阳性克隆测序,经过质量控制和拼接,共得到141个单基因组簇(UniGenes),75个UniGenes与NCBI非冗余蛋白质数据库中登录的蛋白质序列存在显著相似性,其中20个与免疫防御相关,如kazal-like丝氨酸蛋白酶抑制剂类蛋白、kazal-type蛋白酶抑制剂arasin-like蛋白、抗内毒素因子、抗微生肽、金属硫蛋白、铁蛋白、类70kD热休克蛋白等,达总测序数的6.67%。研究结果证实,构建cDNA文库是获得拟穴青蟹免疫相关基因的可靠途径。To obtain the potential immune-related genes and explore their function, the cDNA library of haemolymph from Scylla paramamosain induced by Vibrio parahaemolyticus was constructed by SMART technology. The results show that the titer of the library was 1.04×10^7 CFU/mL, the library capacity reached 1.14×10^7 CFU, the range of the cDNA in-serts was 500-2000bp, and the recombinant rate was about 98%. A total of 300 clones of the library were randomly picked out and sequenced, and 270 raw sequences of the ESTs were processed and then assembled into 141 unigenes. Blast analy-sis show that 75 unigenes had significant homology to genes with known or putative protein function in non-redundant protein database of NCBI. There were 20 unigenes, 6.67% of total ESTs, related with immune defence functions, such as Kazal-like serine protease inhibitor-like protein, Kazal-type protease inhibitor, arasin-like protein, antilipopolysaccharide factor, antimicrobial peptide, metallothionein, ferritin, 70kD heat shock-like protein and so on. All above results confirm that constructing high-quality cDNA library was a reliable tool to screen out immune-related genes of S. paramamosain.

关 键 词：拟穴青蟹 副溶血弧菌 CDNA文库 表达序列标签 

分 类 号：Q785[生物学—分子生物学]