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

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1.04×107 CFU/mL,文库总容量达1.144×107 CFU,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× 107 CFU/mL,the library capacity reached 1.14× 107 CFU,the range of the cDNA inserts 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 analysis 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.