黏质沙雷氏菌诱导的黄喉拟水龟SMART cDNA文库构建及相关基因的鉴定

Construction of a smart cDNA library of Asian yellow pond turtle stimulated with Serratia marcescens and identification of related genes

以致病黏质沙雷氏菌人工感染的黄喉拟水龟肝组织为材料,应用SMART(switching mechanism at 5’end of RNA transcript)技术,构建了黄喉拟水龟的全长cDNA文库。首先用SMARTTM PCR cDNA systhesis kit合成全长的双链cDNA,通过琼脂糖凝胶分级分离技术切除小片段的cDNA,将大于500 bp的cDNA连接到pGEM-T载体中,电转化到J M109感受态细胞。在构建好的文库中,经测定,文库约含有1.8×105个重组子,重组效率达90%,插入片段多在0.5～3.0 kb之间。对库中长度约为1 000 bp的80个基因进行了测序,结果显示大部分首次在龟类发现。测序鉴定的基因包括免疫相关基因9个、信号传导基因6个、催化酶类基因8个、糖代谢相关基因2个、转运相关基因1个、结构基因2个。

To understand anti-infectious response to bacteria in the Asian yellow pond turtle （Mauremys mutica）,a full length cDNA library was constructed for it by SMART technique experimentally infected with Serratia marcescens.Firstly,the double-strand cDNA was synthesized using SMART TM PCR cDNA systhesis kit.Second,the ds cDNA was separated into two parts based on the size distribution of amplified ds cDNA by agarose gel size fractionation.The part shorter than 500 bp was discarded and the other one longer than 500 bp was ligated to the pGEM-T vector.The ligation mixture was transformed into E.coli JM109 by electroporation.The cDNA library contained 1.8×105 independent clones with DNA inserts of 0.5-3.0 kb.The recombination rate was 90.3%.We sequenced 80 cDNA clones about 1 kb and most of the genes were found the first time in reptiles.We classified these clones in functions with 9 in immunity,6 in cell signaling,8 in catalytic activity,2 in sugar/glycolysis metabolism,1in transport metabolism,and 2 in cell structure.The successfully constructed cDNA library will be essential for rapid isolation of differentially expressed genes related to Serratia marcescens infection and useful for understanding the anti-infectious molecular mechanism in the Asian yellow pond turtle.