野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析

目的]构建野桑蚕中肠组织cDNA文库并分离其丝氨酸蛋白酶基因。方法]利用TaKaRa公司生产的cDNA Library Construction Kit构建了野桑蚕中肠组织的cDNA文库,并采用测序法克隆分析丝氨酸蛋白酶基因cDNA。结果]经鉴定,文库的滴度达6.2×105pfu/ml,文库插入片段的平均大小为1.2 kb左右。从文库的测序结果中获得了野桑蚕丝氨酸蛋白酶基因片段(登录号:EU672968),序列分析结果显示:该基因片段由854个核苷酸组成,编码284个氨基酸残基。通过对该基因片段编码的氨基酸和其他10种昆虫的丝氨酸蛋白酶基因编码的氨基酸的同源性分析,发现该氨基酸序列与其他丝氨酸蛋白酶具有一定的同源性。结论]该基因的发现对于研究家蚕和野桑蚕对外来入侵物的抗性具有重要意义。

Construction of Midgut Tissue-specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment

Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene.Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit(TaKaRa),then the serine protease gene was cloned via sequencing of the yielded cDNA library.Result] The titer of cDNA library reached 6.2×105 pfu/ml,average insert size was about 1.2 kb.The serine protease gene cDNA fragment was obtained from colony sequencing(Accession No:EU672968).The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues.Homology analyses show some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects.Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.