家蚕精巢特异性蛋白激酶基因Bm-TSSK的克隆及序列与表达分析

Cloning,Sequence and Expression Analysis of a Testis-specific Protein Kinase Gene Bm-TSSK from Silkworm,Bombyx mori

睾丸特异性丝氨酸/苏氨酸蛋白激酶(testis-specific serine/threonine kinase,TSSK)首先在哺乳动物中被发现,是一类在精子发生过程起重要作用的蛋白激酶。通过EST同源筛选方法,用人(Homo sapiens)的TSSK2、TSSK4、TSSK6基因cDNA和小鼠(Mus musculus)的TSSK1、TSSK3、TSSK5基因cDNA为探针在GenBank家蚕EST数据库中搜索同源基因,通过在家蚕基因组数据库的同源性检索以及电子克隆,获得一条家蚕精巢特异性蛋白激酶基因的序列,命名为Bm-TSSK(GenBank登录号:JN159476)。据此预测序列设计引物,成功地在家蚕精巢组织中克隆了Bm-TSSK基因完整的开放阅读框(ORF),序列分析及同源性比对表明:Bm-TSSK序列长1 402 bp,ORF为1 041 bp,没有内含子,推测编码346个氨基酸残基,在编码氨基酸序列20～284区域有丝氨酸/苏氨酸蛋白激酶的结构域;该基因推导的氨基酸序列在进化上与赤拟谷盗(Tribolium castaneum)、熊蜂(Bombus terrestris)、顶切叶蚁(Acromyrmex echinatior)的亲缘关系较近,相似度分别为81%、86%和85%。基于定量PCR和基因芯片的数据分析显示Bm-TSSK在家蚕5龄第3天幼虫的精巢特异性高表达,推测Bm-TSSK参与了家蚕精子的形成。

Testis-specific serine/threonine kinase（TSSK）,which plays an important role during spermiogenesis,was first found in mammals.In this study,we used cDNAs of TSSK2,TSSK4 and TSSK6 from Homo sapiens and cDNAs of TSSK1,TSSK3 and TSSK5 from Mus musculus as query sequences to search homologous genes in EST database of silkworm in the GenBank by EST homologous screening.Through homologous searching and in silico cloning,a sequence of testis-specific serine kinase gene was obtained from the silkworm genome database and was designated as Bm-TSSK（GenBank accession No.JN159476）.With the primers designed according to the predicted sequence,the complete open reading frame（ORF） of Bm-TSSK was cloned from silkworm testis tissue.Sequence analysis and alignment showed that Bm-TSSK was 1 402 bp long,containing a 1 041 bp ORF which has no intron and encodes 346 amino acid residues.There was a serine/threonine kinase domain in the 20 to 284 amino acids.Phylogenetic analysis showed that Bm-TSSK has close genetic relationship with Tribolium castaneum,Bombus terrestris and Acromyrmex echinatio.The deduced amino acid sequence of Bm-TSSK has 81%,86% and 85% sequence identity with that of Tribolium castaneum,Bombus impatiens and Acromyrmex echinatior,respectively.Quantitative PCR and microarray data analyses showed that Bm-TSSK had high expression level in testis of silkworm larvae at day 3 of the 5th instar,suggesting that Bm-TSSK is involved in spermiogenesis of Bombyx mori.