绿盲蝽丝氨酸蛋白酶基因AlSP3的鉴定及表达谱分析

Identification and expression analysis of serine protease AlSP3 gene in Apolygus lucorum(Meyer-Dür)

从绿盲蝽成虫体内克隆了1个丝氨酸蛋白酶基因,命名为AlSP3(GenBank序列号JQ609681)。经序列分析,该基因开放阅读框长1 041 bp,编码346个氨基酸,预测分子量为3.872×104,理论等电点为9.35,N-末端疏水区包含有19个氨基酸组成的信号肽。蛋白质特征分析结果表明,该基因翻译后的蛋白质具有丝氨酸蛋白酶的典型特征。利用荧光定量PCR技术对绿盲蝽取食不同寄主植物后AlSP3基因的表达谱进行分析,结果表明:相对于其他寄主植物,雌成虫取食转Bt基因棉花后AlSP3基因的表达量最高,比对照上升了11.62倍,并显著高于取食常规棉(P<0.01);雌成虫取食常规棉后,AlSP3基因的表达量上升了6.42倍,仅次于取食Bt棉和豇豆,并显著高于取食其他寄主植物(P<0.01)。雄成虫取食常规棉后,AlSP3基因的表达水平仅次于取食蚕豆,上升了9.37倍,并显著高于取食Bt棉(P<0.01)。由此可见,AlSP3基因是绿盲蝽成虫取食常规棉后的重要消化酶基因,并对雌成虫适应取食Bt棉具有重要作用。

A cDNA encoding serine protease of Apolygus lucorum was cloned,which was named serine protease AlSP3（GenBank accession no.JQ609681）.Sequence analysis showed that the open reading frame（ORF） of AlSP3 was 1 041 bp in length,encoding a 346-amino-acid peptide,with the predicted molecular weight of 3.872 ×104 and the theoretical isoelectric point（pI） of 9.35.The predicted N-terminal hydrophobic region contained 19 amino acid residues carrying the characteristic feature of a signal peptide.Protein signature analysis revealed that protein translated by AlSP3 shared typical structural features of serine proteases.By real-time PCR technique,the expression pattern of AlSP3 of A.lucorum feeding on different hosts was analyzed.The expression of AlSP3 increased the most when female adult A.lucorum fed on Bt cottons,significantly higher than that feeding on conventional cotton（P0.01）,and 11.62 times higher than that feeding on string bean.The expression of AlSP3 also increased significantly when female adult A.lucorum fed on conventional cottons,preceded only by cowpea and Bt cotton（P0.01）.The expression of AlSP3 increased significantly when male adult A.lucorum fed on conventional cottons,preceded only by broad bean,and higher than that on Bt cottons（P0.01）.AlSP3 is the important digestive enzyme gene for adult A.lucorum feeding on conventional cottons,and plays an important role in adaption of female adult A.lucorum to surviving on Bt cottons.