绿盲蝽丝氨酸蛋白酶基因AlSP4的克隆及取食不同寄主植物后的表达谱分析

胰蛋白酶类和胰凝乳蛋白酶类丝氨酸蛋白酶是盲蝽科昆虫消化系统内重要的消化酶。为了更好地了解丝氨酸类蛋白酶在绿盲蝽Apolygus lucorum消化系统中的作用， 本研究首次克隆了绿盲蝽丝氨酸蛋白酶基因AlSP4 (GenBank登录号为JQ609682)。序列分析结果表明， 该基因开放阅读框长999 bp， 编码332个氨基酸， 预测分子量为36.84 kDa， 理论等电点为5.35， N末端疏水区包含有16个氨基酸组成的信号肽。蛋白特征分析表明， 该基因翻译后的蛋白质具有丝氨酸蛋白酶的典型特征， 即氨基酸序列中具有组氨酸(His)、 天门冬氨酸(Asp)以及丝氨酸(Ser)残基组成的酶活性催化中心三元件； 该基因翻译后还具有明显的胰蛋白酶前体的特征， 即此基因具有信号肽、 激活肽以及胰蛋白酶N末端保守的起始氨基酸序列(IVGG)。利用荧光定量PCR技术对绿盲蝽雌、 雄成虫取食不同寄主植物后AlSP4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Bt棉后AlSP4的表达量最高， 并显著高于取食常规棉后的表达量(P＜0.01)。雄成虫取食茼蒿后AlSP4的表达量最高； 雄成虫取食Bt棉后， AlSP4的表达水平仅次于取食茼蒿后的表达量， 也显著高于取食常规棉后的表达量(P＜0.01)。由此可见， AlSP4是绿盲蝽取食Bt棉后的重要消化酶基因， 对绿盲蝽适应Bt棉取食具有重要作用。

Cloning of serine protease gene AlSP4 and its expression patterns after feeding on different host plants in Apolygus lucorum (Hemiptera:Miridae)

Some serine proteases, such as trypsin and chymotrypsin, are important digestive enzymes in the digestive system of mirid bugs. In order to better understand the role of serine proteases in the digestive system of the green plant bug, Apolygus lucorum (Meyer-Dür), we cloned the cDNA encoding serine protease of A. lucorum for the first time in the laboratory, which was named as AlSP4 (GenBank accession no. JQ609682). The results of sequence analysis showed that the open reading frame (ORF) of AlSP4 is 999 bp in length, encoding a 332-amino-acid peptide, with the predicted molecular weight (MW) of 36.84 kDa and the theoretical isoelectric point (pI) of 5.35, and the predicted N-terminal hydrophobic region containing 16 amino acid residues displays the typical feature of a signal peptide. Protein signature analysis revealed that the protein encoded by AlSP4 shares typical structural features of serine proteases with other insects, including His, Asp, and Ser residues for the catalytic amino acid triad of active sites of serine proteases. Putative trypsin precursors from the encoded protein of AlSP4 cDNA contain a signal peptide, activation peptide, and conserved N-termini (IVGG). By the Real-time PCR technique, we determined the expression pattern of AlSP4 in A. lucorum fed on different hosts. The expression level of AlSP4 was the highest in female adult A. lucorum fed on Bt cottons, significantly higher than that fed on conventional cotton (P＜0.01). The expression of AlSP4 increased significantly in male adult A. lucorum fed on Bt cottons, its expression level was only lower than that fed on garland chrysanthemum and significantly higher than that fed on conventional cottons (P＜0.01). Therefore, AlSP4 is the important digestive enzyme gene for adult A. lucorum to feed on Bt cottons, and plays an important role in adaption of A. lucorum to survive on Bt cottons.