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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999bp,编码332个氨基酸,预测分子量为36.84kDa,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。
Trypsin and chymotrypsin-like serine proteases are important digestive enzymes in the digestive system of the Miridae. In order to better understand the role of serine proteases in the digestive system of Apolygus lucorum, we cloned the phytase gene AlSP4 (GenBank accession number JQ609682) for the first time. Sequence analysis showed that the open reading frame of this gene was 999 bp long and encoded 332 amino acids with a predicted molecular weight of 36.84 kDa and a theoretical isoelectric point of 5.35. The N-terminal hydrophobic region contained a signal peptide consisting of 16 amino acids. Protein characterization analysis showed that the translated protein possesses the typical characteristics of serine protease, that is, the active catalytic center consisting of histidine (His), aspartic acid (Asp) and serine residues in the amino acid sequence The gene also has obvious characteristics of trypsin precursor after translation, that is, the gene has a signal peptide, an activating peptide and a starting amino acid sequence (IVGG) which is conserved at the N-terminus of trypsin. The expression patterns of AlSP4 in female and male adults fed on different host plants were analyzed by fluorescence quantitative PCR. The results showed that AlSP4 expression was the highest in female adults when compared to other host plants, Which was higher than that of conventional cotton (P <0.01). The expression level of AlSP4 was the highest in adult male adults fed Artemisia selenges, and the expression level of AlSP4 was only second to that of Artemisia selengensis. The expression level of AlSP4 in male adult was also significantly higher than that in normal cotton (P < 0.01). Thus, AlSP4 is an important digestive enzyme gene after feeding on Bt cotton by Apolygus lucorum, which plays an important role in adapting Apolygus lucorum to Bt cotton feeding.