小贯小绿叶蝉半胱氨酸蛋白酶基因的克隆与表达

Molecular Cloning and Expression of a Cysteine Gene in Empoasca onukii

半胱氨酸蛋白酶是植食性昆虫体不可或缺的重要水解蛋白酶之一。本文从小贯小绿叶蝉的转录组数据库中获得半胱氨酸蛋白酶基因Eocyp的基因片段的转录本序列,利用RACE技术得到其c DNA全长序列,利用生物信息学技术对Eocyp所编码的理论氨基酸序列进行分析,使用q RT-PCR技术检测Eocyp在小贯小绿叶蝉不同发育阶段,雌、雄成虫不同部位以及不同温度和光周期下的表达量。结果表明,小贯小绿叶蝉Eocyp的全长c DNA序列包含一个1 656 bp的开放阅读框,编码551个氨基酸,并具有由24个氨基酸残基组成的信号肽。推测其相对分子量为62.09 k D,预测其分子式为C_(2782)H_(4163)N_(737)O_(835)S_(25);理论等电点为6.02,不稳定系数为36.10。Eocyp具有半胱氨酸蛋白酶基因的典型特征,即催化三联体Cys^(357)-His^(499)-Asn^(519),同时具有ERFNIN,GNFD和GCDGG簇等保守结构域。Eocyp编码氨基酸序列与茶翅蝽CYP基因编码序列相似度最高。表达特征分析结果表明,Eocyp在小贯小绿叶蝉的各个生长时期均有表达,在5龄若虫时期达到最高;Eocyp在小贯小绿叶蝉雌、雄成虫的腹部均有较高的表达量;然而,Eocyp的表达量对于高、低温和不同光周期均无响应。这些结果为进一步研究Eocyp功能提供了理论依据。

Cysteine proteinases are important proteolytic enzymes for herbivorous insects.In this paper,the transcript sequence of Eocyp was obtained from the transcriptome database of Empoasca onukii.The full-length c DNA sequence of Eocyp was then cloned by RACE.The theoretical amino acid sequence of Eocyp was analyzed by bioinformatics.The expression levels of Eocyp in different developmental stages,different parts of female and male adults of E.onukii and nymphs under different temperatures and photoperiods were detected by q RT-PCR.The full-length c DNA sequence of Eocyp contained an open reading frame of 1 656 bp,which encoded 551 amino acids and had a signal peptide consisting of 24 amino acid residues.Its molecular formula was predicted to be C（2782）H（4163）N（737）O（835）S（25）.Its relative molecular weight and the theoretical isoelectric point were estimated to be 62.09k D and 6.02,and the instability coefficient of Eocyp was 36.10.Eocyp had the typical feature of cysteine protease,a catalytic triad Cys（357）-His（499）-Asn（519）,and the conserved domains such as ERFNIN,GNFD amino acid residues and the GCDGG clusters.The amino acid sequence of Eocyp had a high similarity with the CYP gene of Halyomorpha halys.Expression analysis showed that Eocyp was expressed in all developmental stages of E.onukii with its peak in the fifth instar nymphs.Higher expression of Eocyp was observed in the abdomen of adult E.onukii,irrespective of female and male.However,the expression level of Eocyp did not respond to the variation of temperatures and photoperiods.These results provided a theoretical basis for further study of Eocyp.