斜纹夜蛾蛋白二硫键异构酶基因的发育表达分析

Developmental expression pattern analysis of Spodoptera litura protein disulfide isomerase gene

[目的]分析斜纹夜蛾蛋白二硫键异构酶基因(SlitPDI)的序列特征和发育表达模式,为解析SlitPDI蛋白在斜纹夜蛾生长发育过程中的生理功能提供依据。[方法]采用DNASTAR对SlitPDI基因序列特征和进化关系进行分析,并以实时荧光定量PCR研究SlitPDI基因在斜纹夜蛾2龄、4龄、6龄幼虫及蛹和新羽化雌、雄成虫共6个不同发育时期的相对表达量。[结果]SlitPDI基因编码494个氨基酸,预测该蛋白分子量约55.3 kD,理论等电点(pI)为4.52。氨基酸序列分析结果表明,SlitPDI蛋白序列具有PDI家族的典型特征:在序列的N端及C端均具有二硫键/巯基氧化还原位点-CGHC-。系统发育进化树分析结果表明,SlitPDI蛋白与意大利蜜蜂(Apis mellifera)的PDI蛋白序列一致性最低,为50.2%;与家蚕(Bombyx mori)的PDI蛋白序列一致性最高,为83.4%。斜纹夜蛾不同发育时期的实时荧光定量PCR分析结果表明,SlitPDI基因在4龄幼虫中的表达量最高,其表达量是基准含量2龄幼虫的1.27倍;在刚羽化雌、雄成虫的表达量也较高,分别为基准含量2龄幼虫的1.03和1.12倍。[结论]SlitPDI蛋白属于I类PDI家族蛋白,SlitPDI基因在斜纹夜蛾4龄幼虫和新羽化成虫中的表达量相对较高。

[Objective]The sequence characteristics and developmental expression patterns of Spodoptera litura pro- tein disulfide isomerase(SlitPDI)were analyzed to provide reference for the study of the physiological function of SlitPDI protein during S. litura growth.[Method]The sequence characteristics and evolutionary relationship of SlitPDI gene was analyzed by DNAstar software. The relative expression levels of SlitPDI gene in the stages of the 2nd,4th,6th instar larvae, pupa,newly emerged male and female adults were investigated by real-time fluorescence quantitative PCR method.[Re- sult]The SlitPDI gene encoded 494 amino acids with an estimated molecular mass of 55.3 kD,and the theoretical isoelec- tric poin( t pI)was 4.52. Amino acid sequence analysis showed that the SlitPDI protein sequence had the typical characteris- tics of the PDI protein family:it had disulfide bond/sulfhydryl reduction-oxidation site CGHC at the N-terminus and C-ter- minus of the sequence. Phylogenetic analysis indicated that SlitPDI protein had the lowest sequence identity(50.2%)with the PDI protein sequence of Apis mellifera,and it had the highest sequence identity(83.4%)with the PDI protein se- quence of Bombyx mori. Real-time fluorescence quantitative PCR analysis showed that the expression level of SlitPDI in the 4th instar larva reached the highest peak,and the amount was as 1.27 times as that in the 2nd instar larva. The expres- sions of the newly emerged male and female larvae were also high,which were as 1.03 and 1.12 times as that in the 2nd in- star larva respectively.[Conclusion]The SlitPDI protein belongs to the class I PDI family protein,and the SlitPDI gene is expressed relatively high in the 4th instar larvae and newly emerged adults of S. litura.