扶桑绵粉蚧组织蛋白酶B基因的克隆、原核表达和不同发育阶段表达分析

Molecular cloning,prokaryotic expression and expression at different developmental stages of cath.tifin B gene in mealybug Phenacoccus solenopsis Tinsley(Hemiptera:Pseudococcidae)

昆虫组织蛋白酶B在昆虫代谢过程中发挥重要作用。本研究利用RACE技术克隆了扶桑绵粉蚧Phenacoccus solenopsis Tinsley组织蛋白酶B基因的开放阅读框(ORF)序列,命名为PsCb(GenBank登录号:JQ727999)。生物信息学分析表明,该基因的开放阅读框包含927bp的片段,编码308个氨基酸。多序列比对表明,该基因编码的蛋白在N端变异较大,在C端保守性高。组织蛋白酶B基因的系统进化树结果表明扶桑绵粉蚧组织蛋白酶独自成为一支。原核表达电泳检测到一条大约35kDa的目的条带,与预测的蛋白分子量相符。组织蛋白酶B基因在扶桑绵粉蚧各个虫态均有表达,卵期表达量相对较低,2龄若虫期达到最高峰,然后下降。本研究为进一步研究该基因的功能并开发出组织蛋白酶抑制剂,从而研制出扶桑绵粉蚧杀卵剂和胚胎发育抑制剂等提供理论依据。

Cath.tifin B plays an important role in insect metabolism.In this study,cath.tifin B gene was cloned from mealybug Phenacoccus solenopsis and named as PsCb（GenBank accession no.JQ727999）.This cDNA contains an open reading frame（ORF） which is 927 bp in length encoding 308 amino acids.Homology analysis shows that the N-terminal sequence of PsCB has larger variation,while the C-terminal sequence is conserved.Phylogenetic tree shows that PsCB is separated with other branches.Prokaryotic protein expression test showed the expressed product has the MW of 35 kDa,nearly equal to the predicted.Real-time PCR analysis revealed that PsCb mRNA was expressed in various developmental stages of P.solenopsis.The expression level was relatively lower at the egg stage,reached a peak at the 2nd instar nymph and then declined.Our study provides a theoretical basis for further study of the gene function,the development of cath.tifin inhibitors as ovicides and inhibitors of embryonic development of P.solenopsis.