朱砂叶螨β-N-乙酰己糖胺酶基因TecHEX3的克隆及在蜕皮发育中的作用

Cloning of theβ-N-acetylhexosaminase gene TecHEX3 of Tetranychus cinnabarinus(Acarina:Tetranychidae)and its role during molting

【目的】本研究旨在探明朱砂叶螨Tetranychus cinnabarinus几丁质降解通路中的关键酶β-N-乙酰己糖胺酶基因(TecHEX3)在螨蜕皮发育中的功能,为开发新型安全的防治害螨生物农药奠定基础。【方法】利用PCR克隆朱砂叶螨TecHEX3,并进行生物信息学分析。利用RT-qPCR检测TecHEX3在朱砂叶螨不同发育阶段(卵、幼螨、若螨和成螨)以及饲喂蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone,20E)的若螨中的表达量。通过饲喂法进行朱砂叶螨雌成螨和若螨TecHEX3的RNAi,分析沉默效率,统计并观察朱砂叶螨若螨的死亡率和致死表型。【结果】成功克隆了朱砂叶螨TecHEX3(GenBank登录号:OR413561),其编码蛋白具有典型的20糖苷水解酶家族结构特征;系统发育树表明TecHEX3与二斑叶螨T.urticae TuHEX3的亲缘性最高且均属于β-N-乙酰己糖胺酶Ⅳ型。TecHEX3在朱砂叶螨不同发育阶段都有表达,在成螨期表达量最高;饲喂500 ng/μL的20E对TecHEX3的表达有最好的诱导效果。RNAi结果表明饲喂ds TecHEX3时朱砂叶螨若螨TecHEX3表达量较对照组(饲喂ds EGFP)显著下调81%;沉默TecHEX3导致朱砂叶螨蜕皮困难或蜕皮后形态异常,致死率达40.58%。【结论】TecHEX3参与朱砂叶螨几丁质降解过程,并在朱砂叶螨的蜕皮发育过程中发挥重要作用。

【Aim】The aim of this study is to analyze the function ofβ-N-acetylhexosaminase gene TecHEX3 of Tetranychus cinnabarinus during molting,which is the key enzyme gene in the chitin degradation pathway,so as to lay the foundation for the development of a new safe biopesticide against T.cinnabarinus.【Methods】TecHEX3 from T.cinnabarinus was cloned using PCR and analyzed using bioinformatics methods.The expression levels of TecHEX3 in different developmental stages(egg,larva,nymph and adult),and nymphs fed with 20-hydroxyecdysone(20E)were assayed using RT-qPCR.The efficiency of silencing TecHEX3 of female adult and nymph of T.cinnabarinus through RNAi was analyzed using the feeding method,and the mortality and lethal phenotype of nymphs were counted and observed.【Results】TecHEX3(GenBank accession number:OR413561)was cloned from T.cinnabarinus and the encoded protein belongs to the 20-glycoside hydrolase family.Phylogenetic tree indicated its closest relationship to TuHEX3 of T.urticae,placing it in beta-N-acetylhexosaminase group IV.TecHEX3 was expressed in various developmental stages of T.cinnabarinus,with the highest expression level observed in adults.The best induction of TecHEX3 expression was achieved by feeding 500 ng/μL of 20E.RNAi results demonstrated that the expression level of TecHEX3 in T.cinnabarinus nymphs fed with ds TecHEX3 was significantly reduced by 81%as compared to that of the control group(fed with ds EGFP).Silencing TecHEX3 resulted in failure to shed the old cuticle or abnormal morphology of T.cinnabarinus,causing a mortality rate of 40.58%.【Conclusion】TecHEX3 plays an important role in the molting development of T.cinnabarinus by involvement in the chitin degradation process.