小菜蛾基因组中内源性逆转录病毒元件PxERV的发现与特征分析

Discovery and characterization of an endogenous retroviral element, PxERV, in the genome of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

【目的】内源性逆转录病毒(endogenous retroviruses,ERVs)是一类在宿主基因组中世代留存的有类似病毒结构的序列元件。本研究在通过前期小菜蛾Plutella xylostella精巢转录组分析发现一个小菜蛾内源性逆转录病毒元件PxERV的基础上,探究该逆转录病毒元件的序列特征及侵染活性,为探明内源性逆转录病毒对小菜蛾的影响奠定基础。【方法】利用生物信息学方法分析鉴定了PxERV在小菜蛾基因组上的序列及结构特点,并用基因克隆和测序得到PxERV的env基因序列;进一步通过MEGA6软件构建了昆虫env基因编码的氨基酸序列与核型多角体病毒包膜糖蛋白的系统发育树。利用qPCR技术检测了env基因在小菜蛾G88品系不同发育时期虫体和精巢中的表达模式及FZ品系成虫和G88品系的成虫和幼虫中env基因拷贝数的变化。利用CRISPR/Cas9介导小菜蛾G88品系中env基因突变,检测PxERV的独立复制活性。【结果】PxERV有LTR-pol-env-LTR结构,其env基因与果蝇Drosophila buzzatii逆转座子osvaldo和粉纹夜蛾Trichoplusia ni内源性逆转录病毒Ted的env基因进化关系较近。PxERV的env基因在小菜蛾G88品系雄成虫精巢中特异性高表达,env基因的拷贝数在小菜蛾FZ品系中的低于在G88品系中的,但在不同发育时期的G88品系中没有差异。G88品系多代自交,突变型env基因在小菜蛾基因组上不断减少,直至完全恢复野生型env基因。【结论】内源性逆转录病毒元件PxERV在小菜蛾世代间存在独立复制活性,但在小菜蛾发育过程中不会独立复制。这种病毒元件可能通过小菜蛾雄性生殖系统实现侵染活性,但侵染机理还需进一步探究。

【Aim】Endogenous retroviruses(ERVs)are retrovirus-like elements that have been fixed in the host genome for generations.This study aims to characterize the sequence characteristics and infectious ability of an endogenous retroviral element,PxERV,in the diamondback moth(DBM),Plutella xylostella,which we found during an analysis of its testis transcriptome,so as to provide a basis for addressing the role of ERVs in P.xylostella.【Methods】The sequence and structural characteristics of PxERV in the P.xylostella genome were analyzed and identified using bioinformatics methods.The env gene sequence of PxERV was cloned and sequenced.A phylogenetic tree of amino acid sequences coded by env genes and envelope glycoproteins of nucleopolyhedroviruses of insects was constructed using MEGA6 software.The expression levels of env gene in the body and testis of strain G88 of P.xylostella at different developmental stages and the copy numbers of env gene in the adult of strain FZ and the adult and larva of strain G88 of P.xylostella were detected using qPCR technology.The independent replication activity of the PxERV in strain G88 was detected by using CRISPR/Cas9 to mediate the mutation of env gene.【Results】PxERV exhibits a structure of LTR-pol-env-LTR.The env gene of PxERV was evolutionarily related to the env genes of the retrotransposon osvaldo in Drosophila buzzatii and the endogenous retrovirus Ted in Trichoplusia ni.The expression level of env gene of PxERV was specifically high in male adult testis of strain G88,and its copy number was lower in strain FZ than in strain G88 and showed no difference among different developmental stages of strain G88.After multiple generations of self-crossing in strain G88,the mutant env gene was gradually reduced in the P.xylostella genome until the wild-type env gene was completely restored.【Conclusion】PxERV shows the independent replication activity between generations of P.xylostella,but no independent replication activity in different developmental stages of the same generation of P.xylostella.We assume that PxERV might complete inter-generational infection through the male reproductive system of P.xylostella,with further studies required to address the mechanism associated with its infection.