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    林健聪, 孙悦, 刘用垄, 王锦达, 王荣, 张飞萍. 木毒蛾氨肽酶N1基因克隆与表达分析[J]. 北京林业大学学报, 2021, 43(9): 94-100. DOI: 10.12171/j.1000-1522.20210186
    引用本文: 林健聪, 孙悦, 刘用垄, 王锦达, 王荣, 张飞萍. 木毒蛾氨肽酶N1基因克隆与表达分析[J]. 北京林业大学学报, 2021, 43(9): 94-100. DOI: 10.12171/j.1000-1522.20210186
    shu
    Lin Jiancong, Sun Yue, Liu Yonglong, Wang Jinda, Wang Rong, Zhang Feiping. Cloning and expression analysis of APN1 gene from Lymantria xylina[J]. Journal of Beijing Forestry University, 2021, 43(9): 94-100. DOI: 10.12171/j.1000-1522.20210186
    Citation: Lin Jiancong, Sun Yue, Liu Yonglong, Wang Jinda, Wang Rong, Zhang Feiping. Cloning and expression analysis of APN1 gene from Lymantria xylina[J]. Journal of Beijing Forestry University, 2021, 43(9): 94-100. DOI: 10.12171/j.1000-1522.20210186
    shu

    木毒蛾氨肽酶N1基因克隆与表达分析

    Cloning and expression analysis of APN1 gene from Lymantria xylina

      摘要
    • 摘要:
        目的  氨肽酶N（APN）是昆虫中肠一类重要的Bt受体蛋白，Bt细菌产生的Cry毒素对昆虫的毒杀作用机理在学术界存在一定争议，但是普遍认为毒素与Bt受体蛋白的结合是产生毒力的必要环节。本研究通过基因克隆、生物信息学分析以及不同龄期组织中的表达对木毒蛾APN1基因进行研究，为后续研究APN基因家族、其他Bt受体蛋白及Cry毒素作用机制提供有益补充。
        方法  以木毒蛾中肠cDNA为模板，对木毒蛾APN1基因进行克隆并进行生物学分析，利用实时定量PCR（qRT-PCR）技术分析该基因在木毒蛾发育阶段及不同组织中的表达模式。
        结果  克隆获得木毒蛾APN1基因的全长DNA，命名为LxAPN1。LxAPN1序列全长为3 159 bp，ORF为3 054 bp，编码1 017个氨基酸，序列比对和进化树分析表明，LxAPN1与舞毒蛾的LdAPN1高度同源，在N-端都具有信号肽，都具有锌结合位点HEXXH（X18）E以及保守区域GAMENWG，在末端具有GPI结合位点；LxAPN1在木毒蛾卵期无表达，在所有幼虫阶段中均有表达，幼虫期过后LxAPN1表达量锐减；LxAPN1在肠道的表达量明显高于头部和表皮。
        结论  LxAPN1在木毒蛾中肠被成功克隆，LxAPN1与LdAPN1高度同源，并且在进化树的分布上也极其相近，推测两者的APN1功能上近似；LxAPN1在木毒蛾2龄幼虫期表达量最高。并且在6龄幼虫肠道中表达量最高，肠道高表达与LxAPN1作为Bt受体在木毒蛾中肠发挥作用息息相关。

       

      Abstract:
        Objective  Aminopeptidase N(APN) is a class of important kind of BT receptor protein in insect midgut. The mechanism of Cry toxin produced by Bt bacteria to kill insects has been controversial in the academic research, but it is generally believed that the binding of toxin and Bt receptor protein is the necessary link to its virulence. The APN1 gene of Lymantria xylina was studied by gene cloning, biological information analysis and expression patterns, with the purpose to provid a reference for the subsequent study of APN gene family, other Bt receptor proteins and the mechanism of Cry toxin.
        Method  APN1 was cloned by cDNA from midgut of the moth as template in a PCR system. Biological analysis and real time quantitative PCR(qRT-PCR) were performed to analyze the expression pattern of LxAPN1 gene in different ages and tissues of Lymantria xylina.
        Result  The full-length DNA of APN1 gene was cloned from midgut of Lymantria xylina and named LxAPN1. The full-length sequence of LxAPN1 was 3 159 bp, ORF was 3 054 bp, encoding 1 017 amino acids. Sequence alignment and phylogenetic tree analysis showed that LxAPN1 and LdAPN1 were highly homologous, with signal peptide at N-terminal, zinc binding site HEXXH (X18) E and conserved region GAMENWG, and GPI binding site at the end. LxAPN1 was not expressed in egg stage, and expressed in 1−7 instar larvae, the expression of LxAPN1 decreased after larval stage; the expression of LxAPN1 in intestine was significantly higher than that in head and cuticle.
        Conclusion  LxAPN1 was successfully cloned in the midgut of Lymantria xylina. LxAPN1 and LdAPN1 are highly homologous, and the distribution of phylogenetic tree is also very similar, which not only indicates the similarity between Lymantria xylina and Lymantria dispar, but also the similarity of APN1 function between them; The expression of LxAPN1 was the highest in the second instar larvae of Lymantria xylina. And the expression was the highest in the gut from 6 instar larvae, as an Bt receptor in the intestinal of Lymantria xylina.

       

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