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木毒蛾氨肽酶N1基因克隆与表达分析

林健聪 孙悦 刘用垄 王锦达 王荣 张飞萍

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. 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. doi: 10.12171/j.1000-1522.20210186
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. 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. doi: 10.12171/j.1000-1522.20210186

木毒蛾氨肽酶N1基因克隆与表达分析

doi: 10.12171/j.1000-1522.20210186
基金项目: 国家自然科学基金项目(31600522、31601363),福建省自然科学基金项目(2017J0106),福建农林大学林学高峰学科建设项目(61201400820)
详细信息
    作者简介:

    林健聪。主要研究方向:森林昆虫。Email:66470106@qq.com 地址:350002 福建省福州市仓山区上下店路福建农林大学田间914

    责任作者:

    王荣,博士,讲师。主要研究方向:森林昆虫。Email:wangrongbl0501@126.com 地址:同上

  • 中图分类号: S763.3

Cloning and expression analysis of APN1 gene from Lymantria xylina

  • 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。

     

  • 图  1  LxAPN1基因全长

    M. 1 kb marker;1代表LxAPN1基因全长。M, 1 kb marker; 1 represents LxAPN1 full-length gene.

    Figure  1.  Full length of LxAPN1 gene

    图  2  木毒蛾APN1编码区核酸序列及氨基酸序列

    高度保守的残基用深蓝色背景表示;部分保守的残基用白色和灰色背景显示。信号肽用黑色下划线标出;保守结构GAMENWG用红色空心方框标出;锌结合位点HEXXH(X)18E用绿色空心方框标出;GPI结合位点用黑实心三角标出;末端信号肽用红色下划线标出。Highly conserved residues are indicated in deep blue background and only partially conserved residues are indicated in white and gray background; black underline indicates the signal peptite; the gluzincin aminopeptidase GAMENWG is marked with a red hollow box; the characteristic zinc bingding/gluzincin motif HEXXH(X)18E is indicated with green hollow boxes. The GPI anchor point is showed as an black triangles. The C-terminal pro-peptide sequences are marked with a red underline.

    Figure  2.  Nucleotide and amino acid sequences of LxAPN1

    图  3  木毒蛾APN1蛋白质二级结构预测

    蓝色代表α-螺旋;紫色代表无规则卷曲;红色代表延伸链;绿色代表β-转角;横坐标表示蛋白大小(kDa)。Blue represents α-helix; purple represents random coli; red represents extend strand; geen represents β-turn; abscissa indicates protein size(kDa).

    Figure  3.  Prediction of secondary structure of LxAPN1 protein

    图  4  木毒蛾APN1蛋白三级结构预测

    Figure  4.  Prediction of three-dimensional structure of LxAPN1 protein

    图  5  木毒蛾APN1与其他昆虫APN蛋白系统进化树

    Figure  5.  Neighbor-joining phylogenetic tree of LxAPN1 and APN in other insects

    图  6  木毒蛾不同龄期及不同组织中LxAPN1基因的表达

    L1~L7为1龄到7龄幼虫。L1—L7 are the 1st to 7th instar larvae.

    Figure  6.  Expression profiles of LxAPN1 in different ages and tissues of Lymantria xylina

    表  1  基因克隆中使用引物列表

    Table  1.   List of primers in gene cloning

    引物名称 Primer name序列(5′—3′) Sequence (5′−3′)用途 Usage
    LxAPN1-F TTACAATAATCCAGTTGAGGGT LxAPN1基因扩增 LxAPN1 gene amplification
    LxAPN1-R GAACATCAAACTAGATTTCTTAGTAATTGAT LxAPN1基因扩增 LxAPN1 gene amplification
    qAPN1-F CGTGATCCGTCCTCAAGATTAC qRT-PCR
    qAPN1-R TGGATGCGACGTAAAGTAAGG qRT-PCR
    qEF-1a-F CCGTGTTGAAACTGGTATCCT qRT-PCR
    qEF-1a-R TAGAGCTTCGTGGTGCATTT qRT-PCR
    qActin-F GGTAGATAAGGAGGCAAGGATTG qRT-PCR
    qActin-R ACCACAATGTACCCTGGTATTG qRT-PCR
    下载: 导出CSV
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  • 收稿日期:  2021-05-14
  • 修回日期:  2021-07-01
  • 网络出版日期:  2021-07-06

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