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樟树溃疡病菌糖苷水解酶基因LtGH88的克隆与表达

唐文彬 林杉杉 郭建酉 张龙华 陈全助

唐文彬,林杉杉,郭建酉,等. 樟树溃疡病菌糖苷水解酶基因LtGH88的克隆与表达 [J]. 福建农业学报,2023,38(9):1112−1116 doi: 10.19303/j.issn.1008-0384.2023.09.013
引用本文: 唐文彬,林杉杉,郭建酉,等. 樟树溃疡病菌糖苷水解酶基因LtGH88的克隆与表达 [J]. 福建农业学报,2023,38(9):1112−1116 doi: 10.19303/j.issn.1008-0384.2023.09.013
TANG W B, LIN S S, GUO J Y, et al. Cloning and Expression of LtGH88 in Lasiodiplodia theobromae [J]. Fujian Journal of Agricultural Sciences,2023,38(9):1112−1116 doi: 10.19303/j.issn.1008-0384.2023.09.013
Citation: TANG W B, LIN S S, GUO J Y, et al. Cloning and Expression of LtGH88 in Lasiodiplodia theobromae [J]. Fujian Journal of Agricultural Sciences,2023,38(9):1112−1116 doi: 10.19303/j.issn.1008-0384.2023.09.013

樟树溃疡病菌糖苷水解酶基因LtGH88的克隆与表达

doi: 10.19303/j.issn.1008-0384.2023.09.013
基金项目: 福建省林业科技项目(2021FKJ05);三明市林业局项目(h211001);福建省教育厅项目(JAT191131)
详细信息
    作者简介:

    唐文彬(1997 — ),男,硕士研究生,主要从事森林病理学研究,E-mail:2790227015@qq.com

    通讯作者:

    陈全助(1981 — ),男,博士,副教授,主要从事森林病理学研究,E-mail:quanzhu0523@126.com

  • 中图分类号: S763.15

Cloning and Expression of LtGH88 in Lasiodiplodia theobromae

  • 摘要:   目的  可可毛色二孢(Lasiodiplodia theobromae)是樟树溃疡病的主要致病菌。糖苷水解酶是病原菌与寄主植物互作过程中的关键因子。对可可毛色二孢糖苷水解酶基因LtGH88进行克隆与表达分析,为研究LtGH88在樟树溃疡病菌致病机制中的作用奠定基础。  方法  采用PCR方法从可可毛色二孢侵染樟树枝干组织中获得LtGH88基因的CDS序列,运用生物信息学方法分析预测编码蛋白的特征和功能,通过qRT-PCR测定LtGH88在可可毛色二孢侵染樟树过程中的表达量;再利用农杆菌介导的烟草瞬时表达技术初步分析LtGH88基因的功能。  结果  LtGH88基因编码序列长度为1 152 bp。其编码蛋白相对分子质量约为42.8 kDa,等电点(pI)为4.56,二级结构由49.09%的α螺旋、11.23%的延伸链和34.99%的无规则卷曲组成。蛋白N端包含18个氨基酸组成的信号肽,属于糖苷水解酶88(GH88)家族,能酶解果胶物质。LtGH88基因在樟树溃疡病菌侵染早期显著表达,不引起本氏烟叶片细胞坏死,且可抑制Bax引发的烟草叶片过敏性坏死反应。  结论  糖苷水解酶LtGH88可能通过抑制寄主免疫反应促进溃疡病菌可可毛色二孢在樟树枝干中的侵入和定殖。
  • 图  1  LtGH88基因的PCR 扩增结果

    M:DM 2000;1–3:LtGH88基因;4:阴性对照。

    Figure  1.  PCR amplified product of LtGH88

    M: DM 2000; 1-3: LtGH88; 4: negative control.

    图  2  LtGH88蛋白的结构预测

    A:二级结构预测;B:三级结构预测。

    Figure  2.  Prediction of LtGH88 structure

    A: Predicted secondary structure; B: tertiary structure.

    图  3  LtGH88信号肽及保守结构域分析

    Figure  3.  Signal peptide and conserved domain of LtGH88

    图  4  LtGH88基因在病原菌侵染阶段的相对表达量

    Figure  4.  Relative expressions of LtGH88 in L. theobromaeinfection

    图  5  LtGH88蛋白可抑制Bax在烟草中诱导的超敏反应

    A:烟草注射表型;B:台酚蓝染色结果。

    Figure  5.  Bax-induced HR in N. benthamiana suppressed by LtGH88

    A: Phenotype of N. benthamiana after injection; B: after being dyed with trypan blue.

    表  1  本试验所用引物

    Table  1.   Primers applied

    引物名称
    Name
    引物序列
    Primer(5′-3′)
    目的
    Purpose
    LtGH88-FgatcctctagagattgcggccgcATGAAGT
    TCTCTTCCGTTGCCG
    基因全长克隆
    LtGH88-RacatcgtatgggtacgcggccgcAGCAGTGT
    AGGCCTCGTACTCC
    LtGH88-qPCR-FCTACGGCAACAAGACCTAC实时荧光定量
    LtGH88-qPCR-RGAGGATGTCGTCCAGAGA
    Actin-FTGGTCGTACCACCGGTATTGTGTT内参基因
    Actin-RTCACTTGCCCATCAGGAAGCTCAT
    下载: 导出CSV
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出版历程
  • 收稿日期:  2023-02-17
  • 修回日期:  2023-07-13
  • 网络出版日期:  2023-10-25
  • 刊出日期:  2023-09-28

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