樟树溃疡病菌糖苷水解酶基因<i>LtGH88</i>的克隆与表达

唐文彬; 林杉杉; 郭建酉; 张龙华; 陈全助

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

唐文彬^1,,
林杉杉¹,
郭建酉¹,
张龙华²,
陈全助^{1, 3, ,}

1.
福建农林大学林学院，福建　福州　350002
2.
三明市林业有害生物防治检疫站，福建　三明　365000
3.
福建农林大学金山学院，福建　福州　350002

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
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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

Cloning and Expression of LtGH88 in Lasiodiplodia theobromae

1.
Forestry College, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Forest Disease and Insect Pest Control and Quarantine Station of Sanming City, Sanming, Fujian　350002, China
3.
Jinshan College, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

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。
- 樟树溃疡病菌 /
- LtGH88基因 /
- 基因克隆 /
- 表达分析 /
- 过敏性坏死反应
Abstract: Objective The glycoside hydrolase gene LtGH88 of Lasiodiplodia theobromae was cloned to study the mechanism of canker in Cinnamomum camphora, which is the major pathogen that causes the disease. Methods The LtGH88 encoding sequence was cloned by PCR from tissues of C. camphora infected by L. theobromae to determine the characteristics and functions of the protein by bioinformatic methods. Expression of the gene was detected by real-time quantitative PCR, and functions analyzed using the Agrobacterium tumefaciens mediated transient transformation in Nicotianaben thamiana. Results The full open reading frame of LtGH88 was 1 152 bp with a molecular weight of 42.8 kDa and a theoretical isoelectric point of 4.56. The predicted secondary structure of the protein consisted of 49.09% α helix, 11.23% extended chain, and 34.99% random coil. A signal peptide of 1-18 amino acids was located at the N terminus. The protein belonged to the glycoside hydrolase family 88 (GH88) capable of degrading pectin. LtGH88 was significantly expressed in the early stage of the infection. It did not cause cell necrosis in the leaf of N. thamiana but was able to inhibit the hypersensitive response (HR) induced by Bax. Conclusion It was postulated that LtGH88 in L. theobromae inhibited the immune response of C. camphora facilitating the pathogenic invasion and colonization on the host plant.
- Lasiodiplodia theobromae /
- LtGH88 /
- gene cloning /
- expression analysis /
- hypersensitive response

HTML全文

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.

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

图 3 LtGH88信号肽及保守结构域分析

Figure 3. Signal peptide and conserved domain of LtGH88

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

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-F	gatcctctagagattgcggccgcATGAAGT TCTCTTCCGTTGCCG	基因全长克隆
LtGH88-R	acatcgtatgggtacgcggccgcAGCAGTGT AGGCCTCGTACTCC	基因全长克隆
LtGH88-qPCR-F	CTACGGCAACAAGACCTAC	实时荧光定量
LtGH88-qPCR-R	GAGGATGTCGTCCAGAGA	实时荧光定量
Actin-F	TGGTCGTACCACCGGTATTGTGTT	内参基因
Actin-R	TCACTTGCCCATCAGGAAGCTCAT	内参基因

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