松墨天牛GST基因克隆及高温胁迫下的表达特性分析

doi:10.12302/j.issn.1000-2006.202304018

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南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (1): 28-36.doi: 10.12302/j.issn.1000-2006.202304018

• 专题报道：松材线虫病绿色防控研究（执行主编叶建仁骆有庆） • 上一篇下一篇

松墨天牛GST基因克隆及高温胁迫下的表达特性分析

李子纯(), 郝德君^*(), 李慧, 李长燕, 许丹雯仪, 杨华磊, 赵培渊

南京林业大学林草学院,南方现代林业协同创新中心,江苏南京 210037

收稿日期:2023-04-10 修回日期:2024-02-02 出版日期:2025-01-30 发布日期:2025-01-21
通讯作者: * 郝德君(dejunhao@163.com),教授。
作者简介:
李子纯(leezichun@njfu.edu.cn)。
基金资助:
国家自然科学基金项目(32201562);江苏省自然科学基金项目(BK20220412);江苏省大学生创新训练计划重点项目(202310298002Z)

Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress

LI Zichun(), HAO Dejun^*(), LI Hui, LI Changyan, XU Danwenyi, YANG Hualei, ZHAO Peiyuan

Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry and Grassland, Nanjing Forestry University, Nanjing 210037, China

Received:2023-04-10 Revised:2024-02-02 Online:2025-01-30 Published:2025-01-21

摘要/Abstract

摘要：

【目的】克隆松墨天牛(Monochamus alternatus)谷胱甘肽S转移酶(glutathione S-transferases, GST)相关基因,明确GST基因在松墨天牛响应高温胁迫中的效用,为探究亚热带地区松墨天牛的耐热分子机制提供理论依据。【方法】克隆3条松墨天牛GST基因,结合DNAMAN 9.0、I-TASSER等软件分析松墨天牛GST基因的结构特征;利用qRT-PCR技术测定分析松墨天牛成虫和4龄幼虫在不同高温、不同处理时长后GST基因的相对表达量;通过纸盘扩散法验证3条松墨天牛GST基因在保护机体免受氧化应激中的作用。【结果】克隆3条松墨天牛GST基因的cDNA序列,分别命名为MaltGSTe1、MaltGSTe2和MaltGSTt1。MaltGSTe1与MaltGSTe2均属于GST的Epsilon家族,MaltGSTt1属于GST的Theta家族。3条GST基因三维蛋白结构具有指示性结构特征,属于胞质型GST。松墨天牛4龄幼虫在高温胁迫下MaltGSTe1、MaltGSTe2和MaltGSTt1的相对表达量均出现显著变化,MaltGSTe2相对表达水平上调幅度最大;MaltGSTt1在松墨天牛雄虫体内相对表达量出现明显下调;异源表达3条GST基因蛋白的大肠杆菌表现出较强的抗氧化能力,其中,MaltGSTe2具有更强的抗氧化能力。【结论】克隆获得3条松墨天牛GST基因,发现高温胁迫可诱导GST基因表达量上调;纸盘扩散分析结果表明异源表达GST基因蛋白具有抗氧化能力,推测GST基因具有通过保护机体免受氧化应激来参与松墨天牛幼虫的高温胁迫响应机制。

关键词: 松墨天牛, 高温胁迫, 谷胱甘肽S转移酶, GST基因, 氧化应激, 基因克隆, 表达分析, 生物信息学

Abstract:

【Objective】To clone the glutathione S-transferases (GST) genes of Monochamus alternatus (Coleoptera: Cerambycidae) and to determine the expression levels of these genes under heat stress in adult males and females, as well as at different developmental stages of M. alternatus, we used molecular biology and bioinformatics methods. 【Method】 Three GST cDNA sequences were cloned, and their structural characteristics were analyzed using DNAMAN 9.0 and I-TASSER software. qRT-PCR was employed to measure the relative expression levels of GST genes in adult and fourth instar larvae of M. alternatus under varying temperatures and treatment durations. The role of these GST genes in protecting the organism from oxidative stress was assessed through disc diffusion assays. 【Result】Three GST cDNA sequences were cloned and named MaltGSTe1, MaltGSTe2, and MaltGSTt1. MaltGSTe1 and MaltGSTe2 belong to the Epsilon family, while MaltGSTt1 belongs to the Theta family. Analysis of three-dimensional protein structures indicated that these GST are cytoplasmic. The relative expression levels of MaltGSTe1, MaltGSTe2 and MaltGSTt1 in fourth instar larvae significantly changed under heat stress, with MaltGSTe2 showing the greatest change. In males, the expression level of MaltGSTt1 was significantly down-regulated. Escherichia coli expressing the GST genes demonstrated enhanced antioxidant capacity, with MaltGSTe2 exhibiting the strongest activity. 【Conclusion】We successfully cloned three GST genes and investigated their expression characteristics under high temperature stress in M. alternatus. High temperature stress was found to induce up-regulation of GST gene expression. Disc diffusion assays confirmed that heterogeneously expressed GST proteins have antioxidant capabilities. These findings suggest that GST genes play a role in the response mechanism to high temperature stress by protecting against oxidative stress, providing a theoretical foundation for exploring the heat resistance mechanisms of M. alternatus in subtropical regions.

Key words: Monochamus alternatus, heat stress, glutathione S-transferases, GST gene, oxidative stress, gene clone, express analysis, bioinformatics

中图分类号:

S763.38

李子纯,郝德君,李慧,等. 松墨天牛GST基因克隆及高温胁迫下的表达特性分析[J]. 南京林业大学学报（自然科学版）, 2025, 49(1): 28-36.

LI Zichun, HAO Dejun, LI Hui, LI Changyan, XU Danwenyi, YANG Hualei, ZHAO Peiyuan. Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress[J].Journal of Nanjing Forestry University (Natural Science Edition), 2025, 49(1): 28-36.DOI: 10.12302/j.issn.1000-2006.202304018.

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引物名称 primer name	引物序列(5'-3') primer sequence	相关系数(R²) correlation coefficient	扩增效率/% amplification efficiency
GAPDH-qF	TCGAACGCTTCATGCACAAC	0.99	1.09
GAPDH-qR	CCATCACGCCACAAATTTTCC
MaltGSTe-1-qF	AACGTACTAGTGCCCATCGC	0.99	1.10
MaltGSTe-1-qR	AGCCTACTTGATTGGCCTCG
MaltGSTe-2-qF	ATACCAGCCCTGGACGACAA	0.99	1.03
MaltGSTe-2-qR	GCTTTTCAACATCGGCAGGA
MaltGSTt-1-qF	ATATGCTGCCGGAGATCACG	1.00	1.09
MaltGSTt-1-qR	ACCAGCTTTGGACAAGAGGAT

引物名称 primer name	引物序列(5'-3') primer sequence
MaltGSTe-1-F^①	ATGGCGCCTAAATTACACTACG
MaltGSTe-1-R^①	TCAACCAAGCTTGCTCTTGAC
MaltGSTe-2-F^①	ATGGCTCCAAAGTTATATATGACA
MaltGSTe-2-R^①	TTACGTGGATAATGCATTTTGAACT
MaltGSTt-1-F^①	ATGCCACTAACATTATATGCTGT
MaltGSTt-1-R^①	TTATTTTCTAATGGGGTGAATGGGA
MaltGSTe-1-tF^②	cagcaaatgggtcgcggatccATGGCGCCTAAATTACACTACGC
MaltGSTe-1-tR^②	ctcgagtgcggccgcaagcttACCAAGCTTGCTCTTGACCATT
MaltGSTe-2-tF^②	cagcaaatgggtcgcggatccATGGCTCCAAAGTTATATATGACAATAA
MaltGSTe-2-tR^②	ctcgagtgcggccgcaagcttCGTGGATAATGCATTTTGAACTAGT
MaltGSTt-1-tF^②	cagcaaatgggtcgcggatccATGCCACTAACATTATATGCTGTATCTG
MaltGSTt-1-tR^②	ctcgagtgcggccgcaagcttTTTTCTAATGGGGTGAATGGGA

基因名称 gene name	登录号 genbank No.	氨基酸数量 amount of amino acids	相对分子质量 relative molecular weight	等电点 isoelectric point
MaltGSTe1	OQ476235	216	23.67	5.94
MaltGSTe2	OQ476236	216	24.05	5.73
MaltGSTt1	OQ476237	230	26.66	5.84

松墨天牛GST基因克隆及高温胁迫下的表达特性分析

Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress

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[1]	王露露, 耿兴敏, 宦智群, 许世达, 赵晖. 1-MCP预处理对杜鹃花高温胁迫下光合特性及相关基因表达的影响[J]. 南京林业大学学报（自然科学版）, 2023, 47(4): 103-113.
[2]	刘增才, 王淑婷, 佟鑫宇, 邹莉. 暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究[J]. 南京林业大学学报（自然科学版）, 2023, 47(4): 123-130.
[3]	魏祯祯, 宋程威, 郭丽丽, 郭琪, 侯小改. ‘凤丹’PoERF4基因的克隆及表达分析[J]. 南京林业大学学报（自然科学版）, 2023, 47(3): 56-62.
[4]	王立超, 陈凤毛, 董晓燕, 田成连, 王洋. 松墨天牛取食和产卵特性研究[J]. 南京林业大学学报（自然科学版）, 2023, 47(2): 219-224.
[5]	李慧, 郝德君, 徐天, 代鲁鲁. 高温胁迫对植食性昆虫影响研究进展[J]. 南京林业大学学报（自然科学版）, 2022, 46(6): 215-224.
[6]	张婷, 柏杨, 亓希武, 于盱, 房海灵, 李莉, 刘冬梅, 梁呈元, 李维林. 薄荷MhWRKY57基因克隆及响应茉莉酸信号的表达分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(6): 279-287.
[7]	贾展慧, 贾晓东, 许梦洋, 莫正海, 翟敏, 宣继萍, 张计育, 王刚, 王涛, 郭忠仁. 薄壳山核桃原花青素合成关键酶基因的克隆与表达分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(5): 49-57.
[8]	王立超, 苏胜荣, 陈凤毛, 董晓燕, 田成连, 王洋. 黄山马尾松林天牛及携带线虫种类初步调查[J]. 南京林业大学学报（自然科学版）, 2022, 46(4): 29-35.
[9]	王磊, 叶建仁, 史丽娜. 利用腐生线虫加速替代疫木中松材线虫种群数量研究[J]. 南京林业大学学报（自然科学版）, 2022, 46(4): 36-44.
[10]	严志祥, 杨海燕, 樊苏帆, 吴文龙, 闾连飞, 李维林. 黑莓果实发育过程中蔗糖磷酸合成酶基因的表达分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(1): 179-186.
[11]	林莉莉, 胡安琪, 陈钢, 张霁月, 曹光球, 曹世江. 杉木ClWRKY44基因克隆及其表达特性分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(1): 203-209.
[12]	陈宏健, 郝德君, 田敏, 周杨, 夏小洪, 赵欣怡, 乔恒, 谈家金. 室内饲养松墨天牛幼虫不同肠段细菌的群落结构及功能分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 143-151.
[13]	黎梦娟, 朱礼明, 霍俊男, 张景波, 施季森, 成铁龙. 唐古特白刺NtCBL1、NtCBL2基因克隆及表达分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 93-99.
[14]	张腾倩, 张伟溪, 丁昌俊, 张静, 胡赞民, 苏晓华. 欧美杨PdMODD基因克隆与表达特性分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(2): 43-50.
[15]	张馨, 马苗苗, 吕婉秋, LEE Joobin, 杨静莉. 大青杨PuZFP103基因的序列特征及逆境胁迫的表达分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(1): 36-44.