大豆抗花叶病毒病基因GmRHF1的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2024.23.005

摘要/Abstract

摘要：

【目的】大豆花叶病毒（soybean mosaic virus，SMV）引起的大豆花叶病毒病严重制约着大豆的产量和品质，通过序列变异分析、病毒诱导的基因沉默（virus induced gene silencing，VIGS）等技术验证了大豆RING-H2型锌指蛋白基因GmRHF1的抗病功能，利用酵母双杂交试验和荧光素酶互补试验筛选并验证其互作蛋白，为深入探究GmRHF1的抗SMV作用机制奠定基础。【方法】首先，从大豆品种徐豆14（高抗SMV）和石山黑豆（高感SMV）中分别克隆GmRHF1，并在代表性抗、感种质资源中分析序列变异的一致性，挖掘基因抗病优异变异。其次，利用荧光定量PCR分析基因在不同组织的表达量差异及SMV处理下基因的表达量变化趋势；同时，采用病毒诱导的基因瞬时沉默技术验证GmRHF1的抗SMV功能。通过酵母cDNA文库筛选、酵母对点验证和荧光素酶互补试验筛选并验证GmRHF1的互作蛋白。最后，利用病毒诱导的基因瞬时沉默技术验证GmClpP6的抗SMV功能，明确GmRHF1与GmClpP6互作对大豆抗SMV的生物学意义。【结果】 GmRHF1具有E3泛素蛋白连接酶家族RING-H2型保守结构域，属于典型的RING-H2 finger家族成员。序列变异分析发现GmRHF1具有一个自然存在的非同义突变，可能与SMV抗感性相关。荧光定量PCR显示，病毒诱导后，GmRHF1在抗病材料中的表达量水平显著高于感病大豆。病毒诱导的基因沉默试验显示，有效沉默GmRHF1大豆的叶片降低了大豆对SMV的抗病性，说明GmRHF1具有抗SMV功能。通过酵母cDNA文库，筛选到30个GmRHF1的潜在互作蛋白，利用酵母对点验证和荧光素酶互补试验，证实GmRHF1与GmClpP6间的互作关系。同时，病毒诱导的基因瞬时沉默试验表明，在有效沉默GmClpP6的植株叶片中，大豆对SMV的抗病性减弱。【结论】大豆GmRHF1在编码区序列中存在的非同义突变可能与SMV抗感性相关；GmRHF1可与GmClpP6发生相互作用。GmRHF1在SMV抗性反应中具有重要作用。

关键词: 大豆花叶病毒, 锌指蛋白, 表达模式分析, 酵母文库筛选, 荧光素酶互补试验

Abstract:

【Objective】 Soybean mosaic virus (SMV) disease causes by SMV seriously restricts soybean yields and qualities. In this study, we cloned GmRHF1, which encodes a Ring-H2 type zinc finger protein (RHF), and identified its function in SMV resistance through the sequence variation analysis and the virus induced gene silencing (VIGS) assay. Yeast two-hybrid and luciferase complementation assay (LCA) were used to screen and verify the interacting proteins of GmRHF1. The research lay a foundation for further exploring the mechanism of GmRHF1 in soybean resistance to SMV.【Method】 Firstly, GmRHF1 was cloned from soybean varieties of Xudou 14 (highly resistant to SMV) and Shishanheidou (highly susceptible to SMV), and the consistency of sequence variation was then analyzed in representative resistant and susceptible soybean resources, so as to excavate the excellent alleles with SMV resistance. Secondly, the expression abundance of GmRHF1 in different soybean tissues and under SMV treatment were analyzed by real-time quantitative PCR (RT-qPCR), respectively. At the same time, the function of GmRHF1 in SMV resistance was identified by the VIGS assay. The interacting proteins of GmRHF1 were screened and verified by yeast cDNA library screening, point to point verification and LCA experiments. Finally, the function of GmClpP6 on SMV resistance was verified, and the biological significance based on the interaction of GmRHF1 and GmClpP6 for soybean resistance to SMV was clarified.【Result】 GmRHF1 contained a conserved RING-H2 domain of the E3 ubiquitin protein ligase, and belongs to the typical RING-H2 finger family. The results of sequence variation analysis showed that a natural non-synonymous mutation existed in the CDS region of GmRHF1 among various soybean varieties, providing a probable relevance to the SMV resistance or sensibility. After SMV inoculations, the expression level of GmRHF1 in resistant soybean was significantly higher than that in susceptible material by RT-qPCR assay. Through the VIGS experiments, GmRHF1 was effectively silenced and the soybean leaves showed a weakened SMV resistance than the normal plants, indicating that GmRHF1 could enhance soybean resistance to SMV. Based on the yeast cDNA library, 30 potential interacting proteins of GmRHF1 were screened. The interaction between GmRHF1 and GmClpP6 was confirmed by the two-hybrid and LCA experiments. Furthermore, the soybean resistance to SMV was also reduced in soybean leaves with the silenced GmClpP6 by the VIGS experiments.【Conclusion】 The non-synonymous mutation identified in the coding sequence of GmRHF1 may be a key SNP variation related to the SMV resistance and sensibility; and GmRHF1 can interact with GmClpP6. GmRHF1 plays an important role in SMV resistance.

Key words: soybean mosaic virus, zinc finger protein, expression profile analysis, screening of yeast library, luciferase complementary assay

冯雯蜜, 周芳雪, 于哲, 牟可欣, 井妍, 李海燕. 大豆抗花叶病毒病基因GmRHF1的克隆及功能分析[J]. 中国农业科学, 2024, 57(23): 4632-4643.

FENG WenMi, ZHOU FangXue, YU Zhe, MOU KeXin, JING Yan, LI HaiYan. Cloning and Functional Analysis of GmRHF1 Gene Against Soybean Mosaic Virus[J]. Scientia Agricultura Sinica, 2024, 57(23): 4632-4643.

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https://www.chinaagrisci.com/CN/Y2024/V57/I23/4632

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GmRHF1的候选互作蛋白信息"

编号 Number	基因号 Gene identifier	克隆数量 No. of clones	基因描述 Gene description
1	Glyma.11G117100	2	固醇调节元件结合蛋白Sterol regulatory element-binding protein
2	Glyma.18G036400	3	26S蛋白酶体调节复合体，ATP酶RPT4 26S Proteasome regulatory complex, ATPase RPT4
3	Glyma.11G119300	6	酪蛋白酶蛋白水解亚基6 Clp protease proteolytic subunit 6
4	Glyma.03G116600	3	DNA同源物亚家族C成员DNAJ Homolog subfamily C member
5	Glyma.04G017500	4	过氧化氢酶Catalase
6	Glyma.05G048000	1	F型H⁺转运三磷酸腺苷酶亚基DeltaF-type H⁺-transporting ATPase subunit delta
7	Glyma.07G115900	1	核苷二磷酸激酶/核苷二磷酸激酶Nucleoside-diphosphate kinase/Nucleoside diphosphokinase
8	Glyma.08G235000	2	G蛋白偶联受体G-protein coupled receptor
9	Glyma.10G222100	1	叶绿素a/b结合蛋白结构域 Chlorophyll a/b binding protein domain
10	Glyma.12G037400	3	果糖二磷酸醛缩酶1，叶绿体相关Fructose-bisphosphate aldolase 1, chloroplastic-related
11	Glyma.19G046600	1	核酮糖二磷酸羧化酶小链1A，叶绿体相关 Ribulose bisphosphate carboxylase small chain 1A, chloroplastic-related
12	Glyma.04G003700	2	重金属相关异戊二烯化植物蛋白26 Heavy Metal-associated isoprenylated plant protein 26
13	Glyma.08G158600	1	主要促进因子超家族蛋白Major facilitator superfamily protein
14	Glyma.05G007100	1	碳酸酐酶2，叶绿体相关Carbonic anhydrase 2, chloroplastic-related
15	Glyma.09G148100	1	L-艾杜糖醇-2-脱氢酶/山梨醇脱氢酶L-iditol 2-dehydrogenase/Sorbitol dehydrogenase
16	Glyma.06G157500	1	真核天冬氨酸蛋白酶家族蛋白Eukaryotic aspartyl protease family protein
17	Glyma.08G152500	2	碱性螺旋-环-螺旋DNA结合超家族蛋白Basic helix-loop-helix DNA-binding superfamily protein
18	Glyma.15G107900	2	ATP合成酶-γ链，叶绿体相关 ATP Synthase gamma chain 1, chloroplastic-related
19	Glyma.19G215800	1	包含蛋白的干旱响应ATP结合基序Drought responsive ATP-binding motif containing protein
20	Glyma.20G160900	1	U-BOX结构域蛋白32 U-BOX Domain-containing protein 32
21	Glyma.01G010400	1	DEA (D/H)-box RNA解旋酶家族蛋白DEA(D/H)-box RNA helicase family protein
22	Glyma.01G011300	1	四肽重复（TPR）样超家族蛋白Tetratricopeptide repeat (TPR)-like superfamily protein
23	Glyma.02G228200	1	蛋白磷酸酶2C家族蛋白Protein phosphatase 2C family protein
24	Glyma.13G241000	2	乙酰乳酸合成酶/α-乙酰乳酸合成酶Acetolactate synthase/Alpha-acetolactate synthetase
25	Glyma.19G220400	2	核苷酸-糖转运蛋白家族蛋白Nucleotide-sugar transporter family protein
26	Glyma.14G130100	1	泛素羧基末端水解酶家族蛋白Ubiquitin carboxyl-terminal hydrolase family protein
27	Glyma.16G068700	1	GDSL-like脂肪酶/酰基水解酶超家族蛋白GDSL-like Lipase/Acylhydrolase superfamily protein
28	Glyma.16G098100	1	细胞溶质氨基肽酶家族蛋白Cytosol aminopeptidase family protein
29	Glyma.10G209800	2	组氨酸氨裂合酶Histidine ammonia-lyase
30	Glyma.10G213400	1	线粒体F1F0-ATP合酶，亚基d/ATP7 Mitochondrial F1F0-ATP synthase, subunit d/ATP7

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大豆品种 Soybean varieties	Williams 82	徐豆14 Xudou 14	石山黑豆 Shishanheidou
CDS区序列全长 CDS region region sequence (bp)	1044	1035	1038
氨基酸数量 Number of amino acids	347	344	345
编码蛋白的相对分子质量 Molecular weight (kDa)	38.46	38.15	38.25
理论等电点 Theoretical pI	6.27	6.34	6.17
氨基酸组成 Amino acid composition	20	20	20
含量最高氨基酸 Maximum content amino acids	丝氨酸 Ser, S	丝氨酸 Ser, S	丝氨酸 Ser, S
含量最高氨基酸个数 Maximum content amino acids number	38	40	38
含量最高氨基酸占比 Maximum content amino acids proportion (%)	11.00	11.60	11.00