抗大豆花叶病毒基因位点<i>qTsmv-3</i>候选基因克隆及功能分析

doi:10.7668/hbnxb.20195741

摘要/Abstract

摘要：

鉴定抗大豆花叶病毒(SMV)相关基因功能,为培育抗SMV品种提供基因资源。以前期构建抗SMV位点qTsmv-3的近等基因系和转基因拟南芥为材料,对6个MATE候选基因在抗SMV侵染过程中的功能进行分析和鉴定。在大豆基因组中预测到128个MATE家族基因,可分成5个亚家族。qTsmv-3位点的6个MATE候选基因均位于第Ⅰ亚家族,根据公共数据显示它们的表达量或表达部位存在显著差异,Glyma.03G005600在地上部叶和茎中整体表达量最高,Glyma.03G005800在地下部根和根瘤中整体表达量最高。qTsmv-3近等基因系接种SMV后,与#NIL-SMC家系相比,GmICS1和GmPR1在#NIL-NC中的表达量分别上调2.40,15.16倍,SMV浓度降低70%,表明qTsmv-3位点的抗性与水杨酸(SA)介导的抗病通路相关。此外,6个MATE候选基因仅有Glyma.03G005300和Glyma.03G005600的表达量在近等基因系间出现显著差异,分别下调表达61.0%,82.1%。综合考虑6个MATE基因的表达模式和对SMV侵染后的响应,Glyma.03G005600可能为qTsmv-3位点的关键候选基因。进一步研究发现,携带Glyma.03G005600的转基因拟南芥(OE_MATE)受UV-B胁迫后,AtICS1和AtPR1的表达量比野生型(WT)分别显著上调4.22,9.12倍,说明Glyma.03G005600能够显著促进或影响SA信号通路上基因的表达。研究结果初步证实Glyma.03G005600是抗SMV位点qTsmv-3的关键调控基因,为克隆抗SMV关键调控基因奠定了基础。

关键词: 大豆花叶病毒, 抗病候选基因, MATE, qTsmv-3, 功能分析

Abstract:

Soybean mosaic virus(SMV)disease can cause significant yield losses and quality deterioration in soybeans,and breeding resistant cultivars remains the only effective strategy for SMV control.Identifying the functional genes associated with SMV resistance provides essential genetic resources for developing resistant varieties.Six MATE candidate genes involved in SMV resistance were identified using a near-isogenic line(NIL)of the qTsmv-3 locus and a transgenic Arabidopsis thaliana plant.A total of 128 MATE family genes were predicted in the soybean genome,which were classified into five subfamilies.Notably,all six MATE candidate genes located at the qTsmv-3 locus clustered within subfamily Ⅰ,exhibiting significant differences in expression levels and tissue specificity based on public data.Among them,Glyma.03G005600 showed the highest expression in aerial tissues(leaves and stems),while Glyma.03G005800 was predominantly expressed in underground tissues(roots and nodules).Following SMV inoculation,the resistant NIL(#NIL-NC)exhibited a 70% reduction in viral accumulation compared with the susceptible line(#NIL-SMC).Concurrently,the expression levels of GmICS1 and GmPR1,key genes in the salicylic acid(SA)-mediated defense pathway,were upregulated by 2.40,15.16 folds,respectively,in #NIL-NC,indicating that qTsmv-3 confers resistance through SA-dependent signaling.Among the 6 MATE candidate genes,only Glyma.03G005300 and Glyma.03G005600 displayed significant differential expression between NILs,which were down-regulated by 61.0% and 82.1%,respectively.Considering their expression patterns and responses to SMV infection,Glyma.03G005600 was identified as the most promising candidate gene for qTsmv-3.Further,the expression of GmICS1 and GmPR1 in transgenic Arabidopsis thaliana(OE_MATE),which carrying Glyma.03G005600,was significantly up-regulated by 4.22,9.12 folds compared with that of wild type(WT)after UV-B stress.These results strongly indicated that Glyma.03G005600 could significantly enhance or affect the expression of genes in salicylic acid signaling pathway,and preliminarily confirmed that Glyma.03G005600 was a key regulatory gene for qTsmv-3 locus.In all,the results laid a foundation for cloning the key genes regulating SMV resistance and provided gene resources for genetic improvement of SMV resistance in soybean.

Key words: Soybean mosaic virus, Resistance candidate gene, MATE, qTsmv-3, Function analysis

中图分类号:

S435.651

林静, 史晓蕾, 徐俊杰, 于翠红, 曹志敏, 唐晓东, 杨春燕, 张孟臣, 闫龙. 抗大豆花叶病毒基因位点qTsmv-3候选基因克隆及功能分析[J]. 华北农学报, 2025, 40(4): 57-64. doi: 10.7668/hbnxb.20195741.

LIN Jing, SHI Xiaolei, XU Junjie, YU Cuihong, CAO Zhimin, TANG Xiaodong, YANG Chunyan, ZHANG Mengchen, YAN Long. Cloning and Functional Analysis of Candidate Gene for SMV Resistance Locus qTsmv-3 in Soybean[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 57-64. doi: 10.7668/hbnxb.20195741.

http://www.hbnxb.net/CN/Y2025/V40/I4/57

图/表 6

表1 用于基因表达量分析的RT-qPCR引物

Tab.1 RT-qPCR primers for gene expression analysis

基因编号 Gene ID	前引物(5'-3') Forward primer(5'-3')	后引物(5'-3') Revers primer(5'-3')
Glyma.03G005600	TTTCACTTGCCAGAATTTCCTG	CAGGAATCTCGAGCTTGAATTG
Glyma.03G005200	CAGAAGTTGCTGAGCAGAGAGA	TCCACATCACCTTGCTCTCTTC
Glyma.03G005300	AGCACTGTAGCGTATGTGAACA	CGGGCTATAATCACCTGCTCAT
Glyma.03G005400	CACTGTCATCATACGTTTCGCC	CAGGAAGAAGACAGAGTGCAGT
Glyma.03G005500	TGACTCTCTTAGACCAGGATGA	GTCTGGGTAGTGAATGAGACAA
Glyma.03G005800	CGTTCCTGCAATCTCAAAGCAA	CAAAAGCCAGGAGAGAAACACG
GmActin	CGGTGGTTCTATCTTGGCATC	GTCTTTCGCTTCAATAACCCTA
SMV_CP	AATGGCGTGTGGGTGATGAT	GCCTTCATCTGCGCTATTGC
Glyma.15G062400(GmPR1)	GAGTCACGTTGCCAATGCTC	GCAGACACTCTCCACCAACA
Glyma.03G070600(GmISC1)	CCTTCCTATCCCCTTCACTTTT	TTGGTGTCGTAGAAATGAGTGA
AT1G74710(AtICS1)	TCTCAGGTACGAGCTTTTGTCC	AGAACCCCTTATCCCCCATACA
AT2G14610(AtPR1)	TAAGCGATGTTTACGAACCCCA	GAGTCTGCAGTTGCCTCTTAGT
AT4G20890(AtTub9)	CGGAGCTAAGTTCTGGGAAGTT	AGTTATTCCCAGCACCAGACTG

图1 大豆MATE家族基因的系统发育进化分析

Fig.1 Phylogenetic and evolutionary analysis of MATE family genes in soybean

图2 6个MATE候选基因组织表达分析

Fig.2 Tissue expression analysis of 6 MATE candidate genes

图3 近等基因系接种SMV后SA相关基因的表达分析 A.近等系接种SMV后症状;B、C、D.GmICS1、GmPR1和SMV在近等系间的相对表达量。不同小写字母表示差异显著(P<0.05),图4-5同。

Fig.3 Analysis of SA-related gene expression in near-isogenic lines after SMV inoculation A.Symptoms of near-isogenic lines after SMV inoculation;B,C,D.Relative expression levels of GmICS1,GmPR1,and SMV among near-isogenic lines.Different lowercase letters indicate significant differences (P<0.05),the same as Fig.4-5.

图4 近等基因系间MATE候选基因的表达分析

Fig.4 Expression analysis of MATE candidate genes in near-isogenic lines

图5 Glyma.03G005600转基因植株的功能分析 A.正常环境下转基因拟南芥(OE_MATE)和野生型(WT)长势对比;B.OE_MATE和WT对UV-B处理的反应;C.OE_MATE和WT的根长对比;D.拟南芥中ICS1基因的表达分析;E.拟南芥中PR1基因的表达分析。

Fig.5 Functional analysis of Glyma.03G005600 in transgenic plant A.Growth comparison of transgenic Arabidopsis(OE_MATE)and wild-type(WT)under normal conditions;B.Response of OE_MATE and WT to UV-B treatment; C.Root length comparison between OE_MATE and WT;D.Expression analysis of the ICS1 gene in Arabidopsis;E.Expression analysis of the PR1 gene in Arabidopsis.

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抗大豆花叶病毒基因位点qTsmv-3候选基因克隆及功能分析

Cloning and Functional Analysis of Candidate Gene for SMV Resistance Locus qTsmv-3 in Soybean

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抗大豆花叶病毒基因位点qTsmv-3候选基因克隆及功能分析

Cloning and Functional Analysis of Candidate Gene for SMV Resistance Locus qTsmv-3 in Soybean

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