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作物学报 ›› 2019, Vol. 45 ›› Issue (12): 1822-1831.doi: 10.3724/SP.J.1006.2019.94054

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆抗SC3候选基因的克隆及分析

向文扬,杨永庆,任秋燕,晋彤彤,王丽群,王大刚,智海剑()   

  1. 南京农业大学大豆研究所 / 农业部大豆生物学与遗传育种重点实验室 / 国家大豆改良中心 / 作物遗传与种质创新国家重点实验室, 江苏南京 210095
  • 收稿日期:2019-04-02 接受日期:2019-06-22 出版日期:2019-12-12 网络出版日期:2019-07-15
  • 通讯作者: 智海剑
  • 作者简介:向文扬, E-mail: a15295572307@163.com
  • 基金资助:
    本研究由国家转基因生物新品种培育科技重大专项(2016ZX08004-004)(2016ZX08004-004);国家自然科学基金项目(31571690);国家自然科学基金项目(31571687);中央高校基本科研业务费专项(KYT201801);长江学者和创新团队发展计划项目(PCSIRT_17R55);国家现代农业产业技术体系建设专项(CARS-004);江苏省现代作物生产协同创新项目(JCIC-MCP);国家重点研发计划项目(2017YFD0101501)

Cloning and analysis of candidate gene resistant to SC3 in soybean

Wen-Yang XIANG,Yong-Qing YANG,Qiu-Yan REN,Tong-Tong JIN,Li-Qun WANG,Da-Gang WANG,Hai-Jian ZHI()   

  1. Institute of Soybean, Nanjing Agricultural University / Key Laboratory of Soybean Biology and Genetic Breeding, Ministry of Agriculture / National Center for Soybean Improvement / State Key Laboratory of Crop Genetics & Germplasm Innovation, Nanjing 210095, Jiangsu, China
  • Received:2019-04-02 Accepted:2019-06-22 Published:2019-12-12 Published online:2019-07-15
  • Contact: Hai-Jian ZHI
  • Supported by:
    This study was supported by the Fund of Transgenic Breeding for Soybean Resistance to Soybean mosaic virus(2016ZX08004-004);the National Natural Science Foundation of China(31571690);the National Natural Science Foundation of China(31571687);the Fundamental Research Funds for the Central Universities(KYT201801);the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT_17R55);the National Soybean Industrial Technology System of China(CARS-004);Jiangsu Collaborative Innovation Center for Modern Crop Production(JCIC-MCP);the National Key R&D Program of China(2017YFD0101501)

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摘要：

大豆花叶病毒(Soybean mosaic virus, SMV)病是大豆主要的病害之一, 给我国大豆生产带来了巨大的损失。大豆抗病育种是目前防治大豆花叶病毒病最为经济有效的措施, 发掘抗病基因是抗病育种的基础。本文在前期对大豆抗SMV株系SC3基因精细定位的基础上, 克隆了2个具有TIR-NBS-LRR典型抗病结构域的基因(GmR47和GmR51)。生物信息学分析表明, GmR47和GmR51基因均在抗感品种中存在氨基酸位点的突变, 而且突变位点都位于保守结构域内, 这2个基因编码的蛋白质预测为烟草花叶病毒(TMV)抗性N蛋白; 物种间同源比对结果显示, GmR47和GmR51基因与野生大豆亲缘较近。qRT-PCR结果表明, GmR47和GmR51能够响应SMV的侵染增加表达量, 且在抗病品种中的表达量高于感病品种。2个基因存在IN1、IN2和IN3不同的剪接体, 所有的剪接体都能够响应病毒的诱导增加表达量, 且在抗病品种中的表达量高于感病品种, IN1和IN2的表达量随时间的变化较为明显, IN3的表达量则相对稳定, 说明这些剪接体可能参与大豆对SMV的抗病过程。本研究为后续基因功能的研究奠定了基础。

关键词: 大豆花叶病毒, 抗病基因, 诱导表达, 可变剪接

Abstract:

Soybean mosaic virus (SMV) is one of the prevalent pathogens of soybean, causing great reduction of soybean yield worldwide. Soybean disease resistance-breeding is currently the most cost-effective measure to control SMV, and identification of resistance genes is the basis of disease resistance breeding. According to the previous mapping result of resistance gene to SMV strain SC3, two genes (GmR47, GmR51) with TIR-NBS-LRR domain were cloned. Bioinformatics analysis showed that both GmR47 and GmR51 genes have SNP mutations in the susceptible varieties and resistant varieties, and the mutation sites are located in the conserved domain. These two proteins encoded by GmR47 and GmR51 genes are predicted to be Tobacco mosaic virus (TMV) resistant N proteins. The results of homologous alignment between species indicated that GmR47 and GmR51 genes were close to those of wild soybean. The expression of GmR47 and GmR51 was analyzed after inoculation with soybean mosaic virus in soybean, demonstrating that GmR47 and GmR51 could increase the expression level in response to SMV infection, with the higher level in resistant varieties than in susceptible varieties. Analysis of the alternative splicing of GmR47 and GmR51 revealed that the two genes have different splice variants IN1, IN2, and IN3. The response analysis of splices to SMV showed that all splices were able to increase the expression in response to virus induction, with the higher level in resistant varieties than in susceptible varieties. It indicated that these alternative splicing may be involved in the disease resistance process of soybean to SMV. The result of this study lay a foundation for the study of subsequent gene function.

Key words: Soybean mosaic virus (SMV), resistance gene, inducing expression, alternative splicing

表1

基因克隆引物及荧光定量引物"

引物名称
Primer name		 上游引物
Forward primer (5'-3')		 下游引物
Reverse primer (5'-3')
GmR47-1 TTTCAGAATCTATTGATATTAGGG TCTGTGAAACTATGCCTTGC
GmR51-1 GGATTGGAGTGGGAGGA GGGTTGAAGCAACGAAA
GmR47-2 TGGGATTGGAGTGGGAGGAGT CCATCAAAGTTGAATCCCAATTCAC
GmR51-2 GGGAGGAGTTGAATTAGCG CCAGTCTCCACCACTTTGTT
GmR47/51-qPCR GTAGGGGTCTTTGTTGATGTTGATT TCTGAGAAACTCCTATGTCCGCTA
Tubulin-qPCR AGTATGAGGACGAGGAGGACGAT TACGCATCACATAGCATAAGTAAGACAC

表2

可变剪接检测引物及荧光定量引物"

引物名称
Primer name		 引物
Primer (5'-3')
Intron1-Uni F: TTCGGAGGCGATTGAACAT
R: TGCTCAATTTGATCAATTAATTAAACTCA
Intron2-Uni F: AAATTGTTCGACAGGAATCACC
R: TGGCAGAAACTCGACGTAATG
Intron3-Uni F: CTCTTGAATCCACCAAAGCAGG
R: AGTTCATATGATTAGAAGAACATGTAGAG
Intron1-Spe F: ATCTACTAGTCATATGGATTTGCTCAATTTGATCAATTAATTAAACTCA
R: CGGTACCCGGGGATCCGATTTTCGGAGGCGATTGAACAT
Intron2-Spe F: CGGTACCCGGGGATCCGATTAAATTGTTCGACAGGAATCACC
R: ATCTACTAGTCATATGGATTTGGCAGAAACTCGACGTAATG
Intron3-Spe F: CGGTACCCGGGGATCCGATTCTCTTGAATCCACCAAAGCAGG
R: ATCTACTAGTCATATGGATTAGTTCATATGATTAGAAGAACATGTAGAG
Intron1-qPCR F: GAAATGGAGGAACGCACTGC
R: AAGATCGACAAGAAGATATTACCTGAA
Intron2-qPCR F: GGCAAACGCAGTAGGTTATGG
R: ATACCATCAATAGAATGGCAGAAACT
Intron3-qPCR F: ATGTAGGAGAATGCTACTGAAACAGG
R: AGAATATCTCTTATTTCCAGCCTCAT

图1

GmR47和GmR51编码蛋白质的保守结构域预测 A: GmR47编码蛋白质的保守结构域预测。B: GmR51编码蛋白质的保守结构域预测。"

图2

GmR47和GmR51编码的核苷酸序列在抗病品种(PI96983)及感病品种(南农1138-2)之间的比对结果 A: GmR47编码氨基酸序列的比对结果。B: GmR51编码氨基酸序列比对的结果。"

图3

GmR47和GmR51基因在不同物种间的同源进化树"

图4

GmR47 (A)和GmR51 (B)基因编码的氨基酸序列二级结构预测结果"

图5

抗感品种中GmR47和GmR51基因编码合成的蛋白质三维结构预测结果 A: PI96983 GmR47; B: PI96983 GmR51; C: 南农1138-2 GmR47; D: 南农1138-2 GmR51。"

图6

PI96983和南农1138-2接种SMV后GmR47及GmR51基因的总表达量随时间的变化"

图7

可变剪接体的凝胶电泳检测结果 上排条带为常规引物扩增的结果, 下排条带为内含子特异性引物的扩增结果, 相邻的4个条带为4个品种间的扩增结果。A: 齐黄1号; B: PI96983; C: 南农1138-2; D: Williams 82; M: marker。"

图8

可变剪接测序结果 A: GmR47基因可变剪接体示意图; B: IN1序列; C: IN2序列; D: IN3序列。"

图9

PI96983和南农1138-2接种SMV后GmR47及GmR51基因在不同内含子处的剪切体的总表达量随时间的变化 A: 内含子1处的剪接体的总表达量; B: 内含子2处的剪接体的总表达量; C: 内含子3处的剪接体的总表达量。"

图10

候选基因与GFP融合蛋白在烟草细胞中的亚细胞定位结果"

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