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作物学报 ›› 2018, Vol. 44 ›› Issue (05): 642-649.doi: 10.3724/SP.J.1006.2018.00642

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

普通菜豆中烟草水杨酸结合蛋白2同源基因的鉴定及表达特征分析

薛仁风1,**(), 王利2,**, 丰明1, 葛维德1,*()   

  1. 1辽宁省农业科学院作物研究所, 辽宁沈阳 110161
    2农业部农业生态与资源保护总站, 北京 100125
  • 收稿日期:2017-09-10 接受日期:2018-01-08 出版日期:2018-05-20 网络出版日期:2018-01-29
  • 通讯作者: 薛仁风,王利,葛维德
  • 作者简介:

    第一作者联系方式: E-mail: xuerf82@163.com, Tel: 024-31029901

  • 基金资助:
    本研究由国家自然科学基金项目(31401447), 辽宁省博士科研启动基金(201501113), 特色粮油新品种选育及优质高效生产技术集成与示范(2018416023)和国家现代农业产业技术体系建设专项(CARS-09-08Z)资助

Identification and Expression Analysis of Likely Orthologs of Tobacco Salicylic Acid Binding Protein 2 in Common Beans

Ren-Feng XUE1,**(), Li WANG2,**, Ming FENG1, Wei-De GE1,*()   

  1. 1 Crop Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, Liaoning, China
    2 Rural Energy and Environment Agency, Ministry of Agriculture, Beijing 100125, China
  • Received:2017-09-10 Accepted:2018-01-08 Published:2018-05-20 Published online:2018-01-29
  • Contact: Ren-Feng XUE,Li WANG,Wei-De GE
  • Supported by:
    This work was supported by the National Natural Science Foundation of China (31401447), the Liaoning Doctor Startup Foundation (201501113), the New Varieties Breeding, High Quality and Effective Technology Integration and Demonstration of Special Grain and Oil Crop (2018416023), and the China Agriculture Research System (CARS-08-Z8).

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摘要：

水杨酸(salicylic acid, SA)能够诱导植物产生系统抗病性反应, 而水杨酸结合蛋白2 (salicylic acid binding protein 2, SABP2)是植物细胞内调控水杨酸水平的重要酯酶。本研究利用生物信息学方法在普通菜豆中搜索到7个烟草水杨酸结合蛋白2的同源基因, 命名为PvMES1~PvMES7。分别在2个菜豆感病品种(白刀豆和BRB130)和2个抗病品种(黑芸豆和260205)中接种尖镰孢菌FOP-DM01菌株(Fusarium oxysporum f. sp. phaseoli isolate, FOP-DM01)后, 检测寄主根组织中水解水杨酰甲酯(methyl salicylate, MeSA)活性和游离SA含量的变化, 并利用荧光定量PCR技术分析7个PvMES基因表达量变化。结果表明, PvMES1、PvMES3、PvMES4、PvMES5和PvMES6的转录表达受FOP-DM01菌株诱导均呈现不同程度的升高, 其中黑芸豆中PvMES5基因和260205中PvMES1基因表达量变化最显著, 接种3 d后分别升高至0 d表达量的7.6倍和5.6倍。此外, 黑芸豆和260205根中水杨酰甲酯酯酶(methyl salicylate esterase, MES)活性显著提升, 游离SA含量也相应升高, 激活寄主内SA介导的相关防御反应。本研究结果可以为普通菜豆镰孢菌枯萎病抗病分子育种和抗病机理研究提供理论基础。

关键词: 普通菜豆, 镰孢菌枯萎病, 水杨酸结合蛋白2, 直系同源基因, 系统获得性抗性

Abstract:

Salicylic acid can induce the systematic resistant response in plants, and salicylic acid-binding protein 2 is an important esterase in the regulation of salicylic acid content in plant cells. In this study, we used the bioinformatics method to search the orthologous genes of tobacco salicylic acid-binding protein 2 in common bean, which were designated as PvMES1-PvMES7, then analyzed the changes of MES activity and free SA accumulation in plant roots. The expression level of 7 PvMES genes in susceptible varieties BRB130 and Baidaodou, resistant varieties Heiyundou and 260205 infected by Fusarium oxysporum f. sp. phaseoli isolate FOP-DM01 was detected by Real time PCR, showing that the expression of PvMES1, PvMES3, PvMES4, PvMES5, and PvMES6 was significantly increased at three days after FOP-DM01 infection, with most significant increase of 7.6 folds and 5.6 folds for PvMES5 gene in Heiyundou and PvMES1 gene in 260205 at 0 day, respectively. The MES activity and free SA content in roots of Heiyundou and 260205 were also improved, which activated the relevant defense response reaction mediated by SA in the host. The results of this study provide a theoretical basis for the resistance molecular breeding of Fusarium wilt in common beans.

Key words: common bean, Fusarium wilt, salicylic acid binding protein 2, orthologous genes, system acquired resistance

表1

实验中所用的引物"

基因名称
Gene name		 引物名称
Primer name		 引物序列
Sequence for qPCR (5°-3°)
PvMES1 QMES1F GCTCTATCAACTTTCCCCTG
QMES1R CATCGTTGGTAATCCAAAGT
PvMES2 QMES2F CTATCAACTCTCCCCAACTC
QMES2R CAAAGTAATTGCAAGGTCTAT
PvMES3 QMES3F CAGAAAATTGAAGACGTTGG
QMES3R AACATAGGAGGGCTTGCGC
PvMES4 QMES4F GAGTACCTTGGGAGAGAAT
QMES4R TAGAGAAGCTCTTCTGTTGG
PvMES5 QMES5F GCAATTCCCAGAAAAAATTCTG
QMES5R CTTGGGACCAAAGAACATCAA
PvMES6 QMES6F ACCAAGTTTCTGTCCACTGC
QMES6R TACTCTTTTGGAATTGTCAA
PvMES7 QMES7F GGAGTTGAATTTTTGAGATCTA
QMES7R CATCAAATTATCTTTTCCAGC
PvPR1 QPR1F TGCTAAAGACGCCGATACCA
QPR1R GCAACACCTCCAACTATGCT
Actin Act-F GAAGTTCTCTTCCAACCATCC
Act-R TTTCCTTGCTCATTCTGTCCG

表2

PvMES家族基因与烟草水杨酸结合蛋白2核苷酸序列分析"

基因名称
Gene		 基因位点
Genetic locus		 基因序列位置
Sequence location		 E值
E-value		 一致性
Identity (%)		 染色体
Chromosome
PvMES1 Phvul.003G248200.1 48561200-48564505 1.7×10-35 59.2 3
PvMES2 Phvul.003G248300.1 48567955-48569848 7.0×10-34 57.6 3
PvMES3 Phvul.003G248025.1 48530639-48532632 1.6×10-29 63.8 3
PvMES4 Phvul.003G248050.1 48539059-48541466 1.9×10-28 61.4 3
PvMES5 Phvul.003G248075.1 48545538-48547254 1.9×10-28 63.1 3
PvMES6 Phvul.002G022300.1 2389494-2391172 3.4×10-25 58.4 2
PvMES7 Phvul.010G043800.2 6682398-6684451 5.1×10-4 45.3 10

图1

PvMES1~PvMES7和NtSABP2氨基酸序列比对分析相同位点用黑色表示, 相似位点用灰色表示; 水解活性位点用黑色箭头表示。SA结合位点用黑色三角形表示。"

图2

PvMES1~PvMES7和NtSABP2进化树分析"

图3

FOP-DM01菌株诱导下普通菜豆根组织PvMES1~PvMES7和PvPR1表达量相同时间点中标以不同字母的柱值在不同品种间差异显著(P < 0.05)。"

图4

FOP-DM01菌株诱导下普通菜豆根组织MES活性相同时间点中标以不同字母的柱值在不同品种间差异显著(P < 0.05)。"

图5

FOP-DM01菌株诱导下普通菜豆根组织SA含量分析相同时间点中标以不同字母的柱值在不同品种间差异显著(P < 0.05)。"

图6

普通菜豆接种FOP-DM01菌株后的病情评价 A: 接种FOP-DM01菌株3周后发病情况; B: 接种FOP-DM01菌株3周后的病情指数。相同时间点中标以不同字母的柱值在不同品种间差异显著(P < 0.05)。"

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