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作物学报 ›› 2021, Vol. 47 ›› Issue (11): 2134-2146.doi: 10.3724/SP.J.1006.2021.04254

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

受青枯菌诱导的花生根酵母双杂交文库构建和AhRRS5互作蛋白的筛选

陈玉婷1,2(), 刘露1,2, 楚盼盼1,2, 魏嘉贤1,2, 钱慧娜1,2, 陈华1,2,3, 蔡铁城1,2,3, 庄伟建1,2,3,*(), 张冲1,2,3,*()   

  1. 1福建农林大学豆科油料植物遗传与系统生物学研究中心, 福建福州 350002
    2闽台作物有害生物生态防控国家重点实验室 / 福建农林大学植物保护学院, 福建福州 350002
    3福建省作物分子与细胞生物学重点实验室 / 福建农林大学农学院, 福建福州 350002
  • 收稿日期:2020-11-24 接受日期:2021-04-14 出版日期:2021-11-12 网络出版日期:2021-05-14
  • 通讯作者: 庄伟建,张冲
  • 作者简介:E-mail: 1203654525@qq.com
  • 基金资助:
    国家自然科学基金项目(32072103);国家自然科学基金项目(31701463);国家自然科学基金项目(U1705233);福建省科技厅农业引导性(重点)项目(2018N0004)

Construction of yeast two-hybrid cDNA library induced by Ralstonia solanacearum and interaction protein screening for AhRRS5 in peanut

CHEN Yu-Ting1,2(), LIU Lu1,2, CHU Pan-Pan1,2, WEI Jia-Xian1,2, QIAN Hui-Na1,2, CHEN Hua1,2,3, CAI Tie-Cheng1,2,3, ZHUANG Wei-Jian1,2,3,*(), ZHANG Chong1,2,3,*()   

  1. 1Research Center of Legume Genetics and System Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops / College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3Provincial Key Laboratory of Crop Molecular and Cell Biology / College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2020-11-24 Accepted:2021-04-14 Published:2021-11-12 Published online:2021-05-14
  • Contact: ZHUANG Wei-Jian,ZHANG Chong
  • Supported by:
    National Natural Science Foundation of China(32072103);National Natural Science Foundation of China(31701463);National Natural Science Foundation of China(U1705233);Science and Technology Foundation of Fujian Province of China(2018N0004)

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摘要：

前期研究报道超表达花生AhRRS5基因能够显著提高烟草抗青枯病水平, 为进一步探究NBS-LRR类抗病蛋白AhRRS5在花生应答青枯菌胁迫的信号通路, 本研究在构建花生受青枯菌诱导的根部组织均一化三框文库的基础上, 通过酵母双杂交技术筛选AhRRS5的互作蛋白。通过改良的CTAB法提取青枯菌诱导后不同时间点的花生根部组织样品总RNA, 分离纯化mRNA并合成双链cDNA, 并基于同源重组方法分别构建酵母双杂交初级和次级文库。构建的酵母双杂交次级cDNA文库库容为1.44×107 cfu mL-1, 重组率为100%, 插入片段大小在1000 bp以上。通过酶切连接法构建pGBKT7-AhRRS5诱饵载体, 在酵母细胞中无自激活和毒性活性, 与酵母双杂交文库共转酵母Y2H Gold菌株后, 经多次筛库和回转验证, 最终获得12个候选互作蛋白, 这些蛋白涉及到植物生长发育、能量代谢、激素信号转导、胁迫响应等多个方面。通过双分子荧光互补(bimolecular fluorescence complementation, BiFC)验证了AhSBT1.6和AhRRS5的体内互作。转录组数据显示, 花生AhSBT1.6基因在不同组织中表达差异显著; 实时荧光定量PCR显示, 该基因在抗青枯病花生品种中受青枯菌诱导上调表达, 推测AhSBT1.6可能参与调控花生青枯病抗性。研究结果为进一步研究NBS-LRR类抗病蛋白AhRRS5和互作蛋白在花生青枯病抗性防御的作用机制奠定了基础。

关键词: 花生, 青枯病, 酵母双杂交, 互作蛋白, AhSBT1.6, 双分子荧光互补

Abstract:

The overexpression of peanut AhRRS5 gene can significantly improve tobacco resistance to bacterial wilt in previous studies. To further explore signaling pathway of the NBS-LRR resistance protein AhRRS5 responding to Ralstonia solanacearum infection in peanut, the interaction proteins of AhRRS5 were screened by yeast two hybrid technology based on the construction of peanut root normalized three-frame libraries induced by Ralstonia solanacearum. Total RNA was extracted from peanut roots at different time points after Ralstonia solanacearum infection. The mRNA was isolated and purified, then double stranded cDNA was synthesized and normalized. The primary and secondary libraries were constructed by homologous recombination method. The titer of the secondary library was 1.44 × 10 7 cfu mL-1, the recombination rate was 100%, and the average length encoded by the inserted cDNA was more than 1000 bp. The bait vector pGBKT7-AhRRS5 was constructed by enzyme digestion and ligation method. Result showed that there was no toxicity and auto-activation in the yeast cells. The AD library plasmid and bait vector pGBKT7-AhRRS5 were co-transformed into yeast Y2H gold strain. After several screening and rotation verification, 12 candidate proteins were obtained, which were involved in plant growth and development, energy metabolism, hormone signal transduction, stress response and so on. The interaction of AhRRS5 with AhSBT1.6 was further confirmed by bimolecular fluorescence complementation assays (BiFC) in vivo. The relative expression levels of AhSBT1.6 genes revealed that there were significant differences in different tissues based on transcriptome profiling, indicating the potential involvement of AhSBT1.6 in regulating bacterial with resistance in peanut. This study lays a foundation for further study on the mechanism of NBS-LRR resistance protein AhRRS5 and its interaction protein in bacterial wilt resistance defense of peanut.

Key words: peanut, bacterial wilt, yeast-two-hybrid, interaction protein, AhSBT1.6, BiFC

图1

受青枯菌诱导的花生根部RNA(A)、mRNA分离(B)和双链cDNA均一化(C)后的琼脂糖凝胶电泳图 M: DL2000 DNA分子量标准品。"

图2

受青枯菌诱导的花生根酵母双杂交初级和次级文库的插入片段的PCR检测 M: DL2000 DNA 分子量标准品; 1~24: 初级文库中随机挑取的24个克隆; 25~48: 次级文库中随机挑取的24个克隆。"

图3

pGBKT7-AhRRS5诱饵载体转化酵母自激活验证"

图4

花生抗青枯病蛋白AhRRS5互作蛋白的筛选 1~12是筛选获得的互作蛋白阳性克隆; -表示阴性对照, +表示阳性对照。"

表1

酵母双杂交筛选互作蛋白插入片段的序列分析"

克隆编号
Clone ID		 蛋白名称
Protein name		 基因编号
Gene ID		 ORF是否完整
Complete ORF or not		 功能
Function
Y2H-1 类枯草杆菌蛋白酶SBT1.6
Subtilisin-like protease SBT1.6		 AH13G08570.1 是 Yes 调控植物发育和衰老, 应对环境胁迫和病原侵染发挥关键作用。
Regulate plant development and senescence, challenge to environmental stress and pathogen infection.
Y2H-2 半胱氨酸蛋白酶类RD21a
Cysteine proteinase RD21a		 AH16G42150.1 否 No 植物发育, 参与植物逆境胁迫和细胞程序性死亡, 响应病原菌侵染等。
Regulate plant development, involve in plant stress and programmed cell death, and response to pathogen infection, etc.
Y2H-3 类BEL1同源结构域蛋白1
BEL1-like homeodomain protein 1		 AH12G34520.1 是 Yes 调控植物分生组织发育等。
Regulate the development of plant meristem.
Y2H-4 类糊精硫醇蛋白酶
Thiol protease aleurain-like		 AH13G27250.1 是 Yes 植物代谢, 参与植物逆境胁迫和细胞程序性死亡, 响应病原菌侵染等。
Plant metabolism, involve in plant stress and programmed cell death, response to pathogen infection, etc.
Y2H-5 组蛋白赖氨酸N-甲基转移酶SUVR3
Histone-lysine N-methyltransferase SUVR3		 AH10G29030.1 否 No 氨基酸降解。
Amino acid degradation.
Y2H-6 40S核糖体蛋白Sa-1
40S ribosomal protein Sa-1		 AH19G26400.1 否 No 参与DNA修复、细胞凋亡以及基因表达调控等。
Involve in DNA repair, apoptosis and gene expression regulation.
克隆编号
Clone ID		 蛋白名称
Protein name		 基因编号
Gene ID		 ORF是否完整
Complete ORF or not		 功能
Function
Y2H-7 类枯草杆菌蛋白酶SBT1.6
Subtilisin-like protease SBT1.6		 AH13G08570.1 是 No 调控植物发育和衰老, 应对环境胁迫和病原侵染发挥关键作用。
Regulate plant development and senescence, challenge to environmental stress and pathogen infection.
Y2H-8 未知蛋白
Unknown protein		 AH20G21850.1 是 Yes 未知。
Unknown.
Y2H-9 V型质子ATP酶亚基C
V-type proton ATPase subunit C		 AH20G03980.1 否 No 氧化磷酸化。
Oxidative phosphorylation.
Y2H-10 脱水诱导蛋白Di19-3
Dehydration-induced 19 homolog 3		 AH16G09740.1 否 No 响应非生物胁迫。
Response to abiotic stress.
Y2H-11 焦磷酸合酶
Pyrophosphate synthase		 AH02G15900.1 是 Yes 脂质代谢和离子吸收相关。
Lipid metabolism and ion absorption.
Y2H-12 乙酰鸟氨酸脱乙酰酶
Acetylornithine deacetylase		 AH05G03880.1 否 No 氨基酸生物合成相关。
Amino acid biosynthesis.

图5

花生抗青枯病蛋白AhRRS5与部分候选互作蛋白回补验证 - 表示阴性对照, + 表示阳性对照。"

图6

利用BiFC在本氏烟草中验证AhRRS5与AhSBT1.6的互作 标尺为50 μm。"

图7

花生AhSBT1.6表达特征分析 A: 花生AhSBT1.6基因在不同组织器官、激素处理和非生物胁迫中的表达热图; B: AhSBT1.6基因在在抗、感花生品种接种青枯菌后不同时间点的表达模式。R: 根; R tip: 根尖; R nod: 根瘤; S: 茎; S tip: 茎尖; L: 叶; RS: 根茎结合处; Peg: 果针; F: 花; Cot: 子叶; 10PC、30PC、50PC: 分别代表果针入土10 d、30 d和50 d的果皮; 10PE、20PE、30PE、50PE: 分别代表果针入土10 d、20 d、30 d和50 d的胚; 20PT、40PT、60PT: 分别代表果针入土20 d、40 d和60 d的果皮; SA: 水杨酸; ABA: 脱落酸; ETH: 乙烯利; BR: 油菜素内酯; PAC: 多效唑; HorCK: 水对照; Dry: 干旱处理组; DryCK: 干旱对照组; LT: 低温处理组; LTCK: 低温对照组; XHXL-MOCK: 感青枯病花生品种新会小粒接种无菌水对照; XHXL-RS: 感青枯病花生品种新会小粒接种青枯菌处理; YY92-MOCK: 抗青枯病花生品种粤油92接种无菌水对照; YY92-RS: 抗青枯病花生品种粤油92接种青枯菌处理。柱上不同大小写字母分别表示处理在0.01和0.05水平差异显著。误差线代表校准误差, n = 3。"

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