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

doi:10.3724/SP.J.1006.2021.04254

Abstract

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

CHEN Yu-Ting, LIU Lu, CHU Pan-Pan, WEI Jia-Xian, QIAN Hui-Na, CHEN Hua, CAI Tie-Cheng, ZHUANG Wei-Jian, ZHANG Chong. Construction of yeast two-hybrid cDNA library induced by Ralstonia solanacearum and interaction protein screening for AhRRS5 in peanut[J].Acta Agronomica Sinica, 2021, 47(11): 2134-2146.

Figures/Tables 8

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Table 1

Sequence analysis of candidate interacrting protein by yeast two-hybrid system"

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.

Table 1

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

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