欢迎访问作物学报，今天是
作物学报

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)

RichHTML

16

PDF

283

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。"

[1] Wicker E, Grassart L, Coranson-Beaudu R, Mian D, Guilbaud C, Fegan M, Prior P. Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential. Appl Environ Microbiol, 2007, 73: 6790-6801.
doi: 10.1128/AEM.00841-07
[2] Zhang C, Chen H, Cai T C, Deng Y, Zhuang R R, Zhang N, Zeng Y H, Zheng Y X, Tang R H, Pan R L, Zhuang W J. Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco. Plant Biotechnol J, 2017, 15: 39-55.
doi: 10.1111/pbi.2017.15.issue-1
[3] 何礼远, 康耀卫. 植物青枯菌(Pseudomonas solanacearum)致病机理. 自然科学进展, 1995, (1):9-18.
He L Y, Kang Y W. Pathogenic mechanism of Pseudomonas solanacearum in plant. Nat Sci Prog, 1995, (1):9-18 (in Chinese with English abstract).
[4] 张明红. 花生青枯病的发生与综合防治. 农业科技通讯. 2019, (6):290-292.
Zhang M H. Occurrence and integrated control of peanut bacterial wilt. Agricul Sci Tech Commun, 2019, (6):290-292 (in Chinese).
[5] Kalia S, Rathour R. Current status on mapping of genes for resistance to leaf- and neck-blast disease in rice. 3 Biotech, 2019, 9: 209-223.
doi: 10.1007/s13205-019-1738-0 pmid: 31093479
[6] Deslandes L, Pileur F, Liaubet L, Camut S, Can C, Williams K, Holub E, Beynon J, Arlat M, Marco Y. Genetic characterization of RRS1, a recessive locus in Arabidopsis thaliana that confers resistance to the bacterial soilborne pathogen Ralstonia solanacearum. Mol Plant Microbe Interact, 1998, 11: 659-667.
doi: 10.1094/MPMI.1998.11.7.659
[7] Deslandes L, Olivier J, Theulieres F, Hirsch J, Feng D X, Bittner-Eddy P, Beynon J, Marco Y. Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes. Proc Natl Acad Sci USA, 2002, 99: 2404-2409.
doi: 10.1073/pnas.032485099
[8] Deslandes L, Olivier J, Peeters N, Feng D X, Khounlotham M, Boucher C, Somssich I, Genin S, Marco Y. Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proc Natl Acad Sci USA, 2003, 100: 8024-8029.
doi: 10.1073/pnas.1230660100
[9] Bernoux M, Timmers T, Jauneau A, Brière C, de Wit J G M P, Marco Y, Deslandes L. RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. Plant Cell, 2008, 20: 2252.
doi: 10.1105/tpc.108.058685 pmid: 18708476
[10] Tasset C, Bernoux M, Jauneau A, Pouzet C, Brière C, Jacquinod K S, Rivas S, Marco Y, Deslandes L. Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R-mediated immunity in Arabidopsis. PLoS Pathog, 2010, 6: e1001202.
doi: 10.1371/journal.ppat.1001202
[11] Narusaka M, Shirasu K, Noutoshi Y, Kubo Y, Shiraishi T, Iwabuchi M, Narusaka Y. RRS1 and RPS4 provide a dual resistance-gene system against fungal and bacterial pathogens. Plant J, 2010, 60: 218-226.
doi: 10.1111/tpj.2009.60.issue-2
[12] Ma Y, Guo H L, Hu L X, Martinez P P, Moschou N P, Cevik V, Ding P T, Duxbury Z, Sarris F P, Jones D G J. Arabidopsis distinct modes of depression of an immune receptor complex by two different bacterial effectors. Proc Natl Acad Sci USA, 2018, 115: 10218-10227.
doi: 10.1073/pnas.1811858115
[13] Sarris F P, Duxbury Z, Huh U S, Ma Y, Segonzac C, Sklenar J, Derbyshire P, Cevik V, Rallapalli G, Saucet B S, Wirthmueller L, Menke L H F, Sohn K H, Jones D G J. A Plant immune receptor detects pathogen effectors that target WRKY transcription factors. Cell, 2015, 161: 1089-1100.
doi: 10.1016/j.cell.2015.04.024
[14] Ngou P M B, Ahn H K, Ding P T, Redkar A, Brown H, Ma Y, Youles M, Tomlinson L, Jones D G J. Estradiol-inducible AvrRps4 expression reveals distinct properties of TIR-NLR-mediated effector-triggered immunity. J Exp Bot, 2020, 71: 2186-2197.
doi: 10.1093/jxb/erz571
[15] Saucet B S, Ma Y, Sarris F P, Furzer J O, Sohn K H, Jones D G J. Two linked pairs of Arabidopsis TNL resistance genes independently confer recognition of bacterial effector AvrRps4. Nat Commun, 2015, 6: 6338.
doi: 10.1038/ncomms7338 pmid: 25744164
[16] Godiard L, Sauviac L, Torii K U, Grenon O, Mangin B, Grimsley N H, Marco Y. ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt. Plant J, 2003, 36: 353-365.
doi: 10.1046/j.1365-313X.2003.01877.x
[17] Martin G B, Brommonschenkel S H, Chunwongse J, Frary A, Ganal M W, Spivey R, Wu T, Earle E D, Tanksley S D. Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science, 1993, 262: 1432-1436.
pmid: 7902614
[18] Scofield S R, Tobias C M, Rathjen J P, Chang J F, Lavelle D T, Michelmore R W, Staskawicz B J. Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato. Science, 1996, 274: 2063-2065.
pmid: 8953034
[19] Deslandes L, Olivier J, Peeters N, Feng D X, Khounlotham M, Boucher C, Somssich I, Genin S, Marco Y. Physical interaction between RRS1-R, a protein conferring resistance to, bacterial wilt, and PopP2, a type III effector targeted to the plant, nucleus. Proc Natl Acad Sci USA, 2003, 100: 8024-8029.
doi: 10.1073/pnas.1230660100
[20] Mackey D, Belkhadir Y, Alonso J M, Ecker J M, Dangl J L. Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance. Cell, 2003, 112: 380-389.
[21] 刘阳娜. 利用酵母双杂交系统筛选GmDREB5的互作蛋白及核蛋白筛选系统(NTT)的建立. 西北农林科技大学硕士学位论文, 陕西杨凌, 2007.
Liu Y N. Screening of Interactor Protein with Soybean Transcription Factor, GmDREB5, Using Yeast Two-Hybrid System and Construction of a Nuclear Transcription Trap (NTT) System. MS Thesis of Northwest A&F University, Yangling, Shaanxi, China, 2007 (in Chinese with English abstract).
[22] 徐萍萍. 利用酵母双杂交技术筛选与马铃薯StERF5转录因子相互作用的蛋白质. 甘肃农业大学硕士学位论文, 甘肃兰州, 2018.
Xu P P. Screening Proteins Interacting with StERF5 Transcription Factor of Potato by Yeast Two-hybrid Technique. MS Thesis of Gansu Agricultural University, Lanzhou, Gansu, China, 2018 (in Chinese with English abstract).
[23] 刘永惠, 沈一, 沈悦, 陈志德. 利用酵母双杂交系统筛选花生AhMYB44互作蛋白. 植物遗传资源学报, 2020, 21: 201-207.
Liu Y H, Shen Y, Shen Y, Chen Z D. Yeast two-hybrid screening of the proteins interacting with AhMYB44 from peanut. J Plant Genet Resour, 2020, 21: 201-207 (in Chinese with English abstract).
[24] 朱佳慧, 徐秋芳, 袁平平, 倪海平, 周益军, 蒋选利, 杜何为. 水稻幼苗酵母双杂交cDNA文库的构建及鉴定. 江苏农业科学, 2018, 46(9):47-50.
Zhu J H, Xu Q F, Yuan P P, Ni H P, Zhou Y J, Jiang X L, Du W H. Construction and identification of yeast two-hybrid cDNA library of rice seedlings. J Jiangsu Agric Sci, 2018, 46(9):47-50 (in Chinese).
[25] 马芳芳, 蒋明义. 外源ABA处理的玉米叶片酵母双杂交cDNA文库的构建及评价. 江苏农业科学, 2016, 44(4):58-61.
Ma F F, Jiang M Y. Construction and evaluation of yeast two-hybrid cDNA library of corn leaf under exogenous ABA treatment. Jiangsu J Agric Sci, 2016, 44(4):58-61 (in Chinese).
[26] 许向阳, 裴童, 吴泰茹, 王子玉, 赵婷婷, 李景富, 杨欢欢, 姜景彬, 张贺. Cf-19介导的抗番茄叶霉病(Cladosporium fulvum)免疫应答酵母双杂交cDNA文库构建和鉴定. 东北农业大学学报, 2020, 51(5):10-16.
Xu X Y, Pei T, Wu T R, Wang Z Y, Zhao T T, Li J F, Yang H H, Jiang J B, Zhang H. Construction and identification of yeast two-hybrid cDNA library for Cf-19 mediated resistance to Cladosporium fulvum infection in tomato. J Northeast Agric Univ, 2019, 51(5):10-16 (in Chinese with English abstract).
[27] 曹玲珑, 李冬兵, 熊大斌, 牛洪斌, 姜玉梅, 尹钧. 外源ABA处理大麦胚的均一化cDNA文库的构建. 麦类作物学报, 2014, 34: 912-916.
Cao L L, Li D B, Xiong D B, Niu H B, Jiang Y M, Yin J. Construction of a normalized cDNA library of barley embryo treated with exogenous ABA. J Triticeae Crops, 2014, 34: 912-916 (in Chinese with English abstract).
[28] 王玉姣, 陈姗姗, 孙柏华, 艾聪聪, 王中洋, 张修国. 辣椒疫霉菌诱导辣椒酵母双杂交cDNA文库的构建及鉴定. 山东农业大学学报(自然科学版), 2018, 49: 379-382.
Wang Y J, Chen S S, Sun B H, Ai C C, Wang Z Y, Zhang X G. Construction and characterization of yeast two-hybrid cDNA library derived from Capsicum annuum inoculated with Phytophthora capsici. Shandong Agric Univ (Nat Sci Edn), 2018, 49: 379-382 (in Chinese with English abstract).
[29] 张恒, 陈怡名, 张旭, 赵佳, 吴承云, 郝永利, 孙丽, 王海燕, 肖进, 王秀娥. 白粉菌诱导簇毛麦叶片酵母双杂交文库构建及CMPG1-V候选互作蛋白筛选. 南京农业大学学报, 2020, 43: 594-604.
Zhang H, Chen Y M, Zhang X, Zhao J, Wu C Y, Hao Y L, Sun L, Wang H Y, Xiao J, Wang X E. Construction of yeast two-hybrid cDNA library of Haynaldia villosa leaves induced by Blumeria graminis f. sp. tritici and candidate interaction protein screening for CMPG1-V. J Nanjing Agric Univ, 2020, 43: 594-604 (in Chinese with English abstract).
[30] Chen H, Zhang C, Cai T C, Deng Y, Zhou S B, Zheng Y X, Ma S W, Tang R H, Varshney K R, Zhuang W J. Identification of low Ca2+ stress-induced embryo apoptosis response genes in Arachis hypogaea by SSH-associated library lift (SSHaLL). Plant Biotechnol J, 2016, 14: 682-698.
doi: 10.1111/pbi.12415 pmid: 26079063
[31] Zhuang W J, Chen H, Yang M, Wang J P, Pandey K M, Zhang C, Chang W C, Zhang L S, Zhang X T, Tang R H, Garg V, Wang X J, Tang H B, Chow C N, Wang J P, Deng Y, Wang D P, Khan W A, Yang Q, Cai T C, Bajaj P, Wu K C, Guo B Z, Zhang X Y, Li J J, Liang F, Hu J, Liao B S, Liu S Y, Chitilineni A, Yan H S, Zheng Y X, Shan S H, Liu Q Z, Xie D Y, Wang Z Y, Khan A S, Ali N, Zhao C Z, Li X G, Luo Z L, Zhang S B, Zhuang R R, Peng Z, Wang S Y, Mamadou G, Zhuang Y H, Zhao Z F, Yu W C, Xiong F Q, Quan W P, Yuan M, Li Y, Zou H S, Xia H, Zha L, Fan J P, Yu J G, Xie W P, Yuan J Q, Chen K, Zhao S S, Chu W T, Chen Y T, Sun P C, Meng F B, Zhuo T, Zhao Y H, Li C J, He G H, Zhao Y L, Wang C C, Kavikishor B P, Pan R L, Paterson H A, Wang X Y, Ming R, Varshney K R. The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication. Nat Genet, 2019, 51: 865-876.
doi: 10.1038/s41588-019-0402-2
[32] Schmittgen T D. Livak K J. Analyzing real-time PCR data by the comparative CT method. Nat Protoc, 2008, 3: 1101-1108.
pmid: 18546601
[33] Gao X H, Jia R Y, Wang M S, Zhu D K, Chen S, Lin M, Yin Z Q, Wang Y, Chen X Y, Cheng A C. Construction and identification of a cDNA library for use in the yeast two-hybrid system from duck embryonic fibroblast cells post-infected with duck enteritis virus. Mol Biol Rep, 2014, 41: 467-475.
doi: 10.1007/s11033-013-2881-z
[34] Niño M C, Kang K K, Cho Y G. Genome-wide transcriptional response of papain-like cysteine protease-mediated resistance against Xanthomonas oryzae pv. oryzae in rice. Plant Cell Rep, 2020, 39: 457-472.
doi: 10.1007/s00299-019-02502-1
[35] Shindo T, Hoorn R A L V D. Papain-like cysteine proteases: key players at molecular battlefields employed by both plants and their invaders. Mol Plant Pathol, 2008, 9: 119-125.
[36] Shindo T, Misas-Villamil J C, Hörger A C, Song J, Hoorn R A L V. A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14. PLoS One, 2012, 7: e29317.
doi: 10.1371/journal.pone.0029317
[37] Kaschani F, Shabab M, Bozkurt T, Shindo T, Schornack S, Gu C, Ilyas M, Win J, Kamoun S, Hoorn R A L V. An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts. Plant Physiol, 2010, 154: 1794-1804.
doi: 10.1104/pp.110.158030
[38] Bozkurt T O, Schornack S, Win J, Shindo T, Ilyas M, Oliva R, Cano L M, Jones A M, Huitema E, Hoorn R A L V, Kamoun S. Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface. Proc Natl Acad Sci USA, 2011, 108: 20832-20837.
doi: 10.1073/pnas.1112708109
[39] Pogorelko G V, Parijat S J, William B R, Hütten M, Maier T R, Hewezi T, Paulus J, Hoorn R A L V, Grundler F M, Siddique S, Lionetti V, Zabotina O A, Baum T J. Re-targeting of a plant defense protease by a cyst nematode effector. Plant J, 2019, 98: 1000-1014.
doi: 10.1111/tpj.14295
[40] Linde K V D, Hemetsberger C, Kastner C, Kaschani F, van der Hoorn R A L V D, Kumlehn J, Doehlemann G. A maize cystatin suppresses host immunity by inhibiting apoplastic cysteine proteases. Plant Cell, 2012, 24: 1285-1300.
doi: 10.1105/tpc.111.093732
[41] Linde K V D, Mueller A N, Hemetsberger C, Kashani F, Hoorn R A L V D, Doehlemann G. The maize cystatin CC9 interacts with apoplastic cysteine proteases. Plant Signal Behav, 2012, 7: 1397-1401.
doi: 10.4161/psb.21902
[42] Ziemann S, Linde K V D, Lahrmann U, Acar B, Kaschani F, Colby T, Kaiser M, Ding Y, Schmelz E, Huffaker A, Holton N, Zipfel C, Doehlemann G. An apoplastic peptide activates salicylic acid signalling in maize. Nat Plants, 2018, 4: 172-180.
doi: 10.1038/s41477-018-0116-y
[43] Mueller A N, Ziemann S, Treitschke S, Assmann D, Doehlemann G. Compatibility in the stillage maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2. PLoS Pathog, 2013, 9: e1003177.
doi: 10.1371/journal.ppat.1003177
[44] Xu L, Zhu L F, Tu L L, Guo X P, Long L, Sun L Q, Gao W, Zhang X L. Differential gene expression in cotton defense response to Verticillium dahliae by SSH. J Phytopath, 2011, 159: 606-615.
doi: 10.1111/j.1439-0434.2011.01813.x
[45] Zhang S Q, Xu Z P, Sun H, Sun L Q, Shaban M, Yang X Y, Zhu L F. Genome-wide identification of papain-like cysteine proteases in Gossypium hirsutum and functional characterization in response to Verticillium dahliae. Front Plant Sci, 2019, 10: 134.
doi: 10.3389/fpls.2019.00134
[46] Liu Y, Wang K R, Cheng Q, Kong D Y, Zhang X Z, Wang Z B, Wang Q, Xie Q, Yan J J, Chu J F, Ling H Q, Li Q, Miao J M, Zhao B Y. Cysteine protease RD21A regulated by E3 ligase SINAT4 is required for drought-induced resistance to Pseudomonas syringae in Arabidopsis. J Exp Bot, 2020, 71: 5562-5576.
doi: 10.1093/jxb/eraa255 pmid: 32453812
[47] Neuteboom L W, Ng J M, Kuyper M, Clijdesdale O R, Hooykaas P J, van der Zaal B J. Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation. Plant Mol Biol, 1999, 39: 273-287.
pmid: 10080694
[48] Neuteboom L W, Veth-Tello L M, Clijdesdale O R, Hooykaas P J, Zaal B J. A novel subtilisin-like protease gene from Arabidopsis thaliana is expressed at sites of lateral root emergence. DNA Res, 1999, 6: 13-19.
pmid: 10231025
[49] Zhao C, Johnson B J, Kositsup B, Beers E P. Exploiting secondary growth in Arabidopsis. Construction of xylem and bark cDNA libraries and cloning of three xylem endopeptidases. Plant Physiol, 2000, 123: 1185-1196.
pmid: 10889267
[50] Berger D, Altmann T. A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes Dev, 2000, 14: 1119-1131.
[51] Tanaka H, Onouchi H, Kondo M, Haranishimura I H, Nishimura M, Machida C, Machida Y. A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants. Development, 2001, 128: 4681-4689.
pmid: 11731449
[52] Othman R, Nuraziyan A. Fruit-specific expression of papaya subtilase gene. J Plant Physiol, 2010, 167: 131-137.
doi: 10.1016/j.jplph.2009.07.015
[53] D’Erfurth I, Signor C L, Aubert G, Sanchez M, Vernoud V, Darchy B, Lherminier J, Bourion V, Bouteiller N, Bendahmane A, Buitink J, Prosperi J M, Thompson R, Burstin J, Gallardo K. A role for an endosperm-localized subtilase in the control of seed size in legumes. New Phytol, 2012, 196: 738-751.
doi: 10.1111/nph.2012.196.issue-3
[54] Roberts L N, Caputo C, Kade M, Criado M V, Barneix A J. Subtilisin-like serine proteases involved in N remobil-ization during grain filling in wheat. Acta Physiol Plant, 2011, 33: 1997-2001.
doi: 10.1007/s11738-011-0712-1
[55] Ramírez V, López A, Mauch-Mani B, Gil M J, Vera P. An extracellular subtilase switch for immune priming in Arabidopsis. PLoS Pathog, 2013, 9: e1003445.
doi: 10.1371/journal.ppat.1003445
[56] Serrano I, Buscaill P, Audran C, Pouzet C, Jauneau A, Rivas S. A non canonical subtilase attenuates the transcriptional activation of defence responses in Arabidopsis thaliana. eLife, 2016, 5: e19755.
doi: 10.7554/eLife.19755
[57] Pearce G, Yamaguchi Y, Barona G, Ryan C A. A subtilisin-like protein from soybean contains an embedded, cryptic signal that activates defense-related genes. Proc Natl Acad Sci USA, 2010, 107: 14921-14925.
doi: 10.1073/pnas.1007568107
[58] Yamaguchi Y, Barona G, Ryan C A, Pearce G. GmPep914, an eight-amino acid peptide isolated from soybean leaves, activates defense-related genes. Plant Physiol, 2011, 156: 932-942.
doi: 10.1104/pp.111.173096 pmid: 21478368
[59] Fernández M B, Daleo G R, Guevara M G. Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3). Plant Physiol Biochem, 2015, 86: 137-146.
doi: 10.1016/j.plaphy.2014.12.001
[60] Fan T, Bykova N V, Rampitsch C, Tim X. Identification and characterization of a serine protease from wheat leaves. Eur J Plant Pathol, 2016, 146: 293-304.
doi: 10.1007/s10658-016-0914-x
[61] Duan X P, Zhang Z D, Wang J, Zuo K J. Characterization of a novel cotton subtilase gene GbSBT1 in response to extracellular stimulations and its role in Verticillium resistance. PLoS One, 2016, 11: e0153988.
doi: 10.1371/journal.pone.0153988
[62] Ekchaweng K, Khunjan U, Churngchow N. Molecular cloning and characterization of three novel subtilisin-like serine protease genes from Hevea brasiliensis. Physiol Mol Plant Pathol, 2017, 97: 79-95.
doi: 10.1016/j.pmpp.2016.12.007
[63] Saez-Aguayo S, Ralet M C, Berger A, Botran L, Ropartz D, North M P M. PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells. Plant Cell, 2013, 25: 308-323.
doi: 10.1105/tpc.112.106575
[1] 左同鸿, 张贺翠, 曾静, 朱利泉. 甘蓝自交不亲和相关基因BoPUB3L的克隆与表达分析[J]. 作物学报, 2026, 52(6): 1698-1710.
[2] 郑玉珍, 齐飞艳, 孙子淇, 刘华, 秦利, 石磊, 王娟, 汪蒙蒙, 韩锁义, 徐静, 苗利娟, 黄冰艳, 董文召, 郑峥, 张新友. 花生籽仁总超长链脂肪酸和7种脂肪酸组分的QTL定位[J]. 作物学报, 2026, 52(6): 1646-1657.
[3] 陆一楚, 李振影, 买春海, 赵晓蕊, 王利祥. 夜间复合体关键基因AhLUX1正向调控花生结瘤的功能研究[J]. 作物学报, 2026, 52(6): 1658-1668.
[4] 谷春苗, 王润风, 黄璐, 刘浩, 鲁清, 李海芬, 李少雄, 何双呈, 洪彦彬, 陈小平, 谭斌, 余倩霞. 花生WOX基因家族的全基因组分析及不定芽再生候选基因的鉴定[J]. 作物学报, 2026, 52(5): 1326-1340.
[5] 李灿, 张喜伟, 朱博涛, 张沛沛. 小麦GSK激酶TaSK41的功能分析及互作蛋白的筛选[J]. 作物学报, 2026, 52(3): 677-687.
[6] 于天一, 王春晓, 肖丽, 钟召迪, 王宣仓, 赵勇, 路亚, 吴月, 吴正锋. 不同结瘤特性花生品种氮素累积、产量及品质特性对氮肥用量的响应[J]. 作物学报, 2026, 52(3): 881-894.
[7] 张胜忠, 李国卫, 戈立江, 王菲菲, 胡晓辉, 苗华荣, 李燕, 钟文, 陈静. 花生机械脱壳损伤相关农艺指标筛选与QTL定位[J]. 作物学报, 2026, 52(2): 644-652.
[8] 王菲菲, 张胜忠, 杨贵华, 苗华荣, 胡晓辉, 张则林, 刘莎莎, 乔利仙, 单世华, 陈静. 331份花生种质苗期耐盐性综合评价和强耐盐种质鉴选[J]. 作物学报, 2026, 52(1): 279-294.
[9] 金欣欣, 宋亚辉, 苏俏, 杨永庆, 王瑾. 高产高油高油酸花生品种的生长发育及干物质生产特征[J]. 作物学报, 2026, 52(1): 191-201.
[10] 迟晓元, 刘庆, 张君, 赵旭红, 李美, 于天一, 潘丽娟, 许静, 姜骁, 殷祥贞, 马俊卿, 陈娜. 不同花生品种(系)耐盐碱性田间鉴定及各性状指标相关性研究[J]. 作物学报, 2026, 52(1): 85-98.
[11] 孙辰硕, 张月, 田泽锴, 晏立英, 康彦平, 陈玉宁, 王欣, 淮东欣, 王前前, 姜慧芳, 罗怀勇, 黄莉, 廖伯寿, 王志慧, 雷永. 花生种质果柄强度的遗传分化与主要影响因子分析[J]. 作物学报, 2026, 52(1): 118-130.
[12] 万书波, 张佳蕾, 高华鑫, 王才斌. 中国花生高产栽培研究进展与展望[J]. 作物学报, 2025, 51(7): 1703-1711.
[13] 郭腾达, 崔梦杰, 陈琳杰, 韩锁义, 郭敬坤, 吴晨迪, 付留洋, 黄冰艳, 董文召, 张新友. 花生磷脂酰肌醇转运蛋白基因AhSFH的克隆及其响应黄曲霉菌侵染的表达特征分析[J]. 作物学报, 2025, 51(6): 1489-1500.
[14] 李福媛, 杨奕, 马继琼, 许明辉, 林良斌, 孙一丁. 水稻OsPUB4基因克隆、激素诱导表达分析与互作蛋白筛选[J]. 作物学报, 2025, 51(6): 1690-1700.
[15] 李文佳, 廖泳俊, 黄璐, 鲁清, 李少雄, 陈小平, 金晶炜, 王润风. 花生开花时间的全基因组关联分析及候选基因筛选[J]. 作物学报, 2025, 51(5): 1400-1408.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2