陆地棉衰老相关基因GhSAG101的克隆及抗病功能分析

doi:10.11963/cs20210030

摘要/Abstract

摘要：

【目的】挖掘棉花抗病相关基因,为创制抗病棉新品种提供理论依据和候选基因。【方法】以陆地棉（Gossypium hirsutum） TM-1为研究对象,克隆了棉花衰老相关基因GhSAG101（senescence-associated gene 101)。通过实时荧光定量聚合酶链式反应分析GhSAG101在棉花幼苗不同组织中的表达量,以及在黄萎病菌诱导下表达量的变化;通过蛋白序列分析预测了GhSAG101的保守位点和活性位点;利用病毒诱导的基因沉默技术沉默棉花中GhSAG101,以空载体作为对照,通过棉花整株接种和离体叶片接种黄萎病菌研究GhSAG101沉默对黄萎病抗性的影响。【结果】GhSAG101基因编码区长度为1 764 bp,编码587个氨基酸残基的蛋白质,包含水解酶家族、EDS1（enhanced disease susceptibility 1）家族的保守结构域和亲核位点。GhSAG101在根中表达量最高,茎中次之,在叶片中表达量最低,黄萎病菌侵染中后期GhSAG101在根中被诱导上调表达。在棉花中沉默GhSAG101基因降低了黄萎病菌在寄主体内的扩展速度,减缓了病害发生过程。【结论】GhSAG101负调控棉花对黄萎病的抗性,可作为棉花抗病育种的候选基因。

关键词: 陆地棉; GhSAG101; 黄萎病抗性; 衰老相关基因; 抗病育种; 负调控

Abstract:

[Objective] Screening and study of cotton disease resistance genes will provide theoretical basis and genetic resources for cotton disease resistance breeding. [Method] The senescence associated gene 101 (GhSAG101) was cloned from Gossypium hirsutum L. TM-1. The expression levels of GhSAG101 in different organs of cotton seedlings, as well as in cotton roots under Verticillium dahliae inoculation were analyzed by real time quantitative polymerase chain reaction (qRT-PCR). The conserved domains and active sites of GhSAG101 were predicted through protein sequence analysis. Knockdown lines of GbSAG101 generated through virus induced gene silencing (VIGS) system were employed for the examination of Verticillium dahliae resistance by inoculation of the whole plant and detached leaves, and the empty vector transferred plants were used as the negative control. [Result] The coding region of GhSAG101 gene is 1 764 bp in length, encodes a protein with 587 amino acid residues that includes a nucleophilic elbow and the conserved domains belonging to the abhydrolase superfamily and enhanced disease susceptibility 1 (EDS1) family. The highest expression level of GhSAG101 was found in roots, followed by stems, and the lowest expression level was found in leaves. GhSAG101 expression was induced by V. dahliae infection in roots at the middle and late infection stage. Suppression of GhSAG101 expression in cotton reduced the spread of V. dahliae and retarded the disease process. [Conclusion] GhSAG101 negatively regulates cotton resistance to V. dahliae and can be used as a candidate gene in the breeding of cotton with improved disease resistance.

Key words: Gossypium hirsutum; GhSAG101; Verticillium wilt resistance; senescence-associated gene; disease resistance breeding; negative regulation

吴健锋,樊志浩,武连杰,胡晓旺,韩知里,高巍,龙璐. 陆地棉衰老相关基因GhSAG101的克隆及抗病功能分析[J]. 棉花学报, 2022, 34(3): 187-197.

Wu Jianfeng,Fan Zhihao,Wu Lianjie,Hu Xiaowang,Han Zhili,Gao Wei,Long Lu. Cloning and disease-resistant function of a senescence-associated gene GhSAG101 in Gossypium hirsutum[J]. Cotton Science, 2022, 34(3): 187-197.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/cs20210030

https://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2022/V34/I3/187

图/表 6

表1

图1

图2

图3

图4

图5

参考文献 46

[1]	Gao W, Xu F C, Long L, et al. The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton[J/OL]. Plant Biotechnology Journal, 2020, 18(7): 1573-1584[2021-06-29]. https://doi.org/10.1111/pbi.13323.
[2]	Muhammad S, Miao Y H, Abid U, et al. Physiological and molecular mechanism of defense in cotton against Verticillium dahliae[J/OL]. Plant Physiology and Biochemistry, 2018, 125: 193-204[2021-06-29]. https://doi.org/10.1016/j.plaphy.2018.02.011.
[3]	赵丽红, 冯自力, 冯鸿杰, 等. 棉花黄萎病对棉花单株产量和纤维品质的影响[J/OL]. 中国棉花, 2016, 43(7): 19-23[2021-06-29]. http://dx.chinadoi.cn/10.11963/issn.1000-632X.201607006.
	Zhao Lihong, Feng Zili, Feng Hongjie, et al. The effect of cotton Verticillium wilt on yield and fiber quality[J/OL]. China Cotton, 2016, 43(7): 19-23[2021-06-29]. http://dx.chinadoi.cn/10.11963/issn.1000-632X.201607006.
[4]	Fradin E F, Thomma B P. Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum[J/OL]. Molecular Plant Pathology, 2006, 7(2): 71-86.
[5]	Klosterman S J, Atallah Z K, Vallad G E, et al. Diversity, pathogenicity, and management of Verticillium species[J]. Annual Review of Phytopathology, 2009, 47: 39-62. doi: 10.1146/annurev-phyto-080508-081748 pmid: 19385730
[6]	朱荷琴, 宋晓轩, 简桂良. 棉花黄萎病菌致病力变异生理机制的初步研究[J]. 棉花学报, 2004, 16(5): 275-279.
	Zhu Heqin, Song Xiaoxuan, Jian Guiliang. The preliminary study of variation mechanism of pathogenic type of Verticillium dahliae Kleb.[J]. Cotton Science, 2004, 16(5): 275-279.
[7]	Jonge R D, Esse H, Maruthachalam K, et al. Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing[J/OL]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(13): 5110-5115[2021-06-29]. https://doi.org/10.1073/pnas.1119623109.
[8]	刘琳琳, 张文文, 周易, 等. 棉花与番茄抗棉花黄萎病不依赖于Ve1[J/OL]. 中国科学: 生命科学, 2014, 44(8): 803-814[2021-06-29]. http://dx.chinadoi.cn/10.1360/052014-90.
	Liu Linlin, Zhang Wenwen, Zhou Yi, et al. Resistance of cotton and tomato to Verticillium dahliae from cotton is independent on Ve1[J/OL]. Scientia Sinica: Vitae, 2014, 44(8): 803-814[2021-06-29]. http://dx.chinadoi.cn/10.1360/052014-90.
[9]	Fradin E F, Zhang Z, Ayala J C, et al. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1[J]. Plant Physiology, 2009, 150(1): 320-332. doi: 10.1104/pp.109.136762
[10]	Gao X Q, Terry W, Li Z H, et al. Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt[J]. The Plant Journal, 2009, 66(2): 293-305. doi: 10.1111/j.1365-313X.2011.04491.x
[11]	Zhang Y, Wang X, Rong W, et al. Island cotton enhanced disease susceptibility 1 gene encoding a lipase-like protein plays a crucial role in response to Verticillium dahliae by regulating the SA level and H₂O₂ accumulation[J/OL]. Frontiers in Plant Science, 2016, 7: 1830[2021-06-29]. https://doi.org/10.3389/fpls.2016.01830.
[12]	Gao X Q, Li F J, Li M Y, et al. Cotton GhBAK1 mediates Verticillium wilt resistance and cell death[J/OL]. Journal of Integrative Plant Biology, 2013, 55(7): 586-96[2021-06-29]. https://doi.org/10.1111/jipb.12064.
[13]	Ashraf J, Zuo D Y, Wang Q L, et al. Recent insights into cotton functional genomics: progress and future perspectives[J/OL]. Plant Biotechnology Journal, 2018, 16(3): 699-713[2021-06-29]. https://doi.org/10.1111/pbi.12856.
[14]	Gao W, Long L, Zhu L F, et al. Proteomic and virus-induced gene silencing (VIGS) analyses reveal that gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to Verticillium dahliae[J/OL]. Molecular & Cellullar Proteomics, 2013, 12(12): 3690-3703[2021-06-29]. https://doi.org/10.1074/mcp.M113.031013.
[15]	Mo H J, Wang X F, Zhang Y, et al. Cotton polyamine oxidase is required for spermine and camalexin signalling in the defence response to Verticillium dahliae[J/OL]. The Plant Journal, 2015, 83(6): 962-975[2021-06-29]. https://doi.org/10.1111/tpj.12941.
[16]	Xu L, Zhu L F, Tu L L, et al. Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry[J/OL]. Journal of Experimental Botany, 2011, 62(15): 5607-5621[2021-06-29]. https://doi.org/10.1093/jxb/err245.
[17]	张慧, 田新权, 高巍, 等. 陆地棉PPO的全基因组鉴定及对黄萎病菌的响应分析[J/OL]. 棉花学报, 2017, 29(5): 428-436[2021-06-29]. https://doi.org/10.11963/1002-7807.zhll.20170727.
	Zhang Hui, Tian Xinquan, Gao Wei, et al. Genome-wide identification of PPO gene family members and their response to Verticillium dahliae in upland cotton[J/OL]. Cotton Science, 2017, 29(5): 428-436[2021-06-29]. https://doi.org/10.11963/1002-7807.zhll.20170727.
[18]	Guo W F, Jin L, Miao Y H, et al. An ethylene response-related factor, GbERF1-like, from Gossypium barbadense improves resistance to Verticillium dahliae via activating lignin synthesis[J/OL]. Plant Molecular Biology, 2016, 91(3): 305-318[2021-06-29]. https://doi.org/10.1007/s11103-016-0467-6.
[19]	沈吉丽, 肖胜华, 惠慧, 等. GhMYB43负调控木质素的生物合成和茉莉酸信号[J/OL]. 棉花学报, 2020, 32(6): 522-537[2021-06-29]. http://dx.chinadoi.cn/10.11963/1002-7807.sjlzlf.20200907.
	Shen Jili, Xiao Shenghua, Xi Hui, et al. GhMYB43 negatively regulates lignin biosynthesis and jasmonic acid signaling[J/OL]. Cotton Science, 2020, 32(6): 522-537[2021-06-29]. http://dx.chinadoi.cn/10.11963/1002-7807.sjlzlf.20200907.
[20]	Tang Y, Zhang Z N, Lei Y, et al. Cotton WATs modulate SA biosynthesis and local lignin deposition participating in plant resistance against Verticillium dahliae[J/OL]. Frontiers in Plant Science, 2019, 10: 526[2021-06-29]. https://doi.org/10.3389/fpls.2019.00526.
[21]	Xu L, Zhu L F, Tu L L, et al. Differential gene expression in cotton defence response to Verticillium dahliae by SSH[J/OL]. Journal of Phytopathology, 2011, 159(9): 606-615[2021-06-29]. https://doi.org/10.1111/j.1439-0434.2011.01813.x.
[22]	Goicoechea N, Aguirreolea J, Cenoz S, et al. Gas exchange and flowering in Verticillium-wilted pepper plants[J/OL]. Journal of Phytopathology, 2010, 149(5): 281-286.
[23]	Veronese P, Narasimhan M L, Stevenson R A, et al. Identification of a locus controlling Verticillium disease symptom response in Arabidopsis thaliana[J/OL]. The Plant Journal, 2003, 35(5): 574-587.
[24]	Fradin E F, Abd-EI-haliem A, Masini L, et al. Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis[J/OL]. Plant Physiology, 2011, 156(4): 2255-2265[2021-06-29]. https://doi.org/10.1104/pp.111.180067.
[25]	Pieterse C M J, Leon-Reyes A, van der Ent S, et al. Networking by small-molecule hormones in plant immunity[J]. Nature Chemical Biology, 2009, 5: 308-316. doi: 10.1038/nchembio.164 pmid: 19377457
[26]	Robison M M, Griffith M, Pauls K P, et al. Dual role for ethylene in susceptibility of tomato to Verticillium wilt[J/OL]. Journal of Phytopathology, 2001, 149: 385-388. doi: 10.1111/j.1439-0434.2001.tb03867.x
[27]	He Y H, Gan S S. A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis[J/OL]. The Plant Cell, 2002, 14(4): 805-815.
[28]	Dhar N, Caruana J, Erdem I, et al. The Arabidopsis SENESCENCE-ASSOCIATED GENE 13 regulates dark-induced senescence and plays contrasting roles in defense against bacterial and fungal pathogens[J/OL]. Molecular Plant-Microbe Interactions, 2020, 33(5): 754-766[2021-06-29]. https://doi.org/10.1094/MPMI-11-19-0329-R.
[29]	Zhao P, Zhao Y L, Yun J, et al. Colonization process of Arabidopsis thaliana roots by a green fluorescent protein-tagged isolate of Verticillium dahliae[J/OL]. Protein Cell, 2014, 5(2): 94-98[2021-06-29]. https://doi.org/10.1007/s13238-013-0009-9.
[30]	Long L, Yang W W, Liao P, et al. Transcriptome analysis reveals differentially expressed ERF transcription factors associated with salt response in cotton[J/OL]. Plant Science, 2019, 281: 72-81[2021-06-29]. https://doi.org/10.1016/j.plantsci.2019.01.012.
[31]	Gao W, Long L, Xu L, et al. Suppression of the homeobox gene HDTF1 enhances resistance to Verticillium dahliae and Botrytis cinerea in cotton[J/OL]. Journal of Integrative Plant Biology, 2016, 58(5): 503-513[2021-06-29]. https://doi.org/10.1111/jipb.12432.
[32]	中华人民共和国农业部. 棉花抗病虫性评价技术规范第5部分: 黄萎病:GB/T 22101.5-2009[S]. 北京: 中国标准出版社, 2009.
	The Ministry of Agriculture of the People's Republic of China. Technical specification for evaluating resistance of cotton to diseases and insect pests part 5: Verticillium wilt:GB/T 22101.5-2009[S]. Beijing: Standards Press of China, 2009.
[33]	Long L, Xu F C, Zhao J R, et al. GbMPK3 overexpression increases cotton sensitivity to Verticillium dahliae and affects salicylic acid signaling[J/OL]. Plant Science, 2020, 292: 110374[2021-06-29]. https://doi.org/10.1016/j.plantsci.2019.110374.
[34]	Long L, Liu J, Gao Y, et al. Flavonoid accumulation in spontaneous cotton mutant results in red coloration and enhanced disease resistance[J/OL]. Plant Physiology and Biochemistry, 2019, 143(6): 40-49[2021-06-29]. https://doi.org/10.1016/j.plaphy.2019.08.021.
[35]	李周博, 郭楠楠, 古丽罕·如则, 等. 转基因棉花抗黄萎病研究进展[J/OL]. 新疆农业科技, 2019(6): 33-34[2021-06-29]. http://dx.chinadoi.cn/10.3969/j.issn.1007-3574.2019.06.015.
	Li Zhoubo, Guo Nannan, Gulihan·Ruze, et al. Research progress on resistance of transgenic cotton to Verticillium wilt[J/OL]. Xinjiang Agricultural Science and Technology, 2019(6): 33-34[2021-06-29]. http://dx.chinadoi.cn/10.3969/j.issn.1007-3574..2019.06.015.
[36]	陈方圆, 杨洋, 李波, 等. 棉花抗黄萎病分子机制研究进展[J/OL]. 分子植物育种, 2016, 14(10): 2859-2868[2021-06-29]. http://dx.chinadoi.cn/10.13271/j.mpb.014.002859.
	Chen Fangyuan, Li Bo, et al. Research advance on molecular regulation mechanism of Verticillium wilt resistance in cotton[J/OL]. Molecular Plant Breeding, 2016, 14(10): 2859-2868[2021-06-29]. http://dx.chinadoi.cn/10.13271/j.mpb.014.002859.
[37]	Gao W, Long L, Tian X Q, et al. Genome editing in cotton with the CRISPR/Cas9 system[J/OL]. Frontiers in Plant Science, 2017, 8: 1364[2021-06-29]. https://doi.org/10.3389/fpls.2017.01364.
[38]	Wagner S, Stuttmann J, Rietz S, et al. Structural basis for signaling by exclusive EDS1 heteromeric complexes with SAG101 or PAD4 in plant innate immunity cell[J/OL]. Cell Host & Microbe, 2013, 14(6): 619-630[2021-06-29]. https://doi.org/10.1016/j.chom.2013.11.006.
[39]	Zhu S F, Jeong R D, Venugopal S C, et al. SAG101 forms a ternary complex with EDS1 and PAD4 and is required for resistance signaling against turnip crinkle virus[J/OL]. PLoS Pathogens, 2011, 7(11): e1002318[2021-06-29]. https://doi.org/10.1371/journal.ppat.1002318.
[40]	Lapin D, Kovacova V, Sun X, et al. A coevolved EDS1-SAG101-NRG1module mediates cell death signaling by TIR-domain immune receptors[J/OL]. The Plant Cell, 2019, 31(10): 2430-2455[2021-06-29]. https://doi.org/10.1105/tpc.19.00118.
[41]	Reusche M, Truskina J, Thole K, et al. Infections with the vascular pathogens Verticillium longisporum and Verticillium dahliae induce distinct disease symptoms and differentially affect drought stress tolerance of Arabidopsis thaliana[J/OL]. Environmental and Experimental Botany, 2014, 108: 23-37[2021-06-29]. https://doi.org/10.1016/j.envexpbot.2013.12.009.
[42]	Zhang D D, Wang J, Wang D, et al. Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae[J/OL]. New Phytologist, 2019, 222: 1012-1029[2021-06-29]. https://doi.org/10.1111/nph.15672.
[43]	张海娜, 王广恩, 金卫平, 等. 黄萎病对棉花叶片生理特性的影响[J]. 河北农业科学, 2011, 15(2): 50-51, 73.
	Zhang Haina, Wang Guang'en, Jin Weiping, et al. Effects of Verticillium wilt on leaves physiological characteristics of cotton[J]. Hebei Agricultural Sciences, 2011, 15(2): 50-51, 73.
[44]	Michael R, Jana K, Karin T, et al. Stabilization of cytokinin levels enhances Arabidopsis resistance against Verticillium longisporum[J/OL]. Molecular Plant-Microbe Interactions, 2013, 26(8): 850-860[2021-06-29]. https://doi.org/10.1094/MPMI-12-12-0287-R.
[45]	Zhang S Q, Xu Z P, Sun H, et al. Genome-wide identification of papain-like cysteine proteases in Gossypium hirsutum and functional characterization in response to Verticillium dahliae[J/OL]. Frontiers in Plant Science, 2019, 10: 134[2021-06-29]. https://doi.org/10.3389/fpls.2019.00134.
[46]	Swartzberg D, Kirshner B, Rav-David D, et al. Botrytis cinerea induces senescence and is inhibited by autoregulated expression of the IPT gene[J]. European Journal of Plant Pathology, 2008, 120(3): 289-297. doi: 10.1007/s10658-007-9217-6

引物名称 Name	正向引物 Forward primer	反向引物 Reverse primer
SAG101-O	ATGGCAAGCAAGATGAACCAATT	TTAACTGAGTGCCAGCCAGACAC
SAG101-V	AGAAGGCCTCCATGGGGATCCG- GACAGTTAATGTTTACAGCAGGAT	GAGACGCGTGAGCTCGGTACCTCGTAC- CAGCATACAACCACC
SAG101-RT	CTCCGATCACGGTGGTTGTAT	GAGGCTCAATAAGCATCTCATACT
UB7-RT	GAAGGCATTCCACCTGACCAAC	CTTGACCTTCTTCTTCTTGTGCTTG