棉花学报 ›› 2022, Vol. 34 ›› Issue (1): 1-11.doi: 10.11963/cs20210068
• 研究与进展 • 下一篇
李秀青(),王倩(
),胡子曜(
),雷建峰1,代培红2,刘超2,刘晓东2,李月2,*(
)
收稿日期:
2021-12-06
出版日期:
2022-01-15
发布日期:
2022-05-31
通讯作者:
李月
E-mail:549222451@qq.com;WQ960910@126.com;1916938224@qq.com;liyue6905@126.com
作者简介:
李秀青(1993―),男,硕士研究生, 基金资助:
Li Xiuqing(),Wang Qian(
),Hu Ziyao(
),Lei Jianfeng1,Dai Peihong2,Liu Chao2,Liu Xiaodong2,Li Yue2,*(
)
Received:
2021-12-06
Online:
2022-01-15
Published:
2022-05-31
Contact:
Li Yue
E-mail:549222451@qq.com;WQ960910@126.com;1916938224@qq.com;liyue6905@126.com
摘要:
【目的】探索GhMAPKKK2基因在棉花抗黄萎病中的功能。【方法】利用生物信息学方法分析GhMAPKKK2基因的结构特征和进化关系。通过实时荧光定量聚合酶链式反应(Quantitative real-time polymerase chain reaction, qRT-PCR)分析GhMAPKKK2基因在黄萎病菌侵染以及外源茉莉酸(Jasmonic acid, JA)和水杨酸(Salicylic acid, SA)处理下的表达模式。利用病毒诱导的基因沉默技术结合阳性植株表型鉴定以及qRT-PCR技术,对GhMAPKKK2基因在棉花抗黄萎病中的功能及可能参与的抗病机制进行初步分析。【结果】以陆地棉 cDNA为模板克隆得到GhMAPKKK2基因,其编码区全长为1 341 bp,编码446个氨基酸,与雷蒙德氏棉GrMAPKKK2同源性最高。GhMAPKKK2基因的转录水平受黄萎病菌及JA和SA诱导。沉默GhMAPKKK2基因后增强了棉花对黄萎病菌的敏感性,与阴性对照植株相比,沉默植株的病情指数显著增加、维管束褐变程度明显加重、茎段菌丝数量明显增多。qRT-PCR分析表明,沉默植株中JA和SA信号传导途径相关基因的表达量均显著低于阴性对照植株。【结论】GhMAPKKK2基因可能通过参与JA和SA信号通路在棉花抗黄萎病反应中发挥正调控的作用。
李秀青,王倩,胡子曜,雷建峰,代培红,刘超,刘晓东,李月. GhMAPKKK2基因在棉花抗黄萎病中的功能分析[J]. 棉花学报, 2022, 34(1): 1-11.
Li Xiuqing,Wang Qian,Hu Ziyao,Lei Jianfeng,Dai Peihong,Liu Chao,Liu Xiaodong,Li Yue. Functional analysis of GhMAPKKK2 gene in cotton resistance to Verticillium wilt[J]. Cotton Science, 2022, 34(1): 1-11.
表1
试验所用引物"
引物名称 Primer name | 序列 Primer sequence (5’-3’) | 用途 Purpose |
---|---|---|
CM-GhMAPKKK2-F | GAATTCATGTATTTGCCGTTAAGTCCTC | VIGS载体构建 Vector construction for VIGS |
CM-GhMAPKKK2-R | GGTACCCACAACCCAAGTCGGCG | |
q-GhUBQ7-F | GAAGGCATTCCACCTGACCAAC | qRT-PCR |
q-GhUBQ7-R | CTTGACCTTCT TCTTCTTGTGCTTG | |
q-GhMAPKKK2-F | GACACCCAGCAGCGTATTG | |
q-GhMAPKKK2-R | CAAAGAGAAAACCAGATCGTC | |
q-GhCLA1-F | GCCCTTTGTGCATCTTC | |
q-GhCLA1-R | CTCTAGGGGCATTGAAG | |
GhAOC-F | AATAGAGCATAAACCCGAAATGAAAG | |
GhAOC-R | CAAAAATGCCAGACCCACCAGTA | |
GhPR4-F | GAGGGTAAGAAACTCAAGGACTGG | |
GhPR4-R | CTCCATCAGTGTCCAATCGGTT | |
GhPDF1-F | CTGTGGTAGCGGATGGTGATAAG | |
GhPDF1-R | GTGCAGACGCATTTGCGAAGGAA | |
GhNPR1-F | CTAGCTTGCGGAGGGATTGATACC | |
GhNPR1-R | GAGATGGCTGACCTGTCAAACTGC | |
GhPAD4-F | GGATGGAAGAATGGAAAGAAATGAA | |
GhPAD4-R | GAACTAGGAAAGCAGACTAAGGAACCA | |
GhAOC-F | AATAGAGCATAAACCCGAAATGAAAG | |
GhAOC-R | CAAAAATGCCAGACCCACCAGTA |
[1] | 陆光米, 刘国华, 朱再清. 棉花主产区生产比较效益及其影响因素分析[J]. 中国棉花, 2019, 46(1): 1-8, 13. |
Lu Guangmi, Liu Guohua, Zhu Zaiqing. Study on the comparative benefit of cotton production in the main planting regions and its influencing factors[J]. China Cotton, 2019, 46(1): 1-8, 13. | |
[2] |
Zhang Y, Wang X, Rong W, et al. Histochemical analyses reveal that stronger intrinsic defenses in Gossypium barbadense than in G. hirsutum are associated with resistance to Verticillium dahliae[J]. Molecular Plant-Microbe Interactions, 2017, 30(12): 984-996.
doi: 10.1094/MPMI-03-17-0067-R pmid: 28850286 |
[3] |
Meng X, Zhang S. MAPK cascades in plant disease resistance signaling[J]. Annual Review of Phytopathology, 2013, 51: 245-266.
doi: 10.1146/annurev-phyto-082712-102314 |
[4] | 王玫玫. 棉花促丝裂原活化蛋白激酶(GhMAPK)基因的分离与功能分析[D]. 泰安: 山东农业大学, 2007. |
Wang Meimei. Isolation and functional identification of a novel mitogen activated protein kinase gene, GhMAPK, in cotton (Gossypium hirsutum L.)[D]. Tai’an: Shandong Agricultural University, 2007. | |
[5] |
Laethem A V. Activation of p 38 MAPK is required for bax translocation to mitochondria, cytochrome release and apoptosis induced by UVB irradiation in human keratinocytes[J]. Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology, 2004, 18(15): 1946-1948.
doi: 10.1096/fj.04-2285fje |
[6] |
Kieber J J, Rothenberg M, Roman G, et al. CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases[J]. Cell, 1993, 72(3): 427-441.
pmid: 8431946 |
[7] |
Raj S, Saha G, Sasidharan S, et al. Biochemical characterization and chemical validation of leishmania MAP kinase-3 as a potential drug target[J]. Scientific Reports, 2019, 9(1): 2013-2018.
doi: 10.1038/s41598-018-38135-9 |
[8] | Victoria L, Belmiro V, Sami I, et al. MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6[J]. The Plant Journal, 2010, 63(6): 319-334. |
[9] |
Tsuyoshi M, Kazuya I, Kenji I, et al. Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants[J]. FEBS Letters, 1998, 437(1/2): 56-60.
doi: 10.1016/S0014-5793(98)01197-1 |
[10] |
Shoresh M, Gal-On A, Leibman D, et al. Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance[J]. Plant Physiology, 2006, 142(3): 1169-1179.
pmid: 16950863 |
[11] |
Yao D M, Zou C, Shu Y N, et al. WRKY transcription factors in Nicotiana tabacum modulate plant immunity against whitefly via interacting with MAPK cascade pathways[J]. Insects, 2020, 12(1): 16.
doi: 10.3390/insects12010016 |
[12] | 郭慧敏. 分析棉花MAPKs基因家庭并研究MAPK对黄萎病抗性的调控[D]. 临汾: 山西师范大学, 2016. |
Guo Huimin. Analyse the cotton MAPKs gene families and the roles of MAPK family members in the defense of Verticillium wilt disease[D]. Linfen: Shanxi Normal University, 2016. | |
[13] |
Meng J, Gao H, Zhai W B, et al. Subtle regulation of cotton resistance to Verticillium wilt mediated by MAPKK family members[J]. Plant Science, 2018, 272: 235-242.
doi: 10.1016/j.plantsci.2018.05.003 |
[14] | 王倩, 章超, 代培红, 等. 陆地棉GhMAPKKK3基因的克隆及其表达分析[J/OL]. 分子植物育种, 2021: 1-16[2021-10-28]. . |
Wang Qian, Zhang Chao, Dai Peihong, et al. Molecular cloning and expression analysis of GhMAPKKK3 gene in Gossypium hirsutum L.[J/OL]. Molecular Plant Breeding, 2021: 1-16[2021-10-28]. . | |
[15] |
Li Y, Zhou Y J, Dai P H, et al. Cotton Bsr-k1 modulates lignin deposition participating in plant resistance against Verticillium dahliae and Fusarium oxysporum[J]. Plant Growth Regulation, 2021, 95(2): 1-10.
doi: 10.1007/s10725-021-00723-7 |
[16] | 王钰静. GhROP6在棉花抗黄萎病中的功能研究[D]. 武汉: 华中农业大学, 2017. |
Wang Yujing. Functional analysis of GhROP6 in cotton responsive to Verticillium dahliae[D]. Wuhan: Huazhong Agricultural University, 2017. | |
[17] |
袁伟, 万红建, 杨悦俭. 植物实时荧光定量PCR内参基因的特点及选择[J]. 植物学报, 2012, 47(4): 427-436.
doi: 10.3724/SP.J.1259.2012.00427 |
Yuan Wei, Wan Hongjian, Yang Yuejian. Characterization and selection of reference genes for real-time quantitative RT-PCR of plants[J]. Chinese Bulletin of Botany, 2012, 47(4): 427-436.
doi: 10.3724/SP.J.1259.2012.00427 |
|
[18] |
Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J]. Methods, 2001, 25(4): 402-408.
doi: 10.1006/meth.2001.1262 pmid: 11846609 |
[19] | 李秀青, 李月, 刘超, 等. 棉花黄萎病相关基因GhAAT的克隆与功能鉴定[J]. 分子植物育种, 2020, 18(4): 1048-1053. |
Liu Xiuqing, Li Yue, Liu Chao, et al. Cloning and functional identification of cotton Verticillium wilt related gene GhAAT[J]. Molecular Plant Breeding, 2020, 18(4): 1048-1053. | |
[20] | Zhang B, Yang Y, Chen T, et al. Island cotton gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae[J/OL]. PLoS ONE, 2012, 7(12): e51091[2021-10-28]. . |
[21] |
Sun M X, Zhang Z Q, Ren Z Y, et al. The GhSWEET 42 glucose transporter participates in Verticillium dahliae infection in cotton[J]. Frontiers in Plant Science, 2021, 12: 690754.
doi: 10.3389/fpls.2021.690754 |
[22] |
Hu Q, Xiao S H, Wang X R, et al. GhWRKY1-like enhances cotton resistance to Verticillium dahliae via an increase in defense-induced lignification and S monolignol content[J]. Plant Science, 2021, 305: 110833.
doi: 10.1016/j.plantsci.2021.110833 |
[23] | 张坤. 棉花转录因子GhWRKY40-like和GhWRKY70在抗黄萎病中的功能研究[D]. 武汉: 华中农业大学, 2015. |
Zhang Kun. Functional analysis of GhWRKY40-like and GhWRKY70in cotton responsive to Verticillium dahliae[D]. Wuhan: Huazhong Agricultural University, 2015. | |
[24] | 赵付安, 房卫平, 杨晓杰, 等. 陆地棉Dirigent-like基因(GhDIR)的克隆与分析[J]. 华北农学报, 2011, 26(5): 29-33. |
Zhao Fu’an, Fang Weiping, Yang Xiaojie, et al. Cloning and analysis of upland cotton (Gossypium hirsutum) dirigent-like gene (GhDIR)[J]. Acta Agriculturae Boreali-Sinica, 2011, 26(5): 29-33. | |
[25] |
Sun Y D, Zhong M M, Li Y B, et al. GhADF6-mediated actin reorganization is associated with defence against Verticillium dahliae infection in cotton[J]. Molecular plant pathology, 2021, 22(12): 1656-1667.
doi: 10.1111/mpp.13137 |
[26] |
Xiong X P, Sun S C, Zhu Q H, et al. The cotton lignin biosynthetic gene Gh4CL30 regulates lignification and phenolic content and contributes to Verticillium wilt resistance[J]. Molecular Plant-Microbe Interactions, 2021, 34(3): 240-254.
doi: 10.1094/MPMI-03-20-0071-R |
[27] |
Zhao Y P, Shen J L, Li W J, et al. Evolutionary and characteristic analysis of RING-DUF1117 E3 ubiquitin ligase genes in Gossypium discerning the role of GhRDUF4D in Verticillium dahliae resistance[J]. Biomolecules, 2021, 11(8): 1145.
doi: 10.3390/biom11081145 |
[28] |
Xiao X R, Tang Z J, Li X Q, et al. Overexpressing OsMAPK12-1 inhibits plant growth and enhances resistance to bacterial disease in rice[J]. Functional Plant Biology, 2017, 44(7): 694-704.
doi: 10.1071/FP16397 |
[29] | Li Y Z, Qin L, Zhao J J, et al. SlMAPK3 enhances tolerance to tomato yellow leaf curl virus (TYLCV) by regulating salicylic acid and jasmonic acid signaling in tomato (Solanum lycopersicum)[J]. PLoS ONE, 2017, 12(2): e0172466[2021-10-28]. https://doi.org/10.1371/journal.pone.0172466. |
[30] |
Wang C, He X W, Wang X X, et al. Ghr-miR5272a-mediated regulation of GhMKK6 gene transcription contributes to the immune response in cotton[J]. Journal of Experimental Botany, 2017, 68(21/22): 5895-5906.
doi: 10.1093/jxb/erx373 |
[31] |
Wang C, Guo H B, He X W, et al. Scaffold protein GhMORG1 enhances the resistance of cotton to Fusarium oxysporum by facilitating the MKK6-MPK4 cascade[J]. Plant Biotechnology Journal, 2020, 18(6): 1421-1433.
doi: 10.1111/pbi.13307 |
[32] |
Wang C, He X W, Li Y Z, et al. The cotton MAPK kinase GhMPK20 negatively regulates resistance to Fusarium oxysporum by mediating the MKK4-MPK20-WRKY40 cascade[J]. Molecular Plant Pathology, 2018, 19(7): 1624-1638.
doi: 10.1111/mpp.12635 |
[33] | 宁硕, 顾爱星, 张国丽, 等. 大丽轮枝菌侵染棉花幼苗过程中内源水杨酸响应的初步分析[J/OL]. 分子植物育种, 2021: 1-15[2021-11-17]. . |
Ning Shuo, Gu Aixing, Zhang Guoli, et al. Response of endogenous salicylic acid in cotton seedlings infected by Verticillium dahliae[J/OL]. Molecular Plant Breeding, 2021: 1-15[2021-11-17]. . | |
[34] |
Wasternack C, Song S S. Jasmonates: biosynthesis, metabolism, and signaling by proteins activating and repressing transcription[J]. Journal of Experimental Botany, 2017, 68(6): 1303-1321.
doi: 10.1093/jxb/erw443 pmid: 27940470 |
[35] |
Xiong X P, Sun S C, Zhu Q H, et al. Transcriptome analysis and RNA interference reveal GhGDH2 regulating cotton resistance to Verticillium wilt by JA and SA signaling pathways[J]. Frontiers in Plant Science, 2021, 12: 654676.
doi: 10.3389/fpls.2021.654676 |
[36] |
Long L, Xu F C, Zhao J R, et al. GbMPK3overexpression increases cotton sensitivity to Verticillium dahliae by regulating salicylic acid signaling[J]. Plant Science, 2020, 292: 110374.
doi: 10.1016/j.plantsci.2019.110374 |
[37] |
Feng H J, Li C, Zhou J L, et al. A cotton WAKL protein interacted with a DnaJ protein and was involved in defense against Verticillium dahliae[J]. International Journal of Biological Macromolecules, 2021, 167: 633-643.
doi: 10.1016/j.ijbiomac.2020.11.191 |
[38] |
Zhu D D, Zhang X Y, Zhou J L, et al. Genome-wide analysis of ribosomal protein GhRPS6 and its role in cotton Verticillium wilt resistance[J]. International Journal of Molecular Sciences, 2021, 22(4): 1795.
doi: 10.3390/ijms22041795 |
[39] |
Chen B, Zhang Y, Yang J, et al. The G-protein α subunit GhGPA positively regulates Gossypium hirsutum resistance to Verticillium dahliae via induction of SA and JA signaling pathways and ROS accumulation[J]. The Crop Journal, 2021, 9(4): 823-833.
doi: 10.1016/j.cj.2020.09.008 |
[40] |
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]. Molecular & Cellular Proteomics, 2013, 12(12): 3690-3703.
doi: 10.1074/mcp.M113.031013 |
[41] |
Ma Q, Wang N H, Ma L, et al. The cotton BEL1-Like transcription factor GhBLH7-D06 negatively regulates the defense response against Verticillium dahliae[J]. International Journal of Molecular Sciences, 2020, 21(19): 7126.
doi: 10.3390/ijms21197126 |
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