Role of Receptor-like Cytoplasmic Kinase Gene GbRLCK10 from Gossypium barbadense in Disease Resistance

doi:10.13304/j.nykjdb.2021.0809

Abstract

Abstract:

GbRLCK10 gene responsed to Verticillium wilt and hormone stress in Gossypium barbadense. To analysis the function of GbRLCK10 gene， it was introduced into tobacco by Agrobacterium mediated method， and the positive transgenic plants were got. The disease resistances of the transgenic plants were identified in greenhouse， and the expressions of salicylic acid pathway key genes （NtPR1 and NtNPR1）， ethylene pathway genes （NtEIN4 and NtNTHK2）， and jasmonic acid pathway genes （NtJAZ1 and NtAOS） were analyzed by qRT-PCR. The results showed that， before infection， the expressions of NtPR1， NtNPR1 and NtJAZ1 genes in transgenic plants were significantly higher than those in wild type， and the expressions of NtEIN4， NtNTHK2 and NtAOS genes in wild type were significantly higher than those in transgenic plants. After inoculation with Verticillium wilt， the disease index of transgenic plants was significantly higher than that of wild type. And after the wild-type plants were infected， the expressions of NtPR1， NtNPR1 and NtAOS genes were significantly higher than those of the control， while the expressions of NtEIN4， NtNTHK2 and NtJAZ1 genes were significantly lower than those of the control； after transgenic plants were infected， the expression levels of NtPR1， NtNPR1， NtEIN4 and NtAOS genes were significantly higher than those in the control， while the expression levels of NtJAZ1 and NtNTHK2 genes were significantly lower than those in the control. Compared with the wild type， the expressions of NtPR1 and NtAOS genes in transgenic plants were significantly lower， while the expressions of NtNPR1， NtEIN4， NtJAZ1 and NtNTHK2 genes were significantly higher than those in the wild type. In conclusion， GbRLCK10 gene affected tobacco Verticillium wilt resistance by regulating the expressions of key genes in hormone pathway， which provided important basis for further revealing the function of GbRLCK10 gene in response to cotton Verticillium wilt.

Key words: tobacco, GbRLCK10, genetic transformation, function research

摘要：

GbRLCK10为海岛棉响应黄萎病菌和激素胁迫基因。为研究该基因在烟草中的功能，利用农杆菌介导法将其导入烟草。对获得的阳性植株通过室内接种法进行抗病性鉴定，并利用qRT-PCR技术分析水杨酸途径关键基因NtPR1、NtNPR1和乙烯途径关键基因NtEIN4、NtNTHK2及茉莉酸途径关键基因NtJAZ1、NtAOS 的表达特征。结果表明，接种前，转基因植株中NtPR1、NtNPR1、NtJAZ1基因的表达量显著高于野生型植株；而野生型植株中NtEIN4、NtNTHK2、NtAOS基因表达量显著高于转基因植株。接种黄萎病菌后，转基因植株的病情指数显著高于野生型；野生型植株感病后，NtPR1、NtNPR1、NtAOS基因的表达量显著高于发病前，而NtJAZ1、NtEIN4、NtNTHK2基因的表达量显著低于发病前；转基因植株感病后，NtPR1、NtNPR1、NtEIN4、NtAOS基因的表达量均显著高于发病前，而NtJAZ1、NtNTHK2基因的表达量均显著低于发病前；与野生型相比，转基因植株NtPR1、NtAOS基因的表达量显著降低，而NtNPR1、NtEIN4、NtJAZ1、NtNTHK2基因的表达量显著提高。综上所述，GbRLCK10基因通过调控激素途径中关键基因的表达水平影响烟草黄萎病抗性，为进一步揭示GbRLCK10基因对棉花黄萎病响应机制的功能研究提供了重要依据。

关键词: 烟草, GbRLCK10, 遗传转化, 功能研究

CLC Number:

S562

Zengqiang ZHAO, Guoli ZHANG, Panpan MA, Youzhong LI, Zhijun WANG, Zongming XIE, Guoqing SUN. Role of Receptor-like Cytoplasmic Kinase Gene GbRLCK10 from Gossypium barbadense in Disease Resistance[J]. Journal of Agricultural Science and Technology, 2023, 25(3): 57-65.

赵曾强, 张国丽, 马盼盼, 李有忠, 王志军, 谢宗铭, 孙国清. 海岛棉类受体胞质激酶基因GbRLCK10在抗病中的作用[J]. 中国农业科技导报, 2023, 25(3): 57-65.

Figures/Tables 6

Table 1 Sequence of pimers

引物名称 Primer name	引物序列 Primer sequence（5’-3’）
35SF1	TGGCTCCTACAAATGCCATCA
GeneR1	CAGCACCCGAACAACATCACT
GbRLCK-F	GGGGTACCCCATGGGATTCTTGGCTAAACTT
GbRLCK-R	GCTCTAGAGCCTCTACCTTCTTCAATGGACG
NtActin-F	CTCCGAGGGCTGTTTTTCCTA
NtActin-R	GCTGAGGGAAGTGGCGATTTC
NtPR1-F	CCTAGCACATCCAACACGAACC
NtPR1-R	GCAGCAGACGATGTAATGATGG
NtNPR1-F	TCCACAAGCCTAGTGAGCCTC
NtNPR1-R	GTGGTATGTTGAATGTTGCTCCTG
NtPR3-F	GTGGTATGTTGAATGTTGCTCCTG
NtPR3-R	TGATCTAACGAATCCTAGCCTTGG
NtEIN4-F	TGCCTAAATGCACTAGGTCCTC
NtEIN4-R	GCGAAATTTGCGCACTCTCA
NtJAZ1-F	GAGATTGTGGATTCCGGTCGAT
NtJAZ1-F	GAGATTGTGGATTCCGGTCGAT
NtNTHK2-F	TGAGTGCCAAAAGGTGAGTGA
NtNTHK2-R	TGGAAGGAAGGATGAGCACT
NtAOS-F	CAATACGGAAGAGCCAAACGC
NtAOS-R	AACTCATCGGGTCGGTCAAA

Fig. 1 PCR amplification results of GbRLCK10 geneNote： 1 and 2—Recombinant plasmid； M—DL 2000 marker.

Fig. 2 Transformation of GbRLCK10 gene into Nicotiana tabacum and screening of positive plantsA：Co-culture （tobacco leaf disc）； B：Callus culture； C：Selective culture； D：Rooting culture； E and F：Domesticated； G：T0 positive identification of transgenic plants； H and I：T1/2 positive identification of transgenic plants；1~13—Transgenic plants； 14，15 and P—Recombinant plasmid； 16 and 17—Negative control； M—DL2000

Fig. 3 Tobacco disease resistance identificationA：Phenotypic characteristics of transgenic plants and wild-type plants after inoculation with Verticillium dahlia； B：Isolation of Verticillium dahliae from inoculated tobacco； C：Recovery culture of Verticillium wilt； D：Disease index statistics；different lowercase letters in the figure indicate significant differences between different plants at P<0.05 level

Fig. 4 Expression of disease resistance-related genes in WT and transgenic plant before treatment with Verticillium dahliaeNote： WT—Wild-type plant； OE—Transgenic plant； * and ** indicate significant differences between different types at P<0.05 and P<0.01 levels， respectively.

Fig. 5 Expression of disease resistance related genes in wild type and transgenic strains after treatment with Verticillium dahliaeNote： WT—Wild-type plant； OE—Transgenic plant； different lowercase letters indicate significant differences between different types at P<0.05 level； ** indicates significant difference between different quessions in same time at P<0.01 level.

References 39

1	肖武.水稻OsRLCK91基因的功能研究[D].广州:华南农业大学,2018.
	XIAO W. Functional research of receptor like kinase OsRLCK77 in rice (Oryza sativa) [D]. Guangzhou: South China Agricultural University, 2018.
2	COSTA A T, BRAVO J P, KRAUSE-SAKATE R, et al.. The receptor-like kinase SlSOBIR1 is differentially modulated by virus infection but its overexpression in tobacco has no significant impact on virus accumulation [J]. Plant Cell Rep., 2016, 35(1):65-75.
3	LI L, YU Y, ZHOU Z, et al.. Plant pattern-recognition receptors controlling innate immunity [J]. Sci. China Life Sci., 2016, 59(9):878-888.
4	DODDS P N, RATHJEN J P. Plant immunity: towards an integrated view of plant–pathogen interactions [J]. Nat. Rev. Genet., 2010, 11(8):539-548.
5	MACHO A P, ZIPFEL C. Plant PRRs and the activation of innate immune signaling [J]. Mol. Cell., 2014, 54(2):263-272.
6	MONAGHAN J, ZIPFEL C. Plant pattern recognition receptor complexes at the plasma membrane [J]. Curr. Opin. Plant Biol., 2012, 15(4):349-357.
7	WU Y, ZHOU J M. Receptor-like kinases in plant innate immunity [J]. J. Integr. Plant Biol., 2013, 55(12):1271-1286.
8	RAO S, ZHOU Z, MIAO P, et al.. Roles of receptor-like cytoplasmic kinase VII members in pattern-triggered immune signaling [J]. Plant Physiol., 2018, 177(4):1679-1690.
9	BI G, ZHOU Z, WANG W, et al.. Receptor-like cytoplasmic kinases directly link diverse pattern recognition receptors to the activation of mitogen-activated protein kinase cascades in Arabidopsis [J]. Plant Cell, 2018, 30(7):1543-1561.
10	MA X, CLAUS L A N, LESLIE M E, et al.. Ligand-induced monoubiquitination of BIK1 regulates plant immunity [J]. Nature, 2020, 581(7807):1-5.
11	ZHANG J, LI W, XIANG T, et al.. Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector [J]. Cell Host Microbe., 2010, 7(4):290-301.
12	LU D, WU S, GAO X, et al.. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity [J]. Proc. Natl. Acad. Sci. USA, 2010, 107(1):496-501.
13	LIU Z, WU Y, YANG F, et al.. BIK1 interacts with PEPRs to mediate ethylene-induced immunity [J]. Proc. Natl. Acad. Sci. USA, 2013, 110(15):6205-6210.
14	TANG D, WANG G, ZHOU J M. Receptor kinases in plant-pathogen interactions: more than pattern recognition [J]. Plant Cell, 2017, 29(4):618-637.
15	KANDA Y, NAKAGAWA H, NISHIZAWA Y, et al.. Broad-spectrum disease resistance conferred by the overexpression of rice RLCK BSR1 results from an enhanced immune response to multiple MAMPs [J/OL]. Int. J. Mol. Sci., 2019, 20(22): 20225523 [2021-08-10]. .
16	LI K, XIONG X, ZHU S, et al.. MeBIK1, a novel cassava receptor-like cytoplasmic kinase, regulates PTI response of transgenic Arabidopsis [J]. Funct. Plant Biol., 2018, 45(6):658-667.
17	WU T, ZHU X, LYU L, et al.. The wheat receptor-like cytoplasmic kinase TaRLCK1B is required for host immune response to the necrotrophic pathogen Rhizoctonia cerealis [J]. J. Integr. Agric., 2020, 19(11):2616-2627.
18	赵曾强,孙国清,张国丽,等.海岛棉GbRLCK10基因克隆及表达分析[J].西北植物学报,2017,37(11):40-48.
	ZHAO Z Q, SUN G Q, ZHANG G L, et al.. Cloning and expression analysis of the GbRLCK10 gene in Gossypium barbadense L. [J]. Acta Bot. Bor-Occid. Sin., 2017, 37(11):40-48.
19	赵曾强,韩泽刚,李会会,等. GhWRKY44基因在烟草中遗传转化及功能分析[J]. 华北农学报,2016,31(1):117-122.
	ZHAO Z Q, HAN Z G, LI H H, et al.. Genetic transformation and functional analysis of GhWRKY44 gene in tobacco [J]. Acta Agric. Boreali-Sin., 2016, 31(1):117-122.
20	杨占武,杨君,张艳,等.棉花GbRvd的亚细胞定位及其超表达烟草抗病性分析[J].中国农业科学,2016,49(21):4065-4073.
	YANG Z W, YANG J, ZHANG Y, et al.. Subcellular localization and Verticillium wilt resistance analysis of cotton GbRvd in overexpressed tobacco [J]. Sci. Agric. Sin., 2016, 49(21):4065-4073.
21	MONAGHAN J, C ZIP F L. Plant pattern recognition receptor complexes at the plasma membrane [J]. Curr. Opin. Plant Biol., 2012, 15(4):349-357.
22	COUTO D, ZIPFEL C. Regulation of pattern recognition receptor signalling in plants [J]. Nat. Rev. Immunol., 2016, 16(9):537-552.
23	AO Y, LI Z, FENG D, et al.. OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity [J]. Plant J. Cell Mol. Biol., 2015, 80(6):1072-1084.
24	DESAKI Y, TAKAHASHI S, SATO K, et al.. PUB4, a CERK1-interacting ubiquitin ligase, positively regulates MAMP-triggered immunity in Arabidopsis [J]. Plant Cell Physiol., 2019, 60(11): 2573-2583.
25	MONAGHAN J, MATSCHI S, ROMEIS T, et al.. The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst [J]. Plant Signal. Behav., 2015, 10(5):1-5.
26	LEE K J, KIM K. The rice serine/threonine protein kinase OsPBL1 (Oryza sativa PBS1-like 1) is potentially involved in resistance to rice stripe disease [J]. Plant Growth Regul., 2015, 77(1):67-75.
27	KHARE E, KIM K, LEE K J. Rice OsPBL1 (Oryza sativa PBS1-like 1) enhanced defense of Arabidopsis against Pseudomonas syringae DC3000 [J]. Eur. J. Plant Pathol., 2016, 146(4):901-910.
28	YAN Z, XING F W, DING Z G, et al.. Transcriptome profiling of Gossypium barbadense inoculated with Verticillium dahliae provides a resource for cotton improvement [J]. BMC Genomic, 2013, 14(1):637-654.
29	YANG Z, TONG W, YIN K Q, et al.. Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses [J]. New Phytol., 2012, 195(2):450-460.
30	LI J, ZHANGK, MENG Y, et al.. Jasmonic acid/ethylene signaling coordinates hydroxycinnamic acid amides biosynthesis through ORA59 transcription factor [J]. TPJ, 2018, 95(3):444-457.
31	SHABAN M, MIAO Y, ULLAH A, et al.. Physiological and molecular mechanism of defense in cotton against Verticillium dahlia [J]. Plant Physiol. Biochem., 2018, 125:193-204.
32	陈涛,张劲松.乙烯的生物合成与信号传递[J]. 植物学报, 2006,23(5):519-530.
	CHEN T, ZHANG J S. Ethylene biosynthesis and signal pathway model [J]. Chin. Bull. Bot., 2006, 23(5):519-530.
33	ZHANG Z G, ZHOU H L, CHEN T, et al.. Evidence for serine/threonine and histidine kinase activity in the tobacco ethylene receptor protein NTHK2 [J]. Plant Physiol., 2004, 136(2):2971-2981.
34	ZHANG X Q, PENG K Q, WEN Z K U, et al.. Recent research advances on ethylene receptors in tobacco subtropical [J]. Plant Sci., 2007, 36(1):85-88.
35	BARI R, JONES J. Role of plant hormones in plant defence responses [J]. Plant Mol. Biol., 2009, 69(4):473-88.
36	THINES B, KATSIR L, MELOTTO M, et al.. JAZ repressor proteins are targets of the SCF COI1 complex during jasmonate signaling [J]. Nature, 2007, 448(7154):661-665.
37	CHINI A, BOTER M, SOLANO R. Plant oxylipins: COI1/JAZs/MYC2 as the core jasmonic acid signalling module [J]. FEBS J., 2009, 276(17):4682-4692.
38	DEMIANSKI A J, CHUNG K M, KUNKEL B N. Analysis of Arabidopsis JAZ gene expression during Pseudomonas syringae pathogenesis [J]. Mol. Plant Pathol., 2011, 13(1):46-57.
39	ZABALA M T, ZHAI BING, JAYARAMAN S, et al.. Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection [J]. New Phytol., 2016, 209(3):1120-1134.

[1]	Yunfei LIU, Fengjie WEI, Maolin XIA, Zhaojin YU, Hao XIA, Chunyu YI, Jianbo CHANG, Xiaoming JI. Alleviative Effect of New Composite Hydrogels on Cadmium Stress Tobacco Seedlings [J]. Journal of Agricultural Science and Technology, 2023, 25(3): 188-197.
[2]	Yudan ZHANG, Weimin WANG, Bo NI, Xiaohan MA, Junling LI, Zicheng XU, Wei JIA, Jiuchang SHI. Extraction Optimization of Chlorogenic Acid from Tobacco Stalk and Its Antifungal Effect [J]. Journal of Agricultural Science and Technology, 2023, 25(1): 119-127.
[3]	Geng LI, Yuanyuan ZHAO, Yuyuan CHENG, Jiang WU, Weidong DUAN, Guangting YIN, Qian LI, Chen CHEN, Fei ZHENG, Yuan LIU, Hongzhi SHI. Effects of Different Organic-inorganic Nitrogen Ratios on Soil Carbon and Nitrogen and Upper Leaf Quality in Nanyang Tobacco Area [J]. Journal of Agricultural Science and Technology, 2023, 25(1): 175-186.
[4]	Yuqin XIAO, Xiao LEI, Mingjin ZHANG, Yuangai ZHANG, Shan TANG, Hongfei JI, Chuan WANG, Cuiling MA, Yanqiu JING. Effects of Three Bacillus Agents on Seedling Growth of Flue-cured Tobacco [J]. Journal of Agricultural Science and Technology, 2022, 24(5): 85-92.
[5]	Tianwei PENG, Huiya XIE, Sijun LI, Yixuan LIU, Kaifeng SHUAI, Yuanyuan PENG, Qing WANG, Diqin LI. Effects of Sodium Dinitrate with Bacillus Sbutilis Complex on Growth and Physiological Indexes of Tobacco Seedlings [J]. Journal of Agricultural Science and Technology, 2022, 24(4): 154-161.
[6]	Yu WANG, Chunguang LI, Huan LIU, Yuehua ZHANG, Xiaomin FENG, Yaoguang LI, Huaiqi LI, Yanqiu JING, Mi SUN. Study on Chloroplast Ultrastructure of Flue-cured Tobacco Leaves and Its Relationship with Degrading Products of Plastid Pigment [J]. Journal of Agricultural Science and Technology, 2022, 24(3): 67-76.
[7]	Jun LUO, Hong ZHOU, Facong QIAN, Junying LI. Effects of Shading on Nicotine Synthesis of Flue-cured Tobacco [J]. Journal of Agricultural Science and Technology, 2022, 24(2): 115-123.
[8]	Zhuwen LIU, Longfei YANG, Maolin LIU, Guotao JIA, Qian YAO, Yiqiong MA, Ting CUI, Xinling YANG, Yang CHEN, Liangkun CHENG. Effects of Different Soil Amendments on Soil Nutrients and Inherent Quality of Flue-cured Tobacco [J]. Journal of Agricultural Science and Technology, 2022, 24(11): 190-198.
[9]	LIU Yuanbo, WANG Jing, Zhu Xuejie, JIANG Weifeng, LIU Tian, ZHANG Jinzhong, LI Yaoxin, FU Yunpeng. Effects of Different Water and Fertilizer Management Modes on Nutrient Accumulation and Quality of Flue-cured Tobacco Leaves [J]. Journal of Agricultural Science and Technology, 2021, 23(9): 193-201.
[10]	LIU Lijia, XU Zhiqiang, HE Jia, DING Yongle, SUN Jutao. Study on Metabolic Difference of Resistance to Black Shot in Tobacco Induced by Trichoderma harzianum [J]. Journal of Agricultural Science and Technology, 2021, 23(8): 91-105.
[11]	FAN Ningbo1, ZHOU Junxue2, JIANG Kai2, WANG Hong2, SHI Longfei2, GAO Yulong3, CHEN Yi3. Membrane Lipid Peroxidation and Its Relationship with Senescence-Related Genes in Main Veins of Flue-Cured Tobacco at Different Maturity Stages#br# [J]. Journal of Agricultural Science and Technology, 2021, 23(3): 66-72.
[12]	ZHANG Luxiang1, CHEN Simeng1, ZHENG Cong2, JIN Yinan1, HAN Yi3, XU Zicheng1, HUANG Wuxing1, SHAO Huifang1*. Influences of Different Deacclimation Duration on Drought Resistance of Tobacco Seedlings#br# [J]. Journal of Agricultural Science and Technology, 2021, 23(2): 57-64.
[13]	BAO Zhijuan, JIN Rong, YANG Jinqing, ZHANG Qi, ZHU Yongli, ZHAO Zhengxiong*. Effects of Pb and Zn Combination on Antioxidant Enzymes and Carbon-nitrogen Metabolism of Flue-cured Tobacco [J]. Journal of Agricultural Science and Technology, 2021, 23(2): 65-72.
[14]	LUO Yong1, JIAO Guizhen1, LIU Shengbo2, WEI Yuewei1, SHAO Huifang1, JIA Hongfang1. Effects of Cadmium with Different Concentrations on Seeding Growth and Auxin-related Gene Expression of Tobacco [J]. Journal of Agricultural Science and Technology, 2021, 23(1): 58-65.
[15]	YIN Quanyu1, LIU Jianhao1, LIU Guoshun1, YANG Xinling2, LI Xiaofu2, ZHANG Yulan1, LI Yang1,YE Hongchao3*. Effects of Biochar Application for Four Consecutive Years on Microbial Community Structure of Tobacco Cinnamon Soil [J]. Journal of Agricultural Science and Technology, 2021, 23(1): 176-185.