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.

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