陆地棉GhCPR5的克隆及其在抗病中的功能分析

doi:10.3864/j.issn.0578-1752.2023.19.004

摘要/Abstract

摘要：

【目的】通过克隆陆地棉GhCPR5，分析其蛋白结构、表达模式和生物学功能，探究其在棉花响应黄萎病菌与灰霉病菌侵染中的功能和作用机制，为棉花抗病机制研究和育种提供理论基础和候选基因。【方法】根据棉花响应黄萎病菌侵染的转录组数据筛选到GhCPR5，并从陆地棉TM-1中克隆获得GhCPR5的全长编码序列。利用生物信息学技术分析GhCPR5的保守结构域、蛋白结构特征和同源基因的系统进化关系；利用实时荧光定量PCR（qPCR）分析GhCPR5在棉花根、茎、叶、胚珠、纤维、花瓣中的表达模式和黄萎病菌诱导的表达模式；构建病毒诱导的GhCPR5沉默载体TRV:CPR5，利用农杆菌介导的瞬时转化方法创建GhCPR5抑制表达植株，并借助RT-PCR和qPCR技术检测GhCPR5的干涉效率。对TRV:00和TRV:CPR5植株进行黄萎病菌和灰霉病菌接种，观察比较TRV:00和TRV:CPR5植株对病原菌的抗性差异；利用qPCR检测TRV:00和TRV:CPR5植株中防御相关基因的表达量，分析GhCPR5的作用机制。【结果】从陆地棉TM-1中克隆获得GhCPR5，其CDS全长1 683 bp，编码560个氨基酸，蛋白质相对分子量为62.883 kDa，等电点为9.01。多重序列比对和进化树分析显示，GhCPR5与榴莲、可可等物种CPR5同源性较高。此外，GhCPR5与不同物种CPR5的C端蛋白结构高度保守，含有4—5个跨膜结构域。GhCPR5在棉花幼苗真叶中的表达量最高，茎中表达量最低，且其表达受黄萎病菌诱导。在正常条件下，GhCPR5抑制表达植株TRV:CPR5和对照植株TRV:00在发育上无明显差异。但是，接种黄萎病菌后，TRV:CPR5植株对黄萎病菌表现敏感，发病率和病情指数显著高于TRV:00植株。黄萎病菌和灰霉病菌的离体叶片接种和台盼蓝染色结果显示，TRV:CPR5叶片上的病斑面积显著高于TRV:00叶片，说明下调GhCPR5表达降低了棉花对黄萎病菌和灰霉病菌的抗性。此外，GhCPR5抑制植株体内JAZ1表达量显著上升，而PR3、PR4和PR5的表达量显著下降。【结论】GhCPR5正调控棉花抗病性，下调GhCPR5表达可显著降低棉花对黄萎病菌和灰霉病菌的抗性。

关键词: 棉花, 抗病蛋白, 黄萎病菌, 灰霉病菌, 基因表达

Abstract:

【Objective】This study on the function and mechanism of GhCPR5 in response to Verticillium dahliae (V. dahliae) and Botrytis cinerea (B. cinerea) in cotton analysed nucleotide sequence, protein structure, expression pattern, and biological function to provide a theoretical basis and genetic resources for cotton disease resistance and breeding mechanism research. 【Method】GhCPR5 was identified from the unpublished transcriptome data of cotton responses to V. dahliae infection. The full-length coding sequence of GhCPR5 was amplified from upland cotton TM-1. The conserved domain, protein structure, and phylogenetic relationship of GhCPR5 and homologous genes were analysed using bioinformatics techniques. Real-time fluorescence quantitative PCR (qPCR) was used to analyse the GhCPR5 expression patterns in cotton roots, stems, leaves, ovules, fibres, and petals, and the induced expression of GhCPR5 by V. dahliae infection. The silencing fragments of GhCPR5 were amplified and inserted into the VIGS vector to generate the gene silencing construct TRV:CPR5. GhCPR5-silencing plants were created via Agrobacterium-mediated transformation methods. RT-PCR and qPCR were used to analyse the silencing efficiency of GhCPR5 in TRV:CPR5 plants. TRV:00 and TRV:CPR5 plants were inoculated with V. dahliae and B. cinerea, respectively, to analyse the difference in resistance in TRV:00 and TRV:CPR5 plants in response to pathogens. To analyse defence signal pathways involving GhCPR5, the expression levels of defence-related genes in TRV:00 and TRV:CPR5 plants were detected by qPCR. 【Result】The GhCPR5 cloned from G. hirsutum TM-1 is 1 683 bp long and encodes a 560 amino acid protein. The relative molecular weight and isoelectric point of GhCPR5 are 62.883 kDa and 9.01, respectively. Multiple-sequence alignment and phylogenetic analysis showed that GhCPR5 is highly homologous with the CPR5 of Durio zibethinus and Theobroma cacao. Furthermore, the C-terminals of GhCPR5 and CPR5 of other species are highly conserved and contain 4-5 transmembrane domains. The GhCPR5 expression level was induced by V. dahliae infection and is highest in leaves and lowest in stems. Under normal conditions, no significant developmental differences were observed between the GhCPR5-silencing plants, TRV:CPR5, and the TRV:00 control plants. After inoculation with V. dahliae, the rate of disease and the disease index of TRV:CPR5 plants were significantly higher than those of the control plants. Analysis of detached leaves inoculated with V. dahliae and B. cinerea and lactophenol-trypan blue staining showed that the lesions on the leaves of TRV:CPR5 plants were much bigger than those of TRV:00 plants, indicating that silencing GhCPR5 made cotton less resistant to V. dahliae and B. cinerea. In addition, the JAZ1 expression levels in TRV:CPR5 plants were significantly higher than in TRV:00 plants, whereas the PR3, PR4, and PR5 expression levels were markedly lower in TRV:CPR5 plants. 【Conclusion】GhCPR5 positively regulates cotton disease resistance; the downregulated expression of GhCPR5 significantly reduced cotton resistance to V. dahliae and B. cinerea.

Key words: cotton, disease resistance protein, Verticillium dahliae, Botrytis cinerea, gene expression

许福春, 赵静若, 张振楠, 胡改元, 龙璐. 陆地棉GhCPR5的克隆及其在抗病中的功能分析[J]. 中国农业科学, 2023, 56(19): 3747-3758.

XU FuChun, ZHAO JingRuo, ZHANG ZhenNan, HU GaiYuan, LONG Lu. Cloning and Functional Characterization of GhCPR5 in Disease Resistance of Gossypium hirsutum[J]. Scientia Agricultura Sinica, 2023, 56(19): 3747-3758.

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引物名称Primer name	引物序列Primer sequence (5′-3′)
VIGS-GhCPR5-F	CGGGGTACCGATGTTGATCGGCCTTCTGT
VIGS-GhCPR5-R	CGCGGATCCGCAACTCAGCCTGTCCTGT
qRT-GhCPR5-F	TCTGTGGTTTGGCTGGGAAGT
qRT-GhCPR5-R	GGCATAAGACCGCACAATAAGG
qRT-JAZ1-F	AGCCTCAAAAAGGAAGACCTCAAAC
qRT-JAZ1-R	TGGCTGCTCAATCACCATAGTAATC
qRT-JAZ3-F	TGATTTTGCTCAAGGAGATAACGCT
qRT-JAZ3-R	TGATTGCCTACTCGTTGCCTGT
qRT-NPR1-F	CTAGCTTGCGGAGGGATTGATACC
qRT-NPR1-R	GAGATGGCTGACCTGTCAAACTGC
qRT-PR3-F	ACTCCACAATCACCGAAGCCAT
qRT-PR3-R	GCATTCCAACCCTTACCACATTC
qRT-PR4-F	CAGGAGAAGGAGCGACTGTGA
qRT-PR4-R	TCCCACATCCAAATCTAACCC
qRT-PR5-F	AAGGAGTCCACCACAATCACCG
qRT-PR5-R	CTGGCACTGCTATGGCTCGTAT