陆地棉GhBZR1基因的克隆及功能研究

doi:10.11963/cs20200083

摘要/Abstract

摘要：

【目的】BZR1是油菜素内酯（Brassinosteroid,BR）信号通路中关键的转录因子,去磷酸化后可直接调控BR信号通路下游基因的表达,影响植物的生长发育和免疫反应。明确其功能对了解棉花的抗/感病分子机制具有重要意义。【方法】分析未接种和接种黄萎病菌（大丽轮枝菌,Verticilium dahliae）的中植棉KV-3的转录组测序结果,筛选到1个下调表达基因GhBZR1,并通过cDNA末端快速扩增（Rapid amplification of cDNA ends,RACE）、生物信息学分析、实时荧光定量聚合酶链式反应（Real-time quantitative polymerase chain reaction,qRT-PCR）、亚细胞定位、病毒诱导的基因沉默（Virus-induced gene silencing,VIGS）和超表达,对该基因进行了克隆与功能验证。【结果】在陆地棉中克隆出GhBZR1基因全长1 515 bp,开放阅读框为942 bp,编码313个氨基酸。生物信息学分析表明,GhBZR1蛋白与海岛棉中的BZR1蛋白同源性最高,具有BES1_N超家族结构域,属于亲水性蛋白,具有多个磷酸化位点,在其启动子区域存在3个与病原菌抗性相关的顺式作用元件,分别是TC-rich repeats、ABRE和MBSI元件。亚细胞定位显示,GhBZR1蛋白定位在细胞核。qRT-PCR检测结果表明,GhBZR1基因在茎中优势表达;大丽轮枝菌诱导后,该基因在不同抗病、感病品种中的转录水平不同,且能被茉莉酸和水杨酸诱导表达;抑制GhBZR1基因表达能增强感病陆地棉品种86-1对黄萎病菌的抗性;超表达GhBZR1基因使转基因拟南芥抗黄萎病菌能力减弱、病症加重。【结论】GhBZR1基因在棉花抗黄萎病的过程中起负调控作用。

关键词: 陆地棉; 黄萎病; BZR1基因; 克隆; 病毒诱导的基因沉默

Abstract:

[Objective] BZR1 is a key transcription factor in the brassinosteroid (BR) signaling pathway. After dephosphorylation, it can directly regulate the expression of downstream genes in the BR signaling pathway, and then affect the growth and development, and immune responses of plants. It is of great significance to clarify BZR1 gene function for understanding the molecular mechanism of disease resistance/susceptibility in cotton. [Method] Analyzing the transcriptome sequencing results of the KV-3 inoculated and uninoculated with Verticillium dahliae, a down-regulated gene, GhBZR1 was selected. Rapid amplification of cDNA ends, bioinformatics analysis, real-time quantitative polymerase chain reaction (qRT-PCR), subcellular localization technology, virus induced gene silencing and gene overexpression technology were performed to study the characteristics and function of the gene. [Result] The GhBZR1 gene was cloned from Gossypium hirsutum with a total length of 1 515 bp, and the open reading frame is 942 bp and encodes a protein of 313 amino acids. Bioinformatics analysis shows that GhBZR1 has the highest similarity with the BZR1 protein of G. barbadense, and GhBZR1 has a BES1_N superfamily domain, and is a hydrophilic protein with a variety of phosphorylation sites. There are three cis-acting elements related to pathogen resistance in the 1 000 bp promoter region of this gene, which are TC-rich repeats, ABRE and MBSI. Subcellular localization analysis shows that GhBZR1 is localized in the nucleus. And qRT-PCR analysis shows that GhBZR1 gene is preferentially expressed in cotton stems. With infection of V. dahliae, the transcription level of GhBZR1 is different in resistant cotton varieties and susceptible varieties. And the expression of GhBZR1 can be induced by jasmonic acid and salicylic acid. After GhBZR1 silenced, the susceptible cotton variety 86-1 shows enhanced resistance to Verticillium wilt. Overexpression of GhBZR1 gene weakens the disease resistance of transgenic Arabidopsis and aggravates disease symptoms. [Conclusion] The GhBZR1 gene plays a negative regulatory role in the process of cotton resistance to Verticillium wilt.

Key words: Gossypium hirsutum L.; Verticillium wilt; BZR1 gene; clone; virus-induced gene silencing

贺浪,张华崇,司宁,简桂良. 陆地棉GhBZR1基因的克隆及功能研究[J]. 棉花学报, 2021, 33(6): 435-447.

He Lang,Zhang Huachong,Si Ning,Jian Guiliang. Cloning and functional analysis of GhBZR1 in Gossypium hirsutum L.[J]. Cotton Science, 2021, 33(6): 435-447.

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链接本文: https://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/cs20200083

https://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2021/V33/I6/435

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引物名称 Primer name	引物序列 Primer sequence	用途 Purpose
GhBZR1-F	ATGACATCGGATGGGGCGAC	扩增中间片段 Amplification of middle segment sequence
GhBZR1-R	TTAACACTGAGGCTTTCCGCTTC	扩增中间片段 Amplification of middle segment sequence
3′OGhBZR1	TGCCTCCCCAAAATTTGCAC	3′ RACE
GSP Outer	ATGTTTGGGAATATGCAGATCGGA
3′I GhBZR1	CGCGGATCCTCCACTAGTGATTTCACTATAGG
GSP Inner	TGATAATGCCCTTATGGAGGATAG
5′O GhBZR1	GATTACGCCAAGCTTCGGTGGGTAGGACTA- GCTGGGGC	5′ RACE
5′I GhBZR1	GATTACGCCAAGCTTACGACCCAACCAGCC- TCCGAACAA
UPM	CTAATACGACTCACTATAGGGCAAGCAGT- GGTATCAACGCAGAGT
qGhBZR1-F	GCTTCCCTTTTCATCGTTTTCA	扩增基因全长 Amplification of full-length GhBZR1 gene
qGhBZR1-R	GCGATTAAGTTTGGATTGGATTG	扩增基因全长 Amplification of full-length GhBZR1 gene
B-F	TGCCTCCACTCAGAATCTCAAA	棉花GhBZR1表达水平检测 Detection of GhBZR1 expression in cotton
B-R	TGGGGCAGAAACAGCATAAA	棉花GhBZR1表达水平检测 Detection of GhBZR1 expression in cotton
Ubiquitin-F	GCTCGGATACGATTGATAAC	检测Ubiquitin基因表达水平 Detection of Ubiquitin gene
Ubiquitin-R	AAGACGAAGAACAAGGTGAA	检测Ubiquitin基因表达水平 Detection of Ubiquitin gene
YB-F	CC CCCGGGATGACATCGGATGGGGCGAC	构建GhBZR1基因亚细胞定位载体 Vector construction for subcellular localization
YB-R	GC TCTAGAACACTGAGGCTTTCCGCTTC
VIGS-GhBZR1-F	TC TTAATTAAACACCCCCACTTTCATCTCC	构建GhBZR1基因沉默载体 Construction of GhBZR1 gene silencing vector
VIGS-GhBZR1-R	GT ACTAGTCTCTCCTTCCCATGGTTTCAGA
O-GhBZR1-F	CGGGATCCGATGACATCGGATGGGGCGAC	构建GhBZR1超表达载体 Construction of GhBZR1 overexpression vector
O-GhBZR1-R	GCTCTAGATTAACACTGAGGCTTTCCGCTTC
AtBZR1-F	GGCACCACTTATCGCAAGGGATG	检测拟南芥中GhBZR1的表达 Detection of GhBZR1 expression in Arabidopsis
AtBZR1-R	GGAAGGGCTGACTTGACATGAAGG
Atactin-12-F	CCGAAGGCTAACCGTGAGAAGATG	检测Atactin-12的表达 Detection of Atactin-12 expression
Atactin-12-R	GAGGGAGAGAACAGCTTGAATGGC	检测Atactin-12的表达 Detection of Atactin-12 expression

顺式作用元件 Cis-acting element	序列 Sequence	位置 Position/bp	作用 Function
TC-rich repeats	ATTCTCCAAC	（+）481	参与防御和应激反应 Participate in defense and stress response
ABRE	TACGTGTC ACGTG	（-）389 （-）391	响应脱落酸 Participate in abscisic acid reaction
MBSI	AAAAAAC(G/C)GTTA	（+）142	MYB 结合位点,参与类黄酮生物合成基因调控 MYB binding site, involved in the regulation of flavonoid biosynthesis genes