棉花核酸外切酶基因GhWRN的克隆及功能验证

doi:10.11963/1002-7807.xyjysx.20210512

摘要/Abstract

摘要：

【目的】本研究旨在探索棉花GhWRN基因在生长发育中的功能。【方法】用生物信息学方法分析GhWRN基因的结构特征、进化关系及上游1 500 bp的启动子片段并对其功能进行初步分析,利用实时荧光定量-聚合酶链式反应（Quantitative real-time polymerase chain reaction,qRT-PCR）技术分析该基因在不同熟性棉花品种、不同组织及不同激素处理下的表达特性。构建过表达载体转化拟南芥,观察转基因拟南芥的表型。利用qRT-PCR分析拟南芥开花信号途径中标记基因的表达变化。【结果】以陆地棉cDNA为模板克隆得到GhWRN基因,生物信息学分析表明该基因编码产物含有1个保守的WRN-exo结构域,无内含子,不具有跨膜结构,为非分泌蛋白。qRT-PCR表明该基因在早熟棉花品种的二叶期开始上调表达,在植株各组织中均有表达,在雄蕊和苞片表达量较高;外源激素ABA、IAA处理后0.5 h该基因极显著上调表达。与野生型相比转基因拟南芥开花时间提前,莲座叶数量减少,qRT-PCR分析表明花发育相关关键基因AtSOC1、AtFT和AtFUL上调表达。【结论】GhWRN基因与促进植物开花相关,为进一步探究棉花的开花机制提供新线索。

关键词: 陆地棉; 核酸外切酶; 植物激素; 开花; 基因

Abstract:

[Objective] The purpose of this study was to explore the function of GhWRN in the growth and development of cotton. [Methods] GhWRN was analyzed in aspects of gene structure, evolutionary relationship, and gene promoter features through bioinformatic methods. Its expression patterns among different cultivars, tissues, and hormone conditions were gained via quantitative real-time polymerase chain reaction (qRT-PCR). The phenotype of Arabidopsis thaliana with overexpressed GhWRN was observed. The expression pattern of marker genes involved in A. thaliana flowering signal pathway was detected by qRT-PCR. [Results] The GhWRN gene was successfully cloned according to the reference genome of upland cotton. Bioinformatic analysis showed this gene contained a conserved WRN-exo domain, without intron or transmembrane structure, and was not a secretory protein. qRT-PCR showed GhWRN was up-regulated at the second leaf stage in early-maturing cotton cultivars and was expressed in all tissues, especially in stamens and bracts. It was significantly up-regulated at 0.5 h after treatment with exogenous ABA and IAA. The transgenic A. thaliana lines showed early flowering time and decreasing rosettes number compared with the wild-type. The qRT-PCR analysis in A. thaliana indicated that GhWRN could up-regulate the expression of key genes related to flower development, i.e., AtSOC1, AtFT, and AtFUL. [Conclusion] GhWRN could promote plant flowering, which sheds light on further exploration of flowering mechanism in cotton.

Key words: cotton; exonuclease; plant hormone; flowering; gene

薛羽君,魏恒玲,王寒涛,马亮,程帅帅,郝蓬勃,顾丽姣,付小康,芦建华,喻树迅. 棉花核酸外切酶基因GhWRN的克隆及功能验证[J]. 棉花学报, 2021, 33(3): 189-199.

Xue Yujun,Wei Hengling,Wang Hantao,Ma Liang,Cheng Shuaishuai,Hao Pengbo,Gu Lijiao,Fu Xiaokang,Lu Jianhua,Yu Shuxun. Cloning and functional analysis of an exonuclease response gene GhWRN in cotton (Gossypium hirsutum L.)[J]. Cotton Science, 2021, 33(3): 189-199.

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链接本文: http://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/1002-7807.xyjysx.20210512

http://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2021/V33/I3/189

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引物名称 Primer names	引物序列 Primer sequences
GhWRN-F	ATGGAGATCTCAGTGGAGAAGA
GhWRN-R	GGCCATCAAACTCCATAACCAA
qRT-PCR-F	GTTACGGCCTCCTCGAGTTTCC
qRT-PCR-R	GAGGCTTGTAAAGGTCGGGGTC
GhHistone 3-F	TCAAGACTGACTGATTTGCGTTT
GhHistone 3-R	GCGCAAAGGGTTGGTGTCTTC
AtFT-F	CAAGAGTTGAGATTGGTGGAG
AtFT-R	TACACTGTTTGCCTGCCA
AtSOC1-F	TCAGTGCTTTGTGATGCTG
AtSOC1-R	CCTTCTCCCAAGAGTTTACG
AtAP1-F	CTCTGTTCTCTGTGATGCTGA
AtAP1-R	CCAAGATAATGCCTCTGGTT
AtFUL-F	TTGCAAGATCACAACAATTCGCTTCT
AtFUL-R	GAGAGTTTGGTTCCGTCAACGACGAT
At35S-F	CCGGAAACCTCCTCGGATTC
Atactin 2-F	TTCCTCATGCCATCCTCCGTCTT
Atactin 2-R	CAGCGATACCTGAGAACATAGTGG

元件名称 Element	位置 Locus	核心序列 Core sequences	功能 Function
ABRE	1416（+）/1415 （-） /1413 （-）	ACGTG/CACGTG /GACACGTGGC	ABA响应元件 cis-acting element involved in the abscisic acid responsiveness
ARE	1367 （+）/521 （-）	AAACCA	厌氧感应元件 cis-acting regulatory element essential for the anaerobic induction
MBS	1137 （-）	CAACTG	参与干旱诱导的MYB结合位点 MYB binding site involved in drought-inducibility
AuxRR-core	735 （+）	GGTCCAT	生长素响应元件 cis-acting regulatory element involved in auxin responsiveness
TGA-element	1256 （+）/475 （+）	AACGAC	生长素响应元件 Auxin-responsive element
G-Box	1415 （-）	CACGTG	光响应相关元件 cis-acting regulatory element involved in light responsiveness
G-box	1414 （-）	CACGTG/ ACACGTGT	光响应相关元件 cis-acting regulatory element involved in light responsiveness
GT1-motif	1041 （-）/2 （+）	GGTTAA	光响应元件 Light responsive element
TCCC-motif	1433 （+）	TCTCCCT	部分光响应元件 Part of a light responsive element
GATA-motif	1390 （-）	GATAGGG	部分光响应元件 Part of a light responsive element
Box 4	97 （+）	ATTAAT	参与光反应部分DNA保守区 Part of a conserved DNA module involved in light responsiveness
MRE	1043 （+）/173 （+） /630 （-）	AACCTAA	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
TCA-element	1468 （-）	TCAGAAGAGG	水杨酸响应元件 cis-acting element involved in salicylic acid responsiveness
TC-rich repeats	232 （-）	ATTCTCTAAC	防御及应激响应元件 cis-acting element involved in defense and stress responsiveness

株系 Line	植株数量 The number of plants	开花时间（从抽薹计） Flowering time (after bolting) /d	莲座叶数 The number of rosette leaves
1	32	27.7±0.66^*	9.75±0.87^***
2	30	27.3±0.53^*	9.55±0.84^***
3	30	27.0±0.44^*	9.3±0.80^***
WT	28	32.8±0.47	11.93±0.80