陆地棉叶片发育相关基因GhRH39克隆与功能分析

doi:10.11963/cs20200028

摘要/Abstract

摘要：

【目的】研究陆地棉DEAD-box RNA解旋酶基因GhRH39在叶片发育中的功能。【方法】借助生物信息学方法分析GhRH39的基因结构和进化关系,利用实时定量聚合酶链反应PCR（Quantitative real-time polymerase chain reaction, qRT-PCR）分析该基因在棉花不同组织和叶片不同发育时期的表达情况,利用病毒诱导的基因沉默（Virus-induced gene silencing, VIGS）技术对该基因进行沉默。观察阳性植株表型,检测其光合色素含量变化,对阳性植株中叶绿体发育和光合色素合成相关基因进行表达量检测。【结果】 GhRH39编码620个氨基酸,且序列较为保守。qRT-PCR分析表明,该基因在根、茎、叶片、顶芽、花瓣、纤维中均有表达,在叶片中表达量较高,且在叶片中的表达量随着叶片发育进程而变化。利用VIGS技术成功降低了GhRH39的表达水平,基因沉默的阳性棉株出现失绿表型,叶绿素a、叶绿素b、类胡萝卜素3种光合色素含量均降低。阳性沉默棉株的叶绿体发育和光合色素合成相关基因表达量出现一定程度的下降。【结论】 GhRH39基因影响棉花叶绿素和类胡萝卜素合成,同时影响叶绿体发育过程。

关键词: 陆地棉; RNA解旋酶; GhRH39; 叶绿体; 光合色素

Abstract:

[Objective] The aim of this article is to study the function of GhRH39, a DEAD-box RNA helicase gene, in leaf development of upland cotton. [Methode] The gene structure and evolutionary relationships of GhRH39 were analyzed using bioinformatic approaches. The expression patterns of GhRH39 in different tissues and different leaf development stages were analyzed by quantitative real-time polymerase chain reaction. The virus-induced gene silencing (VIGS) was applied to silence the gene. The phenotypes of GhRH39-silenced plants were observead and pigment contents were detected. The expression levels of those genes related to chloroplast development and photosynthetic pigments synthesis in the GhRH39-silenced plants were detected. [Results] Bioinformatic analysis showed that GhRH39 encoding 620 amino acids had conservative sequence. The expressions of GhRH39 were detected in root, stem, leaf, apical bud, petal and fiber. Its expression level is higher in leaf, and changed with the development process of leaf. The expression of GhRH39 was successfully suppressed by VIGS. The virescent leaves were appeared in GhRH39-silenced cotton plants, in which the contents of chlorophyll a, chlorophyll b, and carotenoid all decreased. To some extent, the expressions of those genes related to development of chloroplast and photosynthetic pigments synthesis decreased in the GhRH39-silenced plants. [Conclusion] GhRH39 gene may affect the synthesis of chlorophyll a and b, and carotenoid, and affect the development of chloroplast in cotton.

Key words: upland cotton (Gossypium hirsutum L.); RNA helicase; GhRH39; chloroplast; photosynthetic pigment

卞英杰,王寒涛,魏恒玲,张蒙,李弈,喻树迅. 陆地棉叶片发育相关基因GhRH39克隆与功能分析[J]. 棉花学报, 2021, 33(4): 319-327.

Bian Yingjie,Wang Hantao,Wei Hengling,Zhang Meng,Li Yi,Yu Shuxun. Cloning and functional analysis of GhRH39, a gene related to leaf development, in upland cotton (Gossypium hirsutum L.)[J]. Cotton Science, 2021, 33(4): 319-327.

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链接本文: https://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/cs20200028

https://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2021/V33/I4/319

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引物名称 Primer names	引物序列 Primer sequences
C-GhRH39-F	GGATCTTCCAGAGATATGATGCAGAAAGCTATGATGGC
C-GhRH39-R	CTGCCGTTCGACGATTCAACTTGGTGTAAACGATTCTCTCT
Q-Ghactin-F	ATCCTCCGTCTTGACCTTG
Q-Ghactin-R	TGTCCGTCAGGCAACTCAT
Q-GhRH39-F	GCAGAAAGGATAGAGGCGGCAA
Q-GhRH39-R	TGCACAGATGCAGACACAGCTT
TRV-GhRH39-F	AAGGTTACCGAATTCTCTAGAGCTGGACTTGGGTGTTAAT
TRV-GhRH39-R	CGTGAGCTCGGTACCGGATCCTTTGAAGGTTTGGCTGGAG
Q-CLPR3-F	CTTGCCTCCGTTGCCTCTTCTT
Q-CLPR3-R	TCCACTTGAGCAGGAGTAGCCA
Q-FZL-F	CTCAAGGGGGAATCGTCCACCA
Q-FZL-R	TGTGTATCAGCAATGGCAGGACC
Q-FTSH-F	AACTCCATTGGCGAGATCCGTG
Q-FTSH-R	GACGTGCCTACTCCAACGAACA
Q-SIGF-F	TTCATCATGAGCAAGCGGTCCC
Q-SIGF-R	GCTGGCCAATGTTGTCAGATGC
Q-HEMB1-F	TGCCTCGAACAATTCGTCTGCT
Q-HEMB1-R	CTGGGACACCGCTTGCTTACAT
Q-CHLI-F	TGAACAGGCTCAGAAGGTTGCC
Q-CHLI-R	GTTGTGGACTTCCCAGTTCCCC
Q-CHLG-F	ACAATCACCTGCGACTCCCAAC
Q-CHLG-R	TAGTATCAGTGTCCGCCGCTCT
Q-PDS-F	GGCTGTTGGGATCACCACACTT
Q-PDS-R	CCACATCGATGCCGGCTAAGTT

元件名称 Element	位置 Locus of strand/bp	序列 Sequences	功能注释 Function annotation
ARE	1753(+)/1969(+)	AAACCA	厌氧诱导Anaerobic induction
Box4	375(+)/1563(-)/687(+)/496(+)/1281(-)	ATTAAT	光响应Light responsiveness
CGTCA-motif	722(-)	CGTCA	茉莉酸甲酯响应MeJA-responsiveness
GA-motif	1591(-)	ATAGATAA	部分光响应元件 Part of a light responsive element
GT1-motif	199(+)/1315(-)	GGTTAA	光响应Light responsiveness
LAMP-element	1401(-)	CTTTATCA	部分光响应元件 Part of a light responsive element
MBS	546(+)	CAACTG	干旱诱导的MYB结合位点MYB binding site involved in drought-inducibility
O2-site	125(+)	GATGATGTGG	醇溶蛋白代谢调控 Zein metabolism regulation
TC-rich repeats	230(+)/1372(+)	GTTTTCTTAC	防卫与胁迫响应 Defense and stress responsiveness
WUN-motif	1188(+)	AAATTTCCT	损伤响应Wound responsiveness