陆地棉转录因子基因GhMYB108的克隆及其在抗旱中的作用

doi:10.3864/j.issn.0578-1752.2022.10.001

中国农业科学 ›› 2022, Vol. 55 ›› Issue (10): 1877-1890.doi: 10.3864/j.issn.0578-1752.2022.10.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

陆地棉转录因子基因GhMYB108的克隆及其在抗旱中的作用

刘瑞达^1,²(),葛常伟²,王敏轩^1,²,申延会²,李朋珍²,崔子倩²,刘瑞华²,沈倩²,张思平²,刘绍东²,马慧娟²,陈静²,张桂寅¹(),庞朝友^2,³()

¹河北农业大学/棉花生物学国家重点实验室河北基地,河北保定071001
²中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南安阳455000
³新疆农业科学院经济作物研究所,乌鲁木齐 830000

收稿日期:2021-12-22 接受日期:2022-01-28 出版日期:2022-05-16 发布日期:2022-06-02
联系方式: 刘瑞达,E-mail： 1165667680@qq.com。
基金资助:
2021年度第七师胡杨河市财政科技计划(2021C02);中国农业科学院科技创新工程(CAAS-ASTIP-2020-ICR)

Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum

LIU RuiDa^1,²(),GE ChangWei²,WANG MinXuan^1,²,SHEN YanHui²,LI PengZhen²,CUI ZiQian²,LIU RuiHua²,SHEN Qian²,ZHANG SiPing²,LIU ShaoDong²,MA HuiJuan²,CHEN Jing²,ZHANG GuiYin¹(),PANG ChaoYou^2,³()

¹Hebei Agricultural University/Stare Key Laboratory of Cotton Biology (Hebei Base), Baoding 071001, Hebei
²Institute of Cotton Research of Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology, Anyang 455000, Henan
³Institute of Cash Crops, Xinjiang Academy of Agricultural Sciences, Urumqi 830000

Received:2021-12-22 Accepted:2022-01-28 Published:2022-05-16 Online:2022-06-02

摘要/Abstract

摘要：

【目的】MYB基因家族作为植物中最大的转录因子家族之一,在抵御逆境胁迫中发挥着重要的作用。克隆陆地棉MYB转录因子基因GhMYB108,并进行表达分析,验证其在干旱胁迫响应中的作用,为进一步研究GhMYB108调控陆地棉耐旱的分子机制奠定基础。【方法】根据干旱转录组数据分析,确定GhMYB108为干旱响应基因;运用聚合酶链式反应（PCR）从陆地棉根系cDNA中扩增目的基因;对GhMYB108进行基因结构特征、预测基因序列信息以及系统进化关系等生物信息学分析;利用Plant Care网站对获得的基因启动子序列进行分析;在不同逆境胁迫条件下,对GhMYB108的表达特性进行qRT-PCR分析;通过亚细胞定位确定GhMYB108蛋白在细胞中的位置;利用酵母试验验证其转录活性;使用病毒诱导的基因沉默技术（virus induced gene silencing,VIGS）沉默GhMYB108,并用qRT-PCR检测基因沉默效率。观察沉默株系在干旱处理前后的表型变化,并统计存活率,采用试剂盒测定相关生理生化指标;通过对棉花叶片喷施ABA与氟啶酮试验来分析GhMYB108与ABA的关系。【结果】从陆地棉中克隆了GhMYB108（Gh_A10G1563）,其全长879 bp,编码292个氨基酸,其蛋白质相对分子量为33.288 kD,等电点为6.037,多重序列比对和保守结构域分析,发现GhMYB108含有2个高度保守的MYB结合结构域,属于典型的R2R3型MYB转录因子。不同物种亲缘关系分析发现,GhMYB108与AtMYB108、AtMYB78和AtMYB2的同源性较高,属于同一亚族,且已有研究发现AtMYB108、AtMYB78和AtMYB2与干旱或ABA信号通路相关。GhMYB108定位于细胞核,且具有转录激活活性。在干旱和对照植株中,GhMYB108均在根中表达量最高,茎中表达量最低,并且受自然干旱、18% PEG 6000模拟干旱、盐胁迫和低温等非生物胁迫诱导表达。GhMYB108沉默之后,在自然干旱条件下,沉默植株出现临界表型,与对照相比,其萎蔫更严重,且存活率降低,一些生理生化指标也发生显著变化,如叶片失水率加快,丙二醛含量升高,叶片相对含水量和脯氨酸含量减少,过氧化氢酶（CAT）和过氧化物酶（POD）活性降低,且通过DAB与NBT染色发现植物体积累了更多过氧化氢（H₂O₂）和超氧阴离子（O₂^-）。通过对棉花叶片喷施激素ABA或氟啶酮发现GhMYB108可受ABA信号的正调控。【结论】GhMYB108正调控棉花抗旱性,且受ABA信号的正调控。

关键词: 陆地棉, MYB转录因子, 非生物胁迫, ABA

Abstract:

【Objective】As one of the largest transcription factor families in plants, MYB genes play an important role in resisting stress. The MYB transcription factor GhMYB108 was cloned and analyzed to verify its role in drought stress response, which laid a foundation for further study on the molecular mechanism of GhMYB108 regulating drought tolerance in G. hirsutum.【Method】Through the analysis of unpublished drought transcriptome data, GhMYB108 was identified to drought response. The target gene was amplified from the root cDNA by polymerase chain reaction (PCR). Through bioinformatics analysis of gene structure characteristics, the sequence information and phylogenetic relationship of these genes were predicted. The obtained gene promoter sequences were analyzed by Plant Care website. The genes expression characteristics under different stress conditions were analyzed using Real time fluorescence quantitative PCR (qRT-PCR). The location of GhMYB108 protein was determined by subcellular localization. The transcriptional activity was tested in yeast cell; The GhMYB108 gene was silenced using Virus induced gene silencing (VIGS), and the gene silencing efficiency was detected by qRT-PCR. The phenotypic changes before and after drought treatment were observed and the survival rate was counted. The relevant physiological and biochemical indexes were measured by Solarbio Kit; The relationship between GhMYB108 and ABA was analyzed by spraying ABA and Fluridone on cotton leaves.【Result】GhMYB108 (Gh_A10G1563) was cloned from G. hirsutum, with 879 bp length and 292 amino acids. Its protein relative molecular weight and isoelectric point is 33.288 kD and 6.037, respectively. Multiple sequence alignment and conserved domain analysis showed that GhMYB108 contains two highly conserved MYB binding domains, which belongs to a typical R2R3 MYB transcription factor. Phylogenetic analysis of different species showed that GhMYB108 was highly homology with ATMYB108, ATMYB78 and ATMYB2, belonging to the same subfamily. Previous studies found that ATMYB108, ATMYB78 and ATMYB2 were related to drought and ABA signaling pathway. GhMYB108 located in the nucleus and had transcriptional activation activity. The expression level of GhMYB108 was the highest in roots and the lowest in stems, and was induced by abiotic stresses including natural drought, 18% PEG 6000 simulated drought, salt stress and low temperature. The GhMYB108 silenced plants showed a critical phenotype under natural drought conditions. Compared with the control, the silenced plants showed more serious wilting and decreased survival rate. Some physiological and biochemical indexes also changed significantly, such as accelerated leaf water loss rate, increased malondialdehyde content, decreased leaf relative water content and proline content, and decreased CAT and POD activities. Through DAB and NBT staining, the hydrogen peroxide (H₂O₂) and superoxide anion (O₂^-) were significantly accumulated in plants. By spraying the hormone ABA or Fluridone on cotton leaves, we found that GhMYB108 could be positively regulated by ABA signal.【Conclusion】GhMYB108 positively regulates cotton drought resistance and is positively regulated by ABA signal.

Key words: Gossypium hirsutum Linn, MYB transcription factor, abiotic stress, ABA

刘瑞达, 葛常伟, 王敏轩, 申延会, 李朋珍, 崔子倩, 刘瑞华, 沈倩, 张思平, 刘绍东, 马慧娟, 陈静, 张桂寅, 庞朝友. 陆地棉转录因子基因GhMYB108的克隆及其在抗旱中的作用[J]. 中国农业科学, 2022, 55(10): 1877-1890.

LIU RuiDa, GE ChangWei, WANG MinXuan, SHEN YanHui, LI PengZhen, CUI ZiQian, LIU RuiHua, SHEN Qian, ZHANG SiPing, LIU ShaoDong, MA HuiJuan, CHEN Jing, ZHANG GuiYin, PANG ChaoYou. Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum [J]. Scientia Agricultura Sinica, 2022, 55(10): 1877-1890.

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位点名称 Site name	序列 Sequence	位点功能 Function of site
G-Box	CACGTT	参与光反应的顺式调节元件Cis regulatory elements involved in photoreaction
ABRE	ACGTG	参与脱落酸反应的顺式作用元件Cis acting elements involved in abscisic acid reaction
CGTCA-motif	CGTCA	参与MeJA响应的顺式调控元件Cis regulatory elements involved in MeJA response
TC-rich repeats	GTTTTCTTAC	参与防御和应激反应的顺式作用元件Cis acting elements involved in defense and stress response
TGA-element	AACGAC	生长素响应顺式调控元件Auxin responsive Cis regulatory element

陆地棉转录因子基因GhMYB108的克隆及其在抗旱中的作用

Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum

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