棉花干旱诱导MYB类转录因子GhRAX3的功能分析

doi:10.3864/j.issn.0578-1752.2015.18.001

摘要/Abstract

摘要： 【目的】挖掘、分析响应干旱诱导的MYB转录因子的功能，为棉花抗旱研究和育种提供参考。【方法】以已知受干旱诱导表达的GbMYB5为检索项，Blastx检索NCBI的非冗余蛋白nr数据库中与GbMYB5具有相似序列的棉花MYB转录因子，通过荧光定量PCR验证获得干旱应答相关的MYB转录因子GhRAX3。将GhRAX3-GFP融合蛋白表达载体通过农杆菌注射法在烟草叶片瞬时表达，观察亚细胞定位荧光信号。将棉花曲叶病毒CLCrV介导的基因沉默载体CLCrV﹕GhRAX3通过农杆菌浸润法注射棉花子叶，荧光定量PCR检测棉花GhRAX3表达水平，对GhRAX3沉默的棉花植株进行干旱胁迫处理，观察表型变化，并检测其叶片失水率、叶片含水量、总抗氧化物酶活性、丙二醛含量和离子渗透率等抗旱相关生理指标。【结果】Blastx检索结果发现，与GbMYB5相比，GhRAX3覆盖率达38%，与GbMYB5有79%的相似性，编码一个R2R3-MYB转录因子。GhRAX3响应干旱诱变表达，在18%（v/v）PEG 6000诱导处理0.5 h后即显著上调表达5倍，在48 h后达上调表达33倍。亚细胞定位结果显示GhRAX3-GFP4融合蛋白仅在细胞核有明显的绿色荧光信号，表明GhRAX3定位在细胞核中。CLCrV病毒诱导GhRAX3沉默后，GhRAX3在沉默株的表达量仅为野生型的41%，表明GhRAX3表达已被抑制。取干旱处理前的棉花植株同部位的叶片，测定单位重量叶片在0—7 h内的失水量，发现GhRAX3沉默植株的叶片失水率显著高于野生型和空载体对照植株。在18%（v/v）PEG 6000水溶液处理24 h或在自然干旱15 d后，GhRAX3沉默的棉花植株萎蔫程度比野生型和空载体对照植株严重。自然干旱处理7 d后，检测叶片含水量、总抗氧化物酶活性、丙二醛含量和离子渗透率等抗旱相关生理指标，发现GhRAX3沉默植株的叶片相对含水量为82%，总抗氧化物酶活性为1.29 U·mg^-1，而野生型和空载体对照植株的叶片相对含水量则分别为89%和91%，总抗氧化物酶活性分别为3.44和3.19 U·mg^-1，GhRAX3沉默植株的叶片相对含水量和总抗氧化物酶活性都显著低于野生型和空载体对照植株；相反，GhRAX3沉默植株叶片的离子渗漏率为78.54%，丙二醛含量为74.20 nmol·mg^-1 FW，而野生型和空载体对照植株叶片的离子渗漏率分别为44.98%和47.45%，丙二醛含量分别为44.90和47.29 nmol·mg^-1 FW，GhRAX3沉默植株叶片的离子渗漏率和丙二醛含量均显著高于野生型和空载体对照。【结论】GhRAX3响应干旱胁迫诱导，抑制GhRAX3表达致使棉花在干旱胁迫条件下的叶片相对含水量和总抗氧化物酶活性显著降低，叶片离子渗漏率和丙二醛含量显著升高，使棉花耐干旱胁迫能力下降。

关键词: 棉花, MYB转录因子, 诱导表达, 病毒诱导的基因沉默, 干旱胁迫

Abstract: 【Objective】 Cloning and function analysis of MYB transcription factor in response to drought stress were conducted to understand cotton tolerance to drought stress and its further application in cotton breeding.【Method】With a drought induced GbMYB5 as a query, BLASTX search in the non-redundant protein sequences (nr) database in NCBI was performed to search possible drought induced MYB transcription factors in cotton. Real time PCR confirmed that a MYB transcription factor GhRAX3 responded to drought stress. The agro-infiltration based transient expression of a fused GhRAX3-GFP4 in tobacco cell was used to detect subcellular localization of GhRAX3. A virus induced gene silencing system mediated by cotton leaf crumple virus was used to depress the expression of GhRAX3 in cotton, and the water loss rate, relative water content, total antioxidant activity, malondialdehyde content and electrolyte leakage in leaves of GhRAX3-silenced cotton were subsequently investigated as well as their phenotypes in response to drought stress. 【Result】BLASTX with GbMYB5 as a query revealed that GhRAX3, which encodes an R2R3-MYB transcription factor, had a query cover of 38% and a identity of 79%. GhRAX3 was quickly responded to the induction of 18% (v/v) PEG 6000 by up-regulating 5-fold at 0.5 h post PEG 6000 treatment and up to 33-fold at 24 h post PEG 6000 treatment. The subcellular localization of the fused GhRAX3-GFP4 in tobacco cell indicated that GhRAX3 was located in the cell nucleus. When subjected to virus induced gene silencing, the expression level of GhRAX3 was depressed to 41% of the normal level. The water loss rate in the detached leaves of GhRAX3 silencing (viz. VIGS) cotton plants was significantly higher that those in the wilt type (WT) and the empty vector agro-infiltrated (EM) cotton plantlets during the 0-7 h duration after detaching. Under the 18% (v/v) PEG 6000 treatment for 24 h or water-withhold for 15 d, theVIGS cotton plantlets showed a more severe wilt symptom when compared with the WT and the EM cotton plantlets. At 7 d post water-withhold treatment, the relative water contents, total antioxidant activity, malondialdehyde content and electrolyte leakage in leaves were further investigated in the VIGS, WT and EM plants. The relative water content and total antioxidant activity in the VIGS plants was 82% and 1.29 U·mg^-1protein, respectively. By contrast, the relative water content in the WT and EM plants was 89% and 91%, respectively. And the total antioxidant activity in the WT and EM plants was 3.44 U·mg^-1 protein and 3.19 U·mg^-1 protein, respectively. The relative water content and total antioxidant activity in the VIGS plants were significantly lower than those in the WT and EM plants. The electrolyte leakage and malondialdehyde content in the VIGS plants was 78.54% and 74.20 nmol·mg^-1 FW, respectively. While the electrolyte leakage in the WT and EM plants was 44.98% and 47.45%, respectively. And the malondialdehyde content in the WT and EM plants was 44.90 nmol·mg^-1 FW and 47.29 nmol·mg^-1 FW, respectively. The electrolyte leakage and malondialdehyde content in the VIGS plants were significantly higher than those in the WT and EM plants. 【Conclusion】GhRAX3 was responded to drought stress. The depression of GhRAX3 compromised the tolerance of cotton plants to drought stress as a result of significant decrease in relative water content and total antioxidant activity and a significant increase in the electrolyte leakage and malondialdehyde content under drought stress.

Key words: cotton, MYB transcription factor, induction expression, virus induced gene silencing, drought stress

丁震乾，陈天子，刘廷利，刘小双，张保龙，周兴根. 棉花干旱诱导MYB类转录因子GhRAX3的功能分析[J]. 中国农业科学, 2015, 48(18): 3569-3579.

DING Zhen-qian, CHEN Tian-zi, LIU Ting-li, LIU Xiao-shuang, ZHANG Bao-long, ZHOU Xing-gen. Function Analysis of a Drought Stress Induced MYB Transcription Factor GhRAX3 in Cotton[J]. Scientia Agricultura Sinica, 2015, 48(18): 3569-3579.

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