棉花盐胁迫应答基因GhEXO70B1功能分析

Functional Analysis of a Salt Stress Response Gene, Gh EXO70B1, in Upland Cotton(Gossypium hirsutum L.)

【目的】为了提高棉花的耐盐性,使棉花能够适应盐碱地的生长环境,挖掘耐盐基因迫在眉睫。【方法】利用生物信息学分析了Gh EXO70B1基因的功能和结构特征;利用定量反转录-聚合酶链式反应(Quantitative reverse transcription-polymerase chain reaction,qRT-PCR)分析该基因在不同部位的表达量和盐胁迫下根、茎、叶的表达情况;利用病毒诱导基因沉默技术(Virus-induced gene silencing,VIGS)对该基因进行沉默并进行功能验证,并通过台盼蓝染色观察和生化指标测定综合分析了受胁迫的程度及其可能的生理机制。【结果】本研究从陆地棉中分离了1个新的介导自噬的基因Gh EXO70B1,生物信息学分析表明该基因编码产物含有1个保守的EXO70结构域,不具有跨膜结构,为非分泌蛋白,进化中功能高度保守。qRT-PCR表明该基因在各个组织部位中均有表达,其中根和茎中表达量较高,盐胁迫处理诱导其上调表达。利用VIGS成功沉默GhEXO70B1的表达,基因沉默后的棉株相较于对照更加不耐盐,出现叶片发黄、萎蔫和干枯的现象,脯氨酸含量显著降低,丙二醛含量升高,台盼蓝染色叶片着色范围较大,表明Gh EXO70B1基因沉默后应对盐胁迫的能力降低,细胞死亡的数量显著增加。【结论】陆地棉Gh EXO70B1基因与自噬相关,暗示该基因通过介导细胞自噬应对逆境胁迫。

[Objective]The exploitation of salt-tolerant genes is urgently needed to improve the salt tolerance of cotton and increase its adaptability to saline-alkali soil.[Method]The function and structure of the GhEXO70B1 gene were bioinformatically analyzed.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to analyze the expression of this gene in different cotton plant parts and in salt-stressed roots,stems,and leaves.Virus-induced gene silencing(VIGS)was applied to verify the function of GhEXO70B1.The degree of stress and the possible physiological mechanism of stress response were comprehensively analyzed through trypan blue staining and biochemical measurements.[Result]In this study,the novel gene GhEXO70B1 involved in autophagy was isolated from upland cotton.Bioinformatic analysis showed that GhEXO70B1,which is a non-secretory protein with a conserved EXO70 domain and no transmembrane structure,is evolutionarily highly conserved.In the qRT-PCR analysis,the gene had higher expression levels in roots and stems than in other tissues and was upregulated by salt stress.After VIGS technology-induced silencing of the expression of GhEXO70B1,cotton plants exhibited yellowing,wilting,and drying of leaves and higher salt sensitivity than the control.Proline content decreased significantly,whereas malondialdehyde content increased slightly and the range of colors of trypan blue-stained leaves was larger;these results indicate GhEXO70B1 silencing significantly decreased the salt stress response ability of upland cotton and significantly increased the level of cell death.[Conclusion]The GhEXO70B1 gene is associated with autophagy,which suggests that this gene responds to adversity stress in upland cotton by mediating autophagy.