山葡萄基因VaEXO11及其启动子的克隆与序列分析  

Cloning and sequence analysis of VaEXO11 and its promoter in Vitis amurensis

作　　者：尹晓 许文娣 李娟[1] 马登辉 刘成敏[1] 单守明[1] YIN Xiao;XUWendi;LI Juan;MADenghui;LIU Chengmin;SHAN Shouming(College of Enology and Horticulture,Ningxia University,Yinchuan 750021,Ningxia,China)

机构地区：[1]宁夏大学葡萄酒与园艺学院,银川750021

出　　处：《果树学报》2024年第7期1285-1296,共12页Journal of Fruit Science

基　　金：宁夏科技厅重点研发计划项目(2022BBF03019);宁夏自然科学基金项目(2022AAC03010)。

摘　　要：【目的】探究欧洲葡萄基因VvEXO11在山葡萄中的同源基因VaEXO11的表达及功能,为揭示葡萄抗寒分子通路、培育新的抗寒葡萄品种提供理论依据。【方法】克隆并分析VaEXO11及其启动子PVaEXO11序列,通过瞬时转化烟草探究PVaEXO11与低温胁迫的关系。【结果】从VaEXO11克隆到的cDNA序列全长为957 bp,其中ORF为957 bp,编码318个氨基酸。该基因仅由1个外显子构成,其蛋白含有一个高度保守的Phi_1结构域和一个信号肽,丝氨酸含量丰富,经预测为疏水脂溶性蛋白。对克隆到的P_(VaEXO11)进行顺式作用元件预测,分析结果表明,PVa EXO11不仅含有CAAT-box、TATA-box等核心启动子元件,还具备参与干旱胁迫、昼夜节律调控和创伤响应等功能响应元件。瞬时转化结果表明,低温激活P_(VaEXO11)的活性,VaEXO11的相对表达量迅速上升,直接或间接促进细胞内抗氧化酶的产生。【结论】VaEXO11除了参与低温调控,还可能参与多种逆境相关的调控,从而响应多种生物与非生物胁迫。【Objective】Low temperature is one of the important climatic factors affecting crop yield and quality.It is urgent to study the mechanisms of low temperature at the molecular and genetic levels and to find ways to improve plant cold resistance.According to the previous transcriptome analysis,it was found that six members of the VvEXO family in Muscat were up-regulated under low temperature conditions,among which VvEXO11 was found to be the most significantly up-regulated.The analysis of the expression profile of VvEXO showed that VvEXO11 responded to low temperature stress in grapevines at early stage,and its role in the response to low temperature stress needed to be further studied.Vitis amurensis has been widely studied in cold resistance breeding.In this study,VvEXO11 and its promoter sequence in V.amurensis were cloned and analyzed by bioinformatics methods,and the function of VaEXO11 was preliminarily predicted,thus providing clues for further gene function verification,and revealing the new cold-resistant grape varieties.【Methods】Using V.amurensis as the test material,DNA and total RNA were extracted using DNAsecure novel plant genomic DNA extraction kit and RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit produced by Tiangen Biochemical Technology Co.The total RNA and DNA were extracted according to the product instructions.Using qualified total RNA as template,TransScript one-step gDNA removal and cDNA synthesis kit was used to reverse-transcribe the RNA into cDNA according to the instructions.The cDNA obtained by reverse transcription of V.amurensis RNA and extracted V.amurensis DNA were used as templates,respectively,based on the EXORDIUM sequence fragments obtained by sequencing the V.amurensis transcriptome and combined with the design of primers VaEXO11-F/VaEXO11-R,P_(VaEXO11)-F/P_(VaEXO11)-R for the amplification of ORF and promoter of the target gene.The PCR amplification products were detected by 1.2%agarose gel electrophoresis and DNA Marker DL2000.The amplified D

关 键 词：山葡萄 VaEXO11 启动子 序列分析 

分 类 号：S663.1[农业科学—果树学]