葡萄雌能花相关基因VvMS2的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2013.14.011

摘要/Abstract

摘要： 【目的】从欧亚种葡萄‘魏可’和‘钟山红’的花中分离出VvMS2基因，为研究葡萄雌能花形成的分子机理和分子育种奠定基础。【方法】在NCBI中寻找与拟南芥雄性不育基因MS2同源性最高的葡萄序列（CBI29968），设计特异引物进行克隆测序，通过生物信息学方法分析结构特征，利用实时荧光定量RT-PCR技术研究其在‘魏可’各组织中及‘魏可’和‘钟山红’雄蕊发育不同时期中的表达特性。【结果】VvMS2基因cDNA编码区全长为1 755 bp，编码584个氨基酸，含有NAD结合域和雄性不育C-末端区域。VvMS2对应基因组DNA全长2 776 bp，含有9个外显子和8个内含子。基因编码蛋白质分子量为64.74 kD，等电点为8.84。该基因与GenBank中其它来源的MS2蛋白序列的相似性为40%—71%。VvMS2为花蕾特异表达基因，且仅在花粉发育四分体期到二核期有表达，单核期‘魏可’的表达量显著高于同时期‘钟山红’。【结论】推测该基因异常表达与‘钟山红’葡萄雌能花形成有关。

关键词: 葡萄 , 雌能花 , VvMS2 , 基因克隆 , 特征分析 , 表达模式

Abstract: 【Objective】 This paper aims to isolate the VvMS2 gene from the Vitis vinifera L. cv. Wink and Zhongshanhong, in order to further study the molecular mechanism of grape male sterility and grape molecular breeding. 【Method】 Specific primers were designed based on the grape sequence (CBI29968) in NCBI which is highest homology with Arabidopsis male sterility gene MS2, and the cDNA and genomic DNA of VvMS2 were cloned by PCR. VvMS2 gene structure characteristics were analyzed using the bioinformatics software. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of VvMS2 in different tissues of ‘Wink’ and at different development stages in bud between ‘Zhongshanhong’ and ‘Wink’.【Result】The full length ORF cDNA sequence of VvMS2 with 1 755 bp were cloned. Bioinformatics analysis showed that this gene encodes 584 amino acids, containing NAD-binding-4 structure domains and sterile structure domains. This gene contains 9 exons and 8 introns. The protein molecular weight is 64.74 kD, isoelectric point is 8.84. Compared with other sequences of MS2 in GenBank, the deduced amino acid sequences of VvMS2 shared 40%-71% in homology. VvMS2 only specially expressed in floral buds and at the stages of tetrad to dinucleate. The expression level in button at uninucleate stage in ‘Wink’ is remarkably higher than that in ‘Zhongshanhong’.【Conclusion】The abnormal gene expression is related with male sterility in ‘Zhongshanhong’.

Key words: grape , male sterility , VvMS2 , gene cloning , characterization , expression pattern

郑焕1, 张计育12, 王新卫1, 章镇1, 季晨飞1, 陶建敏1. 葡萄雌能花相关基因VvMS2的克隆及表达分析[J]. 中国农业科学, 2013, 46(14): 2953-2962.

ZHENG Huan-1, ZHANG Ji-Yu-12, WANG Xin-Wei-1, ZHANG Zhen-1, JI Chen-Fei-1, TAO Jian-Min-1. Cloning and Expression Analysis of A Male Sterility-Related Gene VvMS2 from Grapes[J]. Scientia Agricultura Sinica, 2013, 46(14): 2953-2962.

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