中国野生种葡萄及种间杂种VvmybA1基因型和表达分析

doi:10.3864/j.issn.0578-1752.2013.12.012

摘要/Abstract

摘要： 【目的】检测中国野生种葡萄以及山欧杂交品种VvmybA1基因型，并对不同材料VvmybA1基因序列进行比对分析，通过对山欧杂种VvmybA1的基因型和表达分析，揭示白色杂交品种‘熊岳白葡萄’果皮无花色苷合成的分子机理。【方法】设计特异性引物，在DNA水平上利用分子克隆手段和绝对荧光定量PCR方法，检测葡萄属的不同样品VvmybA1基因型。采用相对荧光定量PCR手段，对山欧杂交品种果实转色过程中VvmybA1和UFGT的表达量进行分析。【结果】①检测的中国野生种葡萄材料不存在VvmybA1a等位基因；②与欧亚种葡萄相比，中国野生葡萄VvmybA1基因在启动子、内含子以及第三个外显子区域存在不同程度碱基的缺失、插入和替换，表现出丰富的遗传多样性。而且许多野生种葡萄VvmybA1基因的内含子、外显子和启动子区域存在一些特有序列或突变，这些位点可筛选作为鉴别葡萄种和品种的分子标记；③在‘公酿1号’、‘北红’、‘熊岳白葡萄’等山欧杂交葡萄品种以及分类地位未定的‘宿晓红’葡萄中检测到VvmybA1a等位基因，并且‘熊岳白葡萄’为VvmybA1a纯合类型；④RT-PCR分析结果表明，在葡萄果皮转色过程中，‘公酿1号’葡萄果皮VvmybA1和UFGT的表达量增加，然而在‘熊岳白葡萄’果实成熟期未检测到VvmybA1和UFGT 的表达。【结论】证明VvmybA1a等位基因是欧亚种葡萄以及其杂交种独有的基因型，不存在于中国野生种葡萄中，推测‘宿晓红’葡萄具有欧亚种葡萄血缘；葡萄属不同种的VvmybA1基因序列比对分析揭示，山欧杂交品种‘熊岳白葡萄’果皮不能合成花色苷是由于VvmybA1a纯合，VvmybA1不表达。

关键词: 中国野生葡萄 , VvmybA1 , 花色苷 , 基因型 , 果皮颜色

Abstract: 【Objective】The aim of this study is to detect the genotypes of Chinese wild grapes and the hybrids of V. amurensis and V. vinifera and compare the VvmybA1 gene sequences in different wild grapes, reveal the molecular mechanism of no anthocyanin collected in white-fruited Xiongyuebaiputao by analysising the expression and genotype of VvmybA1 in the interspecific hybrids (V. amurensis ×V. vinifera).【Method】The specific primers were designed to detect the genotype of different wild grapes by the means of molecular cloning and absolute quantitative real-time PCR. Using Real-time PCR methods, analysis the expression of VvmybA1 and UFGT in the the hybrids of V. amurensis and V. vinifera during veraison. 【Result】①There was no VvmybA1a allele detected in Chinese wild grapes. ②Comparing sequences of VvmybA1 gene in the different species of Chinese wild grapes with thoses of V. vinifera, extensive base deletion, insertion and substitution exist in promotor region, intron region and the third coding exon, and exhibits rich genetic diversity between species of Chinese wild grapes. Furthermore，there are several unique bases or mutations in the VvmybA1 gene of wild grapes which could be selected to be the molecule marker of distinguishing different species and cultivars. ③VvmybA1a allele has been discovered in Suxiaohong and the hybrids of V. amurensis × V. vinifera, including Gongniang 1, Beihong and Xiongyuebaiputao. In addition, Xiongyuebaiputao is the homozygous genotype of VvmybA1a. ④ RT-PCR analysis showed high expression of VvmybA1 and UFGT in Gongniang 1 during veraison, but they did not express in Xiongyuebaiputao during the entire mature period.【Conclusion】The result proved that VvmybA1a allele didn’t exist in Chinese wild grapes but existed in V. vinifera and its interspecific hybrids uniquely. High consanguinity was speculated between Suxiaohong and V. vinifera. Different VvmybA1 gene sequences of species in Vitis were obtained and compared with each other. The reason why Xiongyuebaiputao has no anthocyanin collected is that the homozygous genotype of VvmybA1a lead to the no expression of VvmybA1.

Key words: Chinese wild grapes, VvmybA1, anthocyanins, genotyping, skin color

焦健, 刘崇怀, 樊秀彩, 张颖, 孙海生, 姜建福, 李民. 中国野生种葡萄及种间杂种VvmybA1基因型和表达分析[J]. 中国农业科学, 2013, 46(12): 2514-2525.

JIAO Jian, LIU Chong-Huai, FAN Xiu-Cai, ZHANG Ying, SUN Hai-Sheng, JIANG Jian-Fu, LI Min. Expression and Genotype Analysis of VvmybA1 in Chinese Wild Grapes and Interspecific Hybrids[J]. Scientia Agricultura Sinica, 2013, 46(12): 2514-2525.

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