苹果液泡膜葡萄糖转运蛋白基因MdVGT1的克隆与表达分析

Isolation and Expression Analysis of a Vacuolar Glucose Transporter Gene MdVGT1 in Apple

【目的】研究新疆红肉苹果杂种一代优系‘红脆1号’液泡膜葡萄糖转运蛋白基因MdVGT1生物学信息、表达水平及其在糖代谢中的功能,旨在为进一步完善功能型苹果育种的理论与技术体系提供参考。【方法】以新疆红肉苹果杂种一代优系‘红脆1号’为试材,克隆MdVGT1,对其进行生物信息学分析;并采用荧光定量PCR分析该基因在不同组织及不同发育时期的表达,分析‘嘎啦’组培苗中该基因在葡萄糖诱导下的表达,通过酵母双杂交验证其互作关系,并通过原核诱导获得重组蛋白。【结果】在‘红脆1号’中克隆获得MdVGT1全长,测序发现其包含1 506 bp完整的开放阅读框,编码501个氨基酸,预测其编码蛋白质分子量为53.16 kD,等电点为5.92,定位于苹果基因组的1号染色体上,由14个外显子和13个内含子构成;糖代谢相关基因系统进化树分析表明,MdVGT1与AtVGT1、VvVGT1、CsVGT1在同一个进化枝上,功能域分析表明,MdVGT1蛋白含有12个跨膜区域;MdVGT1在苹果的花、叶和幼果中均有较高的表达,在果实发育中,其表达量与葡萄糖含量有显著的相关性;MdVGT1启动子含有与抗逆、糖信号及激素信号相关的顺式作用元件;葡萄糖处理‘嘎啦’组培苗一周后,MdVGT1的表达量明显提高;酵母双杂交试验表明,MdVGT1与MdTMT1在体外能相互作用,并通过原核诱导获得了MdVGT1的重组蛋白。【结论】在‘红脆1号’苹果中克隆获得了液泡膜葡萄糖转运蛋白基因MdVGT1,其可能与MdTMT1互作共同转运葡萄糖进入液泡膜。

【Objective】 In order to develop the theory and breeding technology of functional apple, the bioinformatics, the expression level and the function in the sugar metabolism several aspects of vacuolar glucose transporter gene MdVGT1 in ‘Hongcui No.1＇ of Malus sieversii f. neidzwetzkyana F1 population were studied. 【Method】 The MdVGT1 in ‘Hongcui No.1＇ was cloned and the bioinformation of it was analyzed. The expression level of MdVGT1 in different tissues and different development stages was studied by the q RT-PCR and the expression of MdVGT1 induced by glucose in tissue culture seedlings of ‘gala＇ was also analyzed. Meanwhile, the interaction between MdVGT1 and MdTMT1 was verified by yeast two-hybrid system and the recombinant protein by the prokaryotic induction technology was obtained. 【Result】 The full length of MdVGT1 in ‘Hongcui No.1＇ was cloned, and the gene was 1 506 bp which encoded 501 amino acids. It was predicted that the molecular mass of this protein was 53.16 k D,and p I was 5.92. Furthermore, it was inferred that the gene includes 14 exons and 13 introns, and is located on the chromosome 1 of the apple genome. A phylogenetic tree indicated that MdVGT1, At VGT1, Vv VGT1 and Cs VGT1 are located in the same evolutionary branch. Analysis of functional domain showed that MdVGT1 contains 12 transmembrane regions. MdVGT1 has the higher expression level in flowers, leaves and young fruits of apples, and its expression has a significant correlation with the content of glucose during the development stage of fruits. The promoter of MdVGT1 contains several typical cis-acting elements, including defense responsive elements, sugar signaling responsive elements and phytohormone responsive elements, and the expression of MdVGT1 in tissue culture seedlings of ‘gala＇ was significantly increased after glucose treatment a week later. The yeast two hybrid experiments showed that the MdVGT1 could interact with MdTMT1 in vitro. In addition, the recombinant protein of MdVGT1 was obtained by the prokaryotic induction technology. 【Conclusion】The vacuolar glucose transporter gene MdVGT1 in ‘Hongcui No.1＇ apple was cloned, and it was found that it could interact with MdTMT1 to transport glucose into the vacuole membrane.