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【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】 The objective of this study was to investigate the biological information, expression level and function of MdVGT1, a glycoconjugate glucose transporter gene in Hongshuang apple hybrid F1 generation in Xinjiang, in order to further improve the function of MdVGT1 Theory and technology system for reference. 【Method】 MdVGT1 was cloned by using “Hongbian No.1”, a hybrid generation of red fleshed apple in Xinjiang, and its bioinformatics analysis was carried out. The expression of MdVGT1 in different tissues and different developmental stages was analyzed by real-time PCR The expression of this gene was induced under glucose induction in ’Gala’ tissue culture seedlings. The interaction between these genes was verified by yeast two-hybrid system and the recombinant protein was obtained by prokaryotic induction. 【Result】 The full length of MdVGT1 was cloned from ’Hongbian 1’ and sequenced and found to contain a full open reading frame of 1 506 bp encoding 501 amino acids. The molecular weight of the predicted MdVGT1 protein was 53.16 kD and the isoelectric point was 5.92. Located on chromosome 1 of the apple genome and composed of 14 exons and 13 introns. The phylogenetic tree analysis of the genes related to glucose metabolism showed that MdVGT1 and AtVGT1, VvVGT1 and CsVGT1 are on the same clade, The results showed that the MdVGT1 protein contained 12 transmembrane regions. MdVGT1 was highly expressed in the flowers, leaves and young fruits of apple. The expression of MdVGT1 was significantly correlated with the glucose content during fruit development. The MdVGT1 promoter Containing cis-acting elements related to stress resistance, glucose signal and hormone signaling; the expression of MdVGT1 was significantly increased after glucose treatment of ’Gala’ tissue culture seedlings in one week; yeast two-hybrid assay showed that MdVGT1 and MdTMT1 could interact in vitro , And the recombinant protein of MdVGT1 was obtained by pronuclear induction. 【Conclusion】 The tonoplast glucose transporter gene MdVGT1 was cloned from ’Hongfen 1’ apple, which may interact with MdTMT1 to transport glucose to tonoplast.