猕猴桃GGP1基因启动子区域与抗坏血酸含量的关系研究  

Study on the relationship between the promoter region of GGP1 and ascorbic acid content in kiwifruit

作　　者：魏桦 李凡 宋志娟 原玉林 毛柯 李明军[1] WEI Hua;LI Fan;SONG Zhijuan;YUAN Yulin;MAO Ke;LI Mingjun(College of Horticulture,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《植物生理学报》2021年第12期2357-2365,共9页Plant Physiology Journal

基　　金：国家重点研发计划项目(2018YFD1000200)。

摘　　要：抗坏血酸(AsA)在植物生长发育和抗逆调控中起着重要作用,主要合成途径为l-半乳糖途径, GDP-l-半乳糖磷酸化酶(GDP-l-galactose phosphorylase, GGP)是该途径中AsA合成的限速酶。为了探究猕猴桃(Actinidia spp.)中GGP对抗坏血酸含量的影响,本文以AsA含量差异较大的毛花猕猴桃(A. eriantha)和山梨猕猴桃(A. rufa)为研究对象,分析了猕猴桃中GGP1基因表达差异及其启动子序列变异与AsA含量的关系。结果表明,毛花猕猴桃果实和叶片中GGP1表达水平明显高于山梨猕猴桃, GGP1基因表达水平与AsA含量呈正相关。通过克隆获得了毛花猕猴桃和山梨猕猴桃GGP1启动子的2个单体型,分别为AeGGPp1、AeGGPp2以及ArGGPp1、ArGGPp2,它们之间存在多个单核苷酸多态性(SNP)位点或插入缺失(InDel)标记,特别是与山梨猕猴桃启动子相比,毛花猕猴桃AeGGP启动子在670 bp附近存在一个183 bp的片段缺失。启动子转录活性分析表明,毛花猕猴桃启动子AeGGPp1和AeGGPp2驱动的β-葡萄糖苷酸酶(GUS)活性显著高于山梨猕猴桃启动子ArGGPp1和ArGGPp2,而同种猕猴桃启动子的单体型间活性无显著差异。根据183 bp的片段缺失设计标记引物,对山梨猕猴桃×毛花猕猴桃的F;杂交后代进行研究,结果表明,后代个体中均含有山梨猕猴桃和毛花猕猴桃GGP1启动子的一个单体型;并且杂交后代的果实AsA含量主要集中在双亲平均值附近[5.06~9.40 mg·g;(FW)],相比大多数猕猴桃资源表现出高AsA含量特性。此外,在16个猕猴桃资源种的研究中发现,含有毛花猕猴桃GGP1启动子单体型的资源普遍表现出高AsA含量特性。这些结果表明, GGP1启动子区域的变异是毛花猕猴桃和山梨猕猴桃中GGP的表达和AsA含量差异的原因之一,且毛花猕猴桃启动子区域183 bp的缺失与高AsA含量有关。Ascorbic acid(AsA) plays an important role in plant growth, development and stress resistance regulation. The main synthesis pathway of AsA is l-galactose pathway, and GDP-l-galactose phosphorylase(GGP) is the key rate-limiting enzyme for synthesis of AsA. In order to explore the effect of GGP gene on ascorbic acid content in kiwifruit(Actinidia spp.), A. eriantha and A. rufa with large difference in AsA content were taken as the research objects, for the analysis of the relationship between GGP1 expression differences, GGP1 promoter sequence variation and AsA content in kiwifruit. The results show that the expression level of GGP1 in A. eriantha fruits and leaves were significantly higher than those of A. rufa, and the expression level of GGP1 was positively correlated with AsA content. Two haplotypes of GGP1 promoters of A. eriantha and A. rufa were obtained by cloning, namely AeGGPp1, AeGGPp2, ArGGPp1, and ArGGPp2, which have multiple single nucleotide polymorphism(SNP) or insertion-deletion(InDel). Meanwhile, a 183 bp deletion around 670 bp in AeGGP promoter of A. eriantha was observed, which is significantly different from promoter of A. rufa. The transcriptional activity analysis of the promoter show that β-glucuronidase(GUS) activities driven by AeGGPp1 and AeGGPp2 promoters of A. eriantha were significantly higher than those of ArGGPp1 and ArGGPp2 promoters of A. rufa, but there was no significant difference between the haplotypes of the promoters of the same species. Based on the 183 bp fragment deletion we designed marker primers, and the hybrid F;(A. rufa × A. eriantha) was found to contain a haplotype of GGP1 promoter of A. rufa and A. eriantha, which showed independent allocation. Beisdes, AsA content of the hybrid F;fruits was mainly near the average value of their parents [5.06–9.40 mg·g;(FW)],which showed higher AsA content than those of most kiwifruit resources. In addition, high AsA content resources containing GGP1 promoter haplotype of A. eriantha was observed among 16 kiwifruit species.T

关 键 词：猕猴桃 抗坏血酸 GGP 启动子 序列变异 

分 类 号：S663.4[农业科学—果树学]