毛花猕猴桃果实高糖形成机理及合成相关关键基因的功能型标记开发

As the fruit of Actinidia eriantha usually taste sour and astringent, there is no commercial cultivation of A. eriantha so far. The A. eriantha line ‘MM-11’ with 10.72% of the soluble sugar content and 17.07% of SSC was selected as material with high sugar. Another A. eriantha line ‘MM-3’ with 4.06% of the soluble sugar content and 11.33% of SSC, in addition with closer wild geographic location, nearer genetic similarity coefficient (0.79), was selected as contrast. During the fruit development, sugar compound and content of the fruit will be detected by HPLC. The fruit of significant difference in sugar compound and content will be collected to analyze the transcriptome using the PacBio three generation in combination with BGIseq-500 high-throughput sequencing technology. Besides, the sugar synthesis pathway of other species will be referenced to screen differentially expressed candidate genes. One hundred fifty natural A. eriantha lines with differences in sugar compound and content will be selected to conduct the association analysis based on candidate gene. The candidate genes which shows significant effect after association analysis will be assayed the genetic transformation to identify their function. Then, the allele genes of the key genes will be cloned in ‘MM-11’ and ‘MM-3’, and the sequence differences of the allele genes will be analyzed to design markers. After association analysis, the markers of key genes involving sugar accumulation will be verified. This study aims not only to make clear the molecular mechanism of high sugar formation of A. eriantha but also to lay the foundation for the improvement of flavor quality and molecular genetics breeding of A. eriantha fruit.

毛花猕猴桃(Actinidia eriantha Benth)因鲜食口感普遍酸涩，商业化栽培受限。本研究拟以江西境内发掘的可溶性糖10.72%，SSC17.07%的毛花猕猴桃优株‘MM-11’为高糖型试材，以与之野生地理分布位置近、遗传相似性系数高(0.79)、可溶性糖4.06%，SSC11.33%的单株‘MM-3’为对照，先利用HPLC技术检测果实不同发育时期糖组分与含量，再利用PacBio三代结合BGIseq-500技术进行两者糖含量差异显著时期果实的转录组测序分析获得相关差异表达候选基因。选择自然群体中糖含量差异的150份毛花猕猴桃单株进行候选基因关联分析，达显著效应的候选基因将进行遗传转化鉴定功能。关键基因经等位基因克隆后根据序列差异设计标记，经关联分析验证后开发出功能型标记。从而既阐述毛花猕猴桃高糖形成分子机理，亦为毛花猕猴桃风味品质改良和分子遗传育种奠定基础。

毛花猕猴桃(Actinidia eriantha Benth)是我国特有的野生种质，其果实Vc含量高、易剥皮，且具有较强的抗病性和耐热性等特点，但因其果实鲜食口感普遍酸涩，目前商业栽培的主要是中华猕猴桃和美味猕猴桃，生产中应用的毛花猕猴桃品种极少，绝大部分多处于野生状态。江西省是我国猕猴桃重要产区之一，野生资源十分丰富。课题组前期对江西省境内的毛花猕猴桃资源进行普查搜集时发现了高糖型的优株，经异位高接保存与扩繁，其果实高糖性状表现稳定，遂以此为主要试验材料展开了本项目的研究。项目首先以不同发育时期毛花猕猴桃果实以及不同品种（系）毛花猕猴桃成熟期果实为试材，检测分析了糖组分和含量，发现果糖、葡萄糖和蔗糖是毛花猕猴桃果实中主要的糖，不同品种（系）三种糖的含量与占总糖比例差异显著，但高糖型优株这三种糖的含量均较高。对高糖型与低糖型优株成熟果实进行了转录组与代谢组检测分析，发现淀粉水解相关基因 AeamyA、AeamyB等表现出高表达水平，AeamyB在高糖型毛花猕猴桃优株‘M24’中显著上调；蔗糖合成路径中亦有多个基因在‘M24’中高表达且显著上调。遂检测了‘M24’ 13个不同发育时期果实中以及11个不同品种（系）毛花猕猴桃成熟果实中蔗糖合成酶(SS)等5个糖合成相关酶的活性以及等SPS等15个相关基因的表达情况，发现不同品种（系）的野生毛花猕猴桃果实中SS的活性均较高，且其活性在高糖型毛花猕猴桃优株‘M24’果实整个发育期间活性也较高。SPS1和SPS1基因表达除去花后25d这个时期，其变化趋势与糖在‘M24’果实中的动态积累变化较为一致，即前期较低，后期较高；且‘M24’成熟果实中SPS1的表达高于其它品种（系）。根据相关基因的侧翼序列设计了SSR分子标记引物，并在145份野生毛花猕猴桃中进行了验证分析；发现有3个标记与可溶性糖相关联，即UDK96-035、UDK96-039、A193，其中A193与可溶性糖存在极显著相关关联，解释率为13.95%。本项目的结果将为毛花猕猴桃种质资源的创新以及品种的遗传改良工作奠定了良好基础。