基于转录组测序的甜叶菊Rebaudioside A生物合成关键基因优异单倍型挖掘

项目名称： 基于转录组测序的甜叶菊Rebaudioside A生物合成关键基因优异单倍型挖掘

项目编号： No.31471553

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 食品科学、农学基础与作物学

项目作者： 罗庆云

作者单位： 南京农业大学

项目金额： 82万元

中文摘要： RA(Rebaudioside A)为甜叶菊内30多种甜菊糖苷之一，口味纯正、甜度高、低热安全，广泛应用于食品和饮料中。甜叶菊叶片甜菊糖苷总量可达叶片干重20%以上，而种植品种中RA含量不到叶片干重10％，有必要利用RA生物合成相关基因来促进甜菊糖苷向RA转化，提高其含量。前期研究结果表明，RA生物合成相关基因核苷酸序列上存在多态性位点，有些多态性位点构成的单倍型在高RA含量材料中特异性高表达。为促进相关基因优异单倍型在高RA甜叶菊育种中的应用，本研究以KA13H 和UGT76G1编码序列为对象，转录组分析为手段，在其单倍型多样性调查基础上，分析各单倍型表达水平与甜菊糖苷总量及RA含量间的相关性，获得相关性强的单倍型，在利用RNAi验证其作用的基础上分析其编码蛋白催化特性，明确影响KA13H和UGT76G1催化能力关键位点，阐明调控机制，获得优异单倍型，为高RA甜叶菊分子育种提供依据。

中文关键词： 甜叶菊；莱鲍迪苷A；单倍型；转录组测序

英文摘要： RA(Rebaudioside A) , one of the 30 kind of steviol glycosides (SGs) present in leaf of stevia[Stevia rebuaidana (Bertoni)], due to its pure taste, sweetness and low-calorie, was widely used in food and beverages.There is research result showed that stevia can accumulates SGs up to more than 20% of dry weight in the leaves,after selection breeding for more than 40 years, the content of RA has just increased to less than 10% of the dry weight in leaves, and it is necessary for us to exploit the biosynthesis genes of RA for breeding to increase its content in the leaves. Up to date, all but one gene involved in biosyntheses of RA have been cloned. Our preliminary investigation results showed that there were many polymorphic loci presence on the nucleotide sequence of RA biosynthetic relevant genes, and some haplotypes contain these polymorphic loci were specificly highly expressed in leaves of which characterized of higher content of RA than those of STV. In this study programe, functional elite haplotypes of genes involved in RA biosyntheses will be exploited. Firstly,data of stevia transcriptome were be used to survey the diversity of haplotypes of genes cording KA13H and UGT76G1,two key enzymes in RA biosyntheses. Secondly, correlation between the expressional levels of each haplotypes and the contents of steviosides and RA in leaves will be surveyed to find out the highly related candidate haplotypes. Thirdly, after survey the function of the candidate haplotypes in the biosynthesis of RA by RNAi systems, the catalytic charaterization of enzymes encoded by the haplotypes will be surveyed to find out the key sites those influence the catalytic activate of UGT76G1 and KA13H, and discover the regulatory mechanism of UGT76G1 and KA13H. Finally, obtain the elite haplotypes for breeding of highly RA content stevia.

英文关键词： Stevia;Rebaudioside A;haplotype;RNA-seq