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

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.

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。

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，而种植的甜叶菊品种RA含量不到叶片干重10％，有必要挖掘利用RA生物合成相关基因提高其含量。前期研究表明，RA生物合成相关基因核苷酸序列存在多态性位点，有些多态性位点构成的单倍型在RA含量高材料中特异性高表达。本研究项目以甜菊糖苷总量及RA的绝对和相对含量不同甜叶菊种质为材料，以已登录KA13H和UGT76G1基因编码序列为参照，转录组分析调查了甜菊糖苷总量及RA的绝对和相对含量不同甜叶菊种质间KA13H和UGT76G1基因编码序列的SNPs及其所构成的单倍型多样性，研究了单倍型种类调查中所获各单倍型与甜叶菊叶片中甜菊糖苷总量及RA的绝对和相对含量间相关性，并分析了其编码蛋白催化特性。项目的执行获得了如下结果：1）建立了基于转录组测序数据的材料间差异表达单倍型综合分析系统；2）依据高RA和高STV材料间差异表达单倍型，建立了高RA材料分子检测系统，促进了在高RA甜叶菊育种；3）建立了甜叶菊种质甜菊糖苷特性高效检测技术体系，促进了甜叶菊种质综合评价体系的建立；4）明确了甜叶菊繁育特性，依此提出并发展了基于液体授粉的甜叶菊定向杂交制种技术，有力促进了我国甜叶菊产业升级；5）建立了基于液质联用的化学形态学研究体系，据此实现了甜叶菊全组分分析系统，为促进以甜叶菊为代表的植物全组分综合评价、目标代谢物育种亲本选择和栽培调控体系的建立提供了依据；6）育成高RA、高STV、高RC、高RD、高RM等甜叶菊新品系近50份，新增保存甜叶菊种质近500份； 7）明确了UGT76G1-M1在促进STV转化形成RA中的作用，提出并发展了甜叶菊青贮体系，促进了甜叶菊原料叶生产及调制环节的升级。