基于RNAi技术的杏自交不亲和S-基因沉默表达效果研究

Xinjiang is the one of important apricot origin areas in the world and the central Asia group apricots in South Xinjiang are wealth of genetic diversity and specific and excellent local varieties. But more than 200 apricot cultivars (or types) in Xinjiang exist outstanding problems of slow and unstable production originating in self-incompatibility, which lead to high cost pollination and time-consuming and laborious in commercial apricot cultivation. Our Research team will explore to improve self-incompatible apricot cultivars according to the characteristics of self-compatible mutations known by us for fruits in the Rosaceous Prunus. We aim at providing strategy for breeding self-compatible apricot cultivars. The present study will clone compete cDNA sequence of S-RNase gene and SFB gene at S-locus associated with self-incompatibility in apricot cultivar'Saimaiti'. We plan to design the siRNA aimed at compete cDNA sequence of S-RNase gene and SFB gene at S-locus and construct the hpRNA vector, then transfer apricots and test its efficacy in silencing S-RNase gene and SFB gene. We make an attempt to exploit dsRNA with promoter to effect S-RNase gene and SFB gene expression silencing and further explore the mechanism of self-incompatibility and provide molecular basis for breeding self-compatible cultivars. So the present study has theoretical significance and application prospect.

新疆是世界杏重要的起源中心之一，分布于南疆的中亚生态品种群有许多特异优良的地方品种，但新疆杏资源200多个类型（品种）中，多数自交不亲和、产量低而不稳，由此导致新疆杏商业化生产中授粉成本高且费时费力。本研究团队通过对蔷薇科李属果树亲和突变原因的认识，试图探索借助亲和突变特性改良杏自交不亲和的栽培品种，为未来亲和品种的选育提供可借鉴的育种策略。本研究拟克隆南疆主栽杏品种赛买提的自交不亲和S-位点的S-RNase基因和SFB基因的全长cDNA序列，利用S-RNase基因和SFB基因的cDNA序列设计siRNA，构建其hpRNA介导载体，转化杏，进行基因表达分析后，探索用含有启动子区的dsRNA影响S-RNase基因和SFB基因表达沉默，同时可进一步探索自交不亲和机制，为培育自交亲和品种提供分子研究基础。因此，本项目的开展具有理论意义和应用前景。

新疆是世界杏重要的起源中心之一，分布于南疆的中亚生态品种群有许多特异优良的地方品种，但新疆杏资源 200 多个类型（品种）中，多数自交不亲和、产量低而不稳，由此导致新疆杏商业化生产中授粉成本高且费时费力。本研究团队通过对蔷薇科李属果树亲和突变原因的认识，试图探索借助亲和突变特性改良自交不亲和的南疆主要栽培杏品种，为未来亲和品种的选育提供可借鉴的育种策略。目前已经完成南疆主栽杏品种赛买提自交不亲和S-位点的S-RNase和SFB基因的全长克隆；获得库车小白杏S-RNase和SFB基因全长序列， 构建了小白杏自交不亲和SFB基因和S-RNase基因正义、反义RNA表达载体并转化进入大肠杆菌；构建了自交不亲和SFB基因和S-RNase基因的RNAi表达载体并转化进入大肠杆菌；分别将RNAi表达载体利用叶盘转化法转化至烟草，成功获得了转基因烟草，同时利用花粉管介导法转化至‘小白杏’中，自交座果率有一定提高。建立了轮台小白杏茎段的无菌培养体系，并以段诱导再生的叶片切块为外植体，建立了诱导脱分化形成愈伤组织，及再分化不定芽的配方；建立了轮台小白杏种胚诱导愈伤组织及不定芽的培养体系。本研究为杏自交不亲和基因RNAi表达载体的遗传转化奠定了研究基础，对培育自交亲和杏品种、对其他蔷薇科果树自交不亲和性状的改良具有重要的价值。