空间诱变草地早熟禾矮化突变体矮化机理研究

The one of important approaches to reduce turf mowing frequency and decrease turf management cost is to breed new turfgrass varieties with slowly growth rate and dwarf plants,which is also the emphasis of turfgrass breeding. Traditional breeding methods are the main ways to breed Kentucky bluegrass varieties, and dwarf varieties have not been breeded yet. Genetic engineering can achieve directed breeding and speeding the breeding process, but core of the problem is first clear to key molecular mechanism and regulation mechanism of dwarf feature. The research of dewarf mechanism on crops carried out deeply whilst no researches on turfgrasses. Dwarf mutant is the ideal material to study dwarf mechanism. This project is intended to take advantage of the Kentucky bluegrass dwarf mutant obtained by space mutation, which dwarf feature has be inherited steady, and start with gibberellin regulation, to separate and screen expression differences genes from Kentucky bluegrass dwarf mutant by transcriptiomics and bioinformatics,then test the expression differences genes by real-time quantitative PCR, followed by Western Blotting to verify the obvious expression difference genes on protein level. Completion of this project is contributed to reavel the dwarf mechanism of the Kentucky bluegrass dwarf mutant from molecular and protein level and laid a solid theoretical foundation for breeding Kentucky bluegrass new varieties with growth slowly.

培育生长缓慢、株型低矮的草坪草品种是降低草坪修剪频率、减少管理费用的重要途径之一，也是草坪草育种的重点方向。草地早熟禾育种以传统育种方法为主，目前尚未育成矮化品种。基因工程可以实现定向育种，加快育种进程，但其问题的核心是要先明确矮化性状的关键分子机制和调控机理。矮化机理在农作物上的研究较深入，在草坪草上尚未见报道。矮化突变体是研究矮化机理的理想材料，本项目拟利用通过空间飞行获得的草地早熟禾可稳定遗传的矮化突变体为材料，从赤霉素调控机理入手，应用转录组学和生物信息学等理论和方法，分离和筛选草地早熟禾矮化突变体表达差异基因，采用qRT-PCR方法进行表达差异基因的验证，并通过Western Blotting对表达差异较大的基因进行蛋白水平验证。本项目的完成有助于从分子与蛋白水平上更深层次揭示草地早熟禾矮化突变体矮化表型形成的机制，为进一步培育生长缓慢的草地早熟禾新品种奠定坚实的应用理论基础。

培育生长缓慢、株型低矮的草坪草品种是降低草坪修剪频率、减少管理费用的重要途径之一，也是草坪草育种的重点方向。草地早熟禾育种以传统育种方法为主，目前尚未育成矮化品种。基因工程可以实现定向育种目标，加快育种进程，但其问题的核心是要首先明确矮化性状的关键分子机制和调控机理。矮化机理在农作物上的研究较深入，在草坪草上尚未见报道。矮化突变体是研究矮化机理的理想材料。本项目利用空间飞行诱变获得的草地早熟禾可稳定遗传的矮化突变体为材料，从赤霉素调控机理入手，应用转录组学和生物信息学等理论和方法，分离和筛选草地早熟禾矮化突变体差异表达基因，并通过实时荧光定量和Western blotting等方法对差异表达基因进行验证。内源GA含量测定及外源GA喷施结果表明，矮化突变体GA合成途径受阻，导致内源GA含量降低。转录组测序数据库的生物信息学分析表明，野生型植株与矮化突变体A12之间的差异表达基因为883个，而与A16之间的差异表达基因达到4203个。这些差异表达基因主要涉及到萜类化合物合成、植物激素合成与传导、叶绿素代谢及类黄酮合成等。本研究确定了草地早熟禾空间诱变矮化突变体在GA合成途径上的基因发生了变异，GA合成受阻，导致矮化表型的发生，并明确了与矮化突变体矮化机理相关的GA合成途径几个关键基因如PpGA2OX5、PpGA3OX1、PpGA20OX1等，为利用基因工程技术培育草地早熟禾矮化新品种奠定了基础。