控制花生种子油脂合成的甘油-3-磷酸酰基转移酶基因等位变异和功能研究

Peanut (Arachis hypogaea L.) is an important crop worldwide,being mostly used for edible oil production. There are significant difference in kernel oil contents(32.35%-60.21%）among the peanut germplasms. Searching for the key enzyme genes in oil biosynthesis is the basis of the high oil peanut molecular breeding.Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the fist step in the production of triacylglycerols (TAGs), which are major components of seed storage oils. GPAT genes are important in oil biosynthesis. In this project, we expect to reveal the molecular characteristics of GPAT genes and investigate their polymorphism by cloning GPAT genes from different biological types cultivated peanuts(AABB)with different kernel oil content ,and diploid wild species(AA or BB) of section Arachis.The gene sequence polymorphisms will be analyzed associated to the oil content in 169 cultivated peanut accessions for screening efficient genes, and their specific molecular markers. We will analysis the relationship between gene expression and oil content among the peanut germplasms with different oil content, but their GPAT genes coding regions having the same structure,for screening high oil germplasms having the favorable alleles by non-coding region variation. Research on the effects of overexpression GPAT gene on improving the kernel oil content in transgenic peanut plants. The project's goal is revealing the mechanism of regulation of kernel oil content by GPAT genes, and providing gene resources and functional molecular marker, and new germplasms, for high oil molecular breeding in peanut.

花生是重要的油料作物，种质间籽仁含油量差异显著（32.35%-60.21%），挖掘促进油脂合成的关键酶基因是花生高油分子育种的基础。甘油-3-磷酸酰基转移酶（GPAT）催化植物油脂主要成分三脂酰甘油(TAGs)合成的起始反应，是TAGs合成的关键酶,直接影响种子油脂积累。本研究首先从花生区组二倍体野生种（AA或BB）及栽培种（AABB）分离GPAT基因，探明A、B染色体组基因序列的差异。利用169份栽培种种质组成的自然群体进行基因序列多态性与含油量关联分析，发掘促进油脂合成的等位变异，研发其特异分子标记。并检测含油量差异显著、等位基因编码区相同种质间基因转录水平差异与含油量的关系，筛选该基因非编码区存在有利变异的高油种质。利用转基因技术研究GPAT基因过量表达对提高种子含油量的作用。揭示GPAT基因对花生种子含油量调控的分子基础，为花生高油分子育种储备基因资源、功能性分子标记和新种质。

花生是重要的油料作物和经济作物，挖掘促进油脂合成关键酶基因是花生高油分子育种的基础。甘油-3-磷酸酰基转移酶（GPAT）催化植物油脂主要成分三脂酰甘油(TAGs)合成的起始反应，是 TAGs 合成的关键酶。本项目利用花生栽培种的171份种质为材料，检测其种子的含油量、AhGPAT9-A和AhGPAT9-B 核苷酸序列、及其不同发育时期种子基因表达量，进行等位基因序列多态性与基因表达、含油量的关联分析。同时，构建AhGPAT9基因的植物表达载体，进行遗传转化研究。主要研究结果如下：.AhGPAT9-A DNA全长5538 bp，包含13个外显子和12个内含子，cDNA全长1533 bp， CDS长1131 bp，编码376个aa, 5′UTR长238 bp，3′UTR长163 bp；AhGPAT9-A 和AhGPAT9-B 的DNA序列一致性是90.96%，二者存在155个差异位点,编码蛋白有一个aa的差异。.检测到 AhGPAT9-A多态性位点118个，与表达量显著关联位点7个，与含油量显著关联位点5个，高油种质G82（含油量56.70%）包含3个增效位点；氨基酸改变位点13个，变异种质14份，种质G14变异aa位于酰基转移酶结构域内，效应值为5.69%；种质G44包含2个变异位点，效应值为-7.25%。.检测到AhGPAT9-B基因多态性位点94个，与表达量极显著关联位点20个，与含油量显著关联位点19个，变异效应解释率为2.38～6.19%。氨基酸改变位点6个，变异种质6份，种质G111的多态性位点效应值最高，为6.42 %。.种质G168（a1a1b54b54）和G167（a1a1b51b51）含油量分别比野生基因型（a1a1b1b1）均值降低12.81%和11.69%，G181（a1a1b57b57）和G154（a1a1b40b40）分别比野生基因型（a1a1b1b1）均值增加13.44%和8.85%。.AhGPAT9-B在种子中表达量高于AhGPAT9-A。种质含油量与基因表达模式间无明显相关，相同表达模式的种质间R5期基因表达量与含油量呈正相关。.利用农杆菌介导途径进行遗传转化，获得AhGPAT9 正义和反义的移栽成活正常结果T0代转基因植株8株。初步分析表明AhGPAT9正义和反义转基因材料含油量均有降低趋势。