Identification of rice seed phospholipase D null gene and characterization of seed phospholipase D null rice

米糠油的脂肪、蛋白质和粗纤维含量高，是一种有价值的营养来源，但米糠油的快速降解和水解性酸败的发展速度比其他植物油更快，严重限制了米糠油的人类消费。三酰甘油(TAG)是米糠中的主要脂类，存在于有磷脂膜的油体中。这些膜被磷脂酶D(PLD)分解，释放的标签被脂肪酶降解成游离脂肪酸，导致麸皮质量变差。由于PLD是启动脂肪降解和麸皮品质恶化的触发物，我们用抗PLD的多克隆抗体分离到一个PLD缺失的水稻突变体“03-s108”。虽然PLD缺失型是作为单个隐性性状遗传的，但筛选PLD缺失型菌株的方法需要花费时间和精力的蛋白质印迹杂交和子代测试。为了建立一种简便的PLD缺失菌株的检测方法，我们利用来自“03-s108”和“Koshihikari”的F2代进行了PLD缺失表型与SSR标记的连锁分析，并将PLD缺失表型定位在1号染色体RM3453附近，携带一个可能的PLDS基因(AB001920)。在DNA测序分析中，AB001920的“03-s108”等位基因在第三外显子上显示了从G到A的转换突变。这一改变导致了一个终止密码子，意味着“03-s108”等位基因的无义突变。利用这些DNA序列数据，我们建立了裂解扩增多态序列和斑点印迹-单核苷酸多态分析，用于筛选和选育PLD缺失水稻品种。这些方法是筛选PLD缺失菌株的简便和经济有效的方法。

Rice bran could be a valuable source of nutrition because of its high content of fat, protein, and crude fiber, but its use for human consumption is severely limited by the rapid degradation of rice bran oil and more rapid development of hydrolytic rancidity than occurs in other vegetable oils. Triacylglycerols (TAGs), which are the primary lipids contained in rice bran, occur in oil bodies with phospholipid membranes. These membranes are disintegrated by phospholipase D (PLD) and the released TAGs are degraded by lipase to free fatty acids, resulting in poor bran quality. Since PLD serves as a trigger to initiate the lipid degradation and the deterioration of bran quality, we isolated a PLD-null rice mutant, "03-s108" by using anti-PLD polyclonal antibodies. Although PLD null phenotype is inherited as a single recessive trait, the screening method for PLD null strains involves western blotting with progeny test that consumes time and effort. To develop a convenient PLD null strain detection method, we performed a linkage analysis between PLD null phenotype and SSR markers using F2 progeny from "03-s108" and "Koshihikari" and mapped PLD null phenotype near RM3453 on chromosome 1 carrying one putative PLDs genes (AB001920).In the DNA sequencing analysis, the "03-s108" allele of AB001920 showed a transition mutation from G to A on the third exon. This change resulted in a stop codon, implying a nonsense mutation in the "03-s108" allele. Using this DNA sequence data, we developed cleaved amplified polymorphic sequence and dot-blot-single nucleotide polymorphism analyses for screening and breeding of PLD null rice varieties. These methods are easy and cost-effective for screening PLD null strains.