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

米糠由于其高含量的脂肪、蛋白质和粗纤维而可能是一种有价值的营养来源，但其用于人类消费的用途受到米糠油的快速降解和比其他植物油更快的水解酸败发展的严重限制。三酰甘油（TAG）是米糠中含有的主要脂质，存在于具有磷脂膜的油体中。这些膜被磷脂酶D（PLD）分解，释放的TAG被脂肪酶降解为游离脂肪酸，导致麸皮质量差。由于PLD是启动脂质降解和麸皮品质劣化的触发器，我们利用抗PLD多克隆抗体分离到PLD缺失的水稻突变体“03-s108”。尽管PLD缺失表型作为单一隐性性状遗传，但是PLD缺失菌株的筛选方法涉及消耗时间和精力的后代测试的蛋白质印迹。为了建立一种简便的PLD缺失菌株检测方法，我们利用“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缺失表型定位在第1染色体RM 3453附近，该位点携带一个推测的PLD基因在DNA测序分析中，AB 001920的“03-s108”等位基因在第三外显子上显示从G到A的过渡突变。这种变化导致了一个终止密码子，意味着“03-s108”等位基因中的无义突变。利用这些DNA序列数据，我们开发了切割扩增多态性序列和斑点杂交-单核苷酸多态性分析，用于筛选和培育PLD缺失水稻品种。这些方法对于筛选PLD缺失菌株是简单且成本有效的。

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.