玉米des1突变体粉质胚乳形成的分子机理研究

It has been a long history for the research of maize opaque or floury mutants. To date, most of the characterized opaque or floury mutants are associated with zein proteins. The fatty acid metabolism in plant seed is quite active. Fatty acid metabolism produced an intermediate product called phytanic acid. Defective in phytanic acid degradation in human resulted in phytanic acid over accumulation and caused multiply disorders in different organs, which is known as Refsum disease. Abnormal phytanic acid metabolism pathway has not reported in plant. In previous research, we obtained an opaque mutant with defective endosperm and seedling (des1). Molecular cloning revealed that Des1 was located within a 260-kb interval in the short arm of chromosome 8 with four candidate genes. One of the four genes, encoding a 2-hydroxyphytanoyl-CoA lyase, a key enzyme of phytanic acid metabolism pathway, was down regulated in des1 mutant. In this proposal, we will first confirm the casual gene of des1, and perform detailed observations on des1 endosperm using multiply cytological methods, and measure the zein proteins and starch contents and the expression of related genes quantitatively, analysis the contents of phytanic acid and other intermediate products. We also want to determine the Des1 expression pattern, the sub-cellular localization and biochemical function of DES1 protein. therefore, this project should promote our understanding of the effects of phytanic acid over accumulation on opaque endosperm.

玉米粉质（opaque或floury）的突变已有几十年的研究历史，目前已知的绝大多数粉质突变都与醇溶蛋白相关。植物种子中的脂肪酸代谢非常活跃。植烷酸脂肪酸代谢的中间产物。人体中植烷酸分解异常、过多累积会导致许多器官发生病变，医学上称为雷夫叙姆病。目前植物中还未有植烷酸代谢异常的报道。我们在前期研究中筛选到一个粉质胚乳的突变体des1，图位克隆将Des1定位到8号染色体短臂的260kb区间，包含四个编码基因，其中一个编码植烷酸分解代谢关键酶（植烷羟基辅酶A裂解酶）的编码基因表达明显下降。本申请将构建等位突变验证Des1基因；采用多种细胞生物学手段对des1胚乳进行详细观察、对胚乳组成成分和不同组织植烷酸及中间产物的累积进行分析；确定Des1基因的表达模式和编码蛋白的亚细胞定位和生化功能；因此，本项目的完成将有助于推进我们对植烷酸累积如何引物粉质胚乳的认识。

小分子有机酸草酸广泛存在于微生物、动物和植物体内，体内过量的草酸积累对细胞生长有害，还会降低某些蔬菜的营养品质。然而，植物体内草酸代谢途径的关键酶我们目前还尚未完全知晓。本项目从一个粉质胚乳突变体出发，基因克隆发现其突变基因编码草酰辅酶A脱羧酶1oxalyl-CoA decarboxylase1 (EC4.1.1.8; OCD1)。Ocd1基因可以特异的被草酸诱导，表达广泛，在玉米的多个组织均能检测到其表达，ocd1突变体种子中的草酸含量比野生型高，说明该突变体的草酸代谢途径出现异常。玉米经典突变体o7是最早发现的高赖氨酸突变体之一，O7基因已经被克隆了，但其具体生化功能还未知。O7在拟南芥中的同源蛋白编码草酰辅酶A合成酶（oxalyl-CoA synthetase ；EC 6.2.1.8), 能够催化草酸和辅酶A形成草酰辅酶A。体外体内的酶活实验证明 OCD1能后催化O7蛋白的产物草酰辅酶A经过脱羧作用形成甲酰辅酶A和二氧化碳。Ocd1基因突变还引起了玉米胚乳多个代谢途径的显著改变。该项目的完成表明玉米OCD1蛋白作用于O7的下游，催化草酸降解途径，影响胚乳发育、代谢和营养品质。项目相关研究申请人作为第一作者发表于The Plant Cell。