OsIPCS基因在水稻生长发育和逆境适应中的生物学功能研究

Inositolphosphorylceramide synthase (IPCS) is a key enzyme in the metabolic pathway of sphingolipids, which plays significant roles in organism growth and adversity adaptation. In this project, three OsIPCS genes have been cloned and over-expression, silencing-expression or loss-expression plasmids for these OsIPCS genes, which will be transformed into japonica rice, have been or will be constructed using techniques such as CRISPR. To identify the phenotypic functions and the interaction of the three OsIPCS genes, comparison of the phenotypic differences will be performed between wild-type and homozygous transgenic plants under stress treated and untreated conditions. To further clarify the specific tissue expression characteristics of the three OsIPCS genes, the relative expression level of each of the three genes in roots, stems, leaves and panicles of the wild-type and homozygous transgenic plants under stress treated and untreated conditions will be tested using Quantitative Real-Time PCR. The contents of ceramide and OsIPCS enzyme activities in specific tissues of the wild-type and homozygous transgenic plants under stress treated and untreated conditions will then be analyzed to reveal the physiological and biochemical functions of the three OsIPCS genes. Investigation of the putative metabolic pathways involving the 3 OsIPCS genes will be followed through identification of relevant up-regulated or down-regulated genes in specific tissues of wild-type and homozygous transgenic rice lines under stress treated and untreated conditions using transcriptome sequencing and Quantitative Real-time PCR. Furthermore, fusion expression vectors carrying YFP gene and the three OsIPCS genes will be constructed and transformed into rice protoplasts to determine the subcellular localization of the proteins encoded by these OsIPCS genes. Moreover, the yeast expression vectors for the three OsIPCS genes will also be constructed and introduced into the yeast aur1∆ mutant to further test the functions and enzyme activities of the three OsIPCS proteins. These studies will provide useful reference for further studies on the biological functions of the three OsIPCS genes during the plant growth and adversity adaptation in rice, and lay foundation for molecular breeding in crops.

肌醇磷酸神经酰胺合酶（IPCS）是鞘脂代谢途径中的关键酶，对生物正常生长发育和逆境适应意义重大。本项目拟克隆3个水稻OsIPCS基因，利用CRISPR等技术构建其过量、下调和缺失表达载体，转化粳稻后，比较正常和逆境下野生型植株和转基因纯合植株的表型差异，确定其表型功能及其相互间的关系；荧光定量PCR检测它们在水稻根、茎、叶和穗中的表达，确定其组织表达特性；通过检测特定组织中的神经酰胺含量、OsIPCS活性，确定其生化功能；利用转录组测序等方法，获得正常与逆境下野生型和转基因纯合水稻特定组织中表达显著上/下调的基因，确定OsIPCS基因功能发挥涉及的调控途径。另外，还将对OsIPCS蛋白在酵母中表达的功能特性进行分析，对其在水稻原生质体中的表达进行亚细胞定位。为深入剖析这3个水稻OsIPCS基因在生物学功能上的相互关系，及其生物学功能发挥涉及的遗传途径奠定基础，也为作物分子育种提供依据。

IPCS（肌醇磷酸神经酰胺合酶）是鞘脂代谢途径中的关键酶，但植物中IPCS的表型功能及其作用机制研究鲜有报道。我们克隆了3个水稻OsIPCS基因，明确了它们的时空表达模式和蛋白亚细胞定位情况，发现它们对多种非生物胁迫均具有显著的响应，暗示OsIPCSs可能在水稻逆境适应中具有作用，但有待证实。重点创制了3个OsIPCS基因的单敲、双敲和三敲缺失表达突变体，表型观测和细胞学分析结果表明，OsIPCSs的缺失表达可能引起了水稻茎秆等细胞变短，导致突变体各节间长度以及穗长变短，使得突变体的株高降低，OsIPCSs在影响株高的过程中表现出功能冗余现象，效应大小可能是OsIPCS3>OsIPCS2>OsIPCS1。通过转录组和代谢组测序分析，获得了许多与水稻株高调控相关的差异表达基因和脂质代谢物，发现OsIPCS基因可能参与了糖基磷脂酰肌醇-锚生物合成和磷脂酰肌醇代谢途径。初步揭示了水稻OsIPCSs的表型功能，为深入理解水稻生长发育机理提供了全新的知识。目前已在Plant Mol Biol等期刊上发表了标注本项目基金号资助的学术论文5篇，资助硕士生8名和博士生1名。