小麦植酸代谢相关基因的克隆及功能研究

Phytic acid is the major storage form of phosphorus in cereal grains. The existence of phytic acid in cereal grains will cause environmental phosphorus pollution and reduce bioavailability of micronutrients,therefore low phytic acid breeding and researches on phytic acid biosynthesis in plants have aroused wide attention in recent years. At present, researchers know relatively little about the process of wheat phytic acid biosynthesis, only one gene TaMIPS has been reported. This project aims to clone four wheat genes which are homologous to the known rice genes involved in phytic acid biosynthesis by homologous cloning method. Gene structure and expression pattern of the four genes were analyzed by comparing between genomic DNA and cDNA sequence and quantitative real-time polymerase chain reaction; then the gene which is outstanding up-regulation in seeds will be chosen as target gene, seed-specific gene silencing technology will be used to culture low phytic acid wheat. To evaluate the target gene in cultivating of low phytic acid wheat, the expression level of target gene, phytic acid content of seeds and the main agronomic traits will be compared between transgenic and non-transgenic plants. The research results will greatly enrich the knowledge about structure and function of genes involved in wheat phytic acid bioysynthsis, and provide theoretical and technical basis for breeding of low phytic acid wheat .

植酸是禾谷科作物种子内磷的主要储存形式，其存在易造成环境磷污染和人类微量元素缺乏症，因此低植酸作物培育和植物植酸代谢途径研究成为近年来的研究热点。目前，对小麦植酸代谢途径了解还十分有限，已报道的基因仅有TaMIPS。本项目拟选择4个水稻植酸代谢相关基因，采用同源克隆的方法获得小麦同源基因；并通过基因组扩增、荧光定量RT-PCR分析4个基因的结构和组织时空表达特性；选择在种子内显著表达上调的基因作为靶基因，采用种子特异性基因沉默技术培育低植酸小麦，通过对低植酸转基因小麦种子内靶基因表达水平、植酸含量和主要农艺性状分析，评价相应基因在培育低植酸小麦中的应用价值。研究结果将大幅提升对小麦植酸代谢相关基因结构和功能的认识，并为小麦植酸育种提供理论和技术基础。

植酸是植物体内磷的主要储存形式，在禾谷类、豆科作物中广泛存在，是植物体内的主要抗营养因子。有关小麦植酸代谢途径的研究仅有较少报道。本项目以水稻植酸代谢相关基因作参考，通过电子克隆和PCR扩增的方法获得TaMIK、Ta2-PGK、TaIPK1、TaMRP5 四个小麦植酸代谢相关基因，进一步分析它们基因组序列明确了基因结构，与水稻、玉米、拟南芥同源基因比较，它们在基因结构和序列上都具有较高的保守性。实时荧光定量RT-PCR分析显示，克隆的4个植酸代谢相关基因在幼苗时期的根茎叶和发育种子内都有表达，但表达模式并不相同，其中在胚内的表达强度明显高于胚乳。为明确四个小麦植酸代谢相关基因的功能和培育低植酸小麦，项目应用种子特异性启动子（Em）和hpRNAi基因沉默技术构建了4个基因的基因沉默表达载体，通过农杆菌介导的成熟胚转化法转化小麦，分别获得了独立转基因株系，通过PCR扩增检测除草剂抗性基因Bar和半粒种子无机磷检测法，最终每个载体都获得了低植酸转基因植株，下一步将对它们的它们的种子磷组分、农艺性状、分子表征进行分析。项目对阐明小麦植酸生物合成代谢过程具有重要的意义，为小麦低植酸育种提供了科学理论依据。