荞麦属植物籽粒Waxy酶活性调控途径的发掘及育种应用

Buckwheat is one of the main minor coarse cereals originated in China, and it plays important roles in Guizhou province. Starch accounted for about 70% of the buckwheat seed storage materials. The amylose, one of the main components of starch, synthesized by Waxy, is one of the key factors to buckwheat nutrition, total yield and processing quality. However, there's still unclear about control factors on the enzyme activity. In order to find these possible influencing factors of Waxy enzyme activity and provide more details for Waxy regulation, capillary electrophoresis and high performance size exclusion chromatography identification systems were used to scanning the collected materials, including common buckwheat, tartary buckwheat and their wild relatives in the present study. The results could provide the basic information about Chinese buckwheat germplasm based on the following key parameters: the content and degree of polymerization of seed amylose, the activity and possible types of Waxy enzymes. Gene cloning, promoter sequence analysis and isolating of alternative splicing of mRNAs were used to character these core germplasm with extreme Waxy enzyme activity. In order to find the possible reasons on the levels of -omics, transcriptome and proteome analysis were carried out using the selected two materials from RILs populations during their grain filling period. The integration algorithm based on optimal Steiner tree will be used to construct the regulation networks among transcriptome and proteomics data, combined with the molecular interaction data and the key candidate factors which affects Waxy activity will be verify at the transcriptional level. These results will provide theoretical basis for the efficient utilization of buckwheat Waxy germplasm and amylose molecular breeding.

荞麦是起源于我国的小杂粮之一，也是贵州的特色作物。淀粉占荞麦籽粒贮藏物质的70%左右，其中的直链淀粉则是营养、产量及加工品质的基本构成要素，由Waxy酶活决定，而有关该酶活的控制因素至今尚无定论。项目拟以晋荞2号、甜荞Homo及金荞麦为模式，构建出荞麦籽粒直链淀粉含量的毛细管(CE)鉴定体系、直链淀粉聚合度的高效排阻色谱鉴定体系、Waxy亚基类型及含量的CE鉴定体系，并基于上述体系对800余份荞麦种质、RILs群体及三体系进行扫描，获得我国荞麦直链淀粉合成的关键指纹数据；以具有极端Waxy酶活差异的种质为材料，克隆其Waxy基因、上游启动子区及潜在的可变剪接体；同时，对具有极端Waxy酶活差异的RILs单株进行灌浆期转录组及差异蛋白组测序；采用基于最优斯坦纳树整合算法发掘并标记Waxy酶活的关键候选因子，并在转录水平进行表达验证。研究将为荞麦Waxy种质的利用及直链淀粉的分子育种提供参考。

荞麦属植物具有类似于禾谷类的淀粉籽粒而被广泛食用，同时富含芦丁、槲皮素等次生代谢物而被药用。栽培荞麦在我国的重要小杂粮之一，也是贵州省的特色作物。籽粒淀粉占种子贮藏物质的70%左右，而其中的直链淀粉则是荞麦营养、产量及加工品质的核心构成要素，由Waxy酶专一控制。项目以贵州省荞麦工程技术研究中心收集的500余份种质为材料，通过SDS-PAGE电泳法、2-DE、MALDI-TOF MS/MS、基因组de novo拼接产物及转录组RNA-Seq数据，获得了荞麦直链淀粉编码基因的序列特征，获得了该基因的有效标记，构建了针对该酶的ＣＥ鉴定体系，初步揭示了该基因在其他作物的同源基因；此外，项目构建了基于酶标仪的直链淀粉微量快速鉴定体系，构建了适合于室内栽培的种植装置，构建了基于整合数据库的直链淀粉研究平台，并创制了一批不同直链淀粉含量的荞麦种质。项目研究结果为荞麦直链淀粉育种应用提供基础。