调控小麦籽粒硬度的分子遗传基础及其相关基因的功能分析

The Chinese wheat cultivars from different periods and elite germplasms recently introduced from other countries are used to identify SKCS hardness,Puroindoline alleles and Pinb-2 variants as well as novel alleles or variants by some techniques of gene cloning, molecular marker and bioinformatics etc.Interaction relationships of PINA, PINB and 4 PINB-2 proteins are examined by Yeast-two-hybrid. Type I and II more recently identified in Chinese wheat germplasms are characterized at the DNA level and expression level, and potential genes with interaction to Puroindoline in Type I are screened by Yeast-two-hybrid and will be confirmed by Co-immunoprecipitation. Molecular characterization of unknown PINA-null genotypes are identified by the Primer Walking strategy and functional molecular marker spanning deletions will be developed for identification of PINA-null cultivars at the DNA level. Pinb-2 variants in common wheat are silenced by RNA interference and function of Pinb-2 will be illustrated by analysis of phenotypes of transgenic plants. Meanwhile, copy numbers of Pinb-2 are investigated in diploid, tetraploid and hexaploid wheat cultivars by Southern blot and real-time quantitative PCR. In additon, the identified novel alleles or variants encompassing Type I and Type II will be used to analyze their influence on wheat milling and processing quality by taking advantage of wheat populations, and relatively superior alleles and variants will be crossed into current popular bread wheat cultivars for improvement of wheat quality.

以我国不同时期小麦品种和国外引进优异种质资源为材料，进行SKCS籽粒硬度表型、Puroindoline基因型和Pinb-2基因类型的鉴定和新基因挖掘。采用酵母双杂技术对小麦及其近缘种属中的Pina和Pinb基因间以及4种Pinb-2基因间的互作关系进行研究，并分析前期研究中发现的类型I和类型II分子特征和表达量，筛选类型I中与 Puroindoline互作的基因。采用引物步移技术分析PINA蛋白缺失类型的分子机制，开发出相应的功能标记。采用RNAi技术将普通小麦中Pinb-2基因进行沉默后，通过转基因植株的表型分析，明确其功能。同时利用Southern杂交和荧光定量技术对不同倍性小麦中Pinb-2基因的拷贝数进行鉴定。然后对包括类型I和类型II在内的挖掘出的新基因（型）进行加工品质分析，找出品质性状相对优良的基因（型），开发出标记，并将其转入到当前主栽小麦品种中进行改良。

籽粒硬度是最为重要的小麦品质性状之一，严重影响小麦的磨粉和加工品质。本项目利用包括dCAPS标记在内的一系列分子标记和SKCS单籽粒谷物硬度测试仪对来自5个国家和地区的493份普通小麦品种进行了籽粒硬度主效基因型鉴定，发掘出了3个PINA蛋白缺失新类型，并利用引物步移技术阐明了其蛋白缺失的分子遗传机理，开发出了相应的分子标记；采用测序技术对新引进的来自国际玉米小麦改良中心（CIMMYT）的六倍体小黑麦进行了籽粒硬度Sina-R1和Sinb-R1位点基因型和表型分析，发现了三种新的Sina-R1基因型和两种新的Sinb-R1基因型，并阐明了其与籽粒硬度的关系；阐明了Pinb-2基因变异对小麦农艺性状的影响，找到了农艺性状相对优良的基因型；利用偃展4110为材料，采用EMS诱变技术构建了突变体库，并筛选出了籽粒硬度突变体。在国内外学术期刊发表论文7篇，其中SCI论文3篇，申请国家发明专利2项；培养研究生5名，其中博士生2名；获得了河南省科技进步二等奖。项目主持人陈锋入选了国家万人计划科技创新领军人才、科技部创新人才支持计划中青年科技创新领军人才等。