野生二粒小麦强化小麦面筋性能的贮藏蛋白特性及其分子基础

Wheat flour can be processed into various foods, which is determined by the unique gluten properties of storage protein. However, in the modern wheat cultivars, vast genetic materials controlling high protein content and strong gluten properties have been losted with the breeding goal for high yield. In our previous work, the wild emmer wheats (Triticum dicoccoides, AABB, 2n=4x=28) possessing excellent storage protein composition including high-molecular-weight glutenin subunits (HMW-GSs)、low-molecular-weight glutenin subunits (LMW-GSs) and gliadins, were selected as male parents, and crossed with the bread wheat cultivar ‘Chuannong16’ with both low protein conten and weak gluten properties. And, the HMW-GS gene 1Ay which is always silent in common wheat, firstly was successfully transferred into common wheat from wild emmer wheat, and created the common wheat germplasm having total of 6 HMW-GSs. At the same time, the stable introgration lines with high protein content and strong gluten properties were obtained. However, we discovered that some lines with high protein conten and strong gluten properties are without both 1Ay gene on 1AL chromosome and the transcriptional factors gene NAM-B1 associated with high protein content and flour processing properties on 6BS chromosome of wild emmer wheat. It implied the genetic system is multiple and intricate. So, the modern technologies including proteome, DArT (Diversity Arrays Technology) and RNA-seq transcriptome will be combinedly used in this project. The objective is to analyze the storage protein properties and its molecular bases in wild emmer wheat and its introgration lines, and to explore the candidate genes associated closely with high protein content and gluten strengthening properties, and their distribution sites on the chromosomes, as well as to create the germplasm resources database of common wheat with the genetic materials controlling high protein content and strengthening gluten properties from wild emmer wheat, and the system of molecular mark assistant selection.

小麦籽粒贮藏蛋白独特的面筋特性使之具有加工成多样化面食品的能力。可惜长期追求高产的育种目标致使现代小麦品种的高蛋白与强筋特性遗传物质匮乏。我们用具有优异贮藏蛋白组成的野生二粒小麦与高产弱筋小麦品种‘川农16’杂交，首次成功创制出含野生二粒小麦1Ay基因能稳定表达全部6个HMW-GS的普通小麦以及高蛋白、强筋/中强筋特性的稳定渐渗系。但是，一些高蛋白、强筋特性的渐渗系并不携带野生二粒小麦的高蛋白含量基因NAM-B1。据此推测，调控野生二粒小麦及其渐渗系高蛋白、面筋强化特性的遗传系统是多元复杂的。本项目通过贮藏蛋白组学和高通量DArT标记及RNA-seq转录组分析相结合的方法，深入解析野生二粒小麦及其渐渗系贮藏蛋白的分子基础，发掘高蛋白含量和面筋强化特性的候选基因及其分布位点，为建立携带野生二粒小麦高蛋白、强筋/中强筋特性的普通小麦种质库及其分子标记辅助选择系统提供理论依据与技术指导。

本项目研究材料涉及野生二粒小麦及其渗入系、普通小麦亲本及对照500余份。通过凯氏定氮等方法对籽粒全麦粉和去麸面粉蛋白含量及其清蛋白、球蛋白、谷蛋白、醇溶蛋白、不可溶性谷蛋白等蛋白组分含量，以及面筋含量、沉降值、吸水率、形成时间、稳定时间等品质参数进行了分析，选取典型材料既对面条又对面包进行了加工品质研究，进而探讨了上述所有性状间的相关性。利用DArT标记对源于野生二粒小麦D1和D97的共2套渗入系，进行了蛋白质含量（GPC）的GWAS解析，分别发掘出在2B、3B、6A、6B、7B上和4A、1B、7B上与高GPC关联的新位点，有的PVE高达21.79%甚至32.69%。利用中国春和野生二粒小麦2套参考基因组，对代表性材料灌浆期籽粒GPC含量进行了RNA-seq转录组分析，筛选出高GPC和贮藏蛋白候选基因。通过SDS-PAGE、2-DE、RP-HPLC、MALDI-TOF-MS、分子标记，以及基因克隆与结构分析，解析了HMW-GS全部4个位点如Glu-1Ax2.2、Glu-1Ax1.2、Glu-B1x、Glu-1Ay和Glu-1By8.1，LMW-GS，α、γ、ω三种醇溶蛋白在内的贮藏蛋白，以及高蛋白含量对面粉加工品质效应的分子结构基础。这些结果充分证明了我们前期提出的“引起渐渗系高蛋白含量和面筋强化特性的野生二粒小麦及其与普通小麦杂交重组系的遗传基础是多元而复杂的”推测，而且也有助于理解NAM-B1基因的高GPC却低粒重问题。所创建的携带野生二粒小麦面筋强化特性和高GPC特性且与高粒重相容的载体种质库，对面筋强化与高产并举的普通小麦育种目标具有重要应用价值，现已满足国内多个单位对我们所创制种质资源的育种引用需求，而且育成了深受种养大户欢迎且获国家植物新品种保护权受理的品种。因此，整体上完成了项目研究计划任务。