【作者】 银永安；

【导师】 齐军仓； 李卫华；

【作者基本信息】 石河子大学 ， 作物遗传育种， 2011， 博士

【摘要】 小麦是世界上最重要的粮食作物之一,其产量和品质关乎国际粮食安全与区域局势的稳定。在我国,小麦成为仅次于水稻的第二大粮食作物。随着人民生活水平的不断提高和食品工业的迅猛发展,小麦生产对品质的要求逐年提高。但长期以来,科技工作者对小麦品质的研究都主要集中在籽粒蛋白和优质氨基酸上,往往忽略了淀粉对小麦品质及食品加工品质的作用。淀粉是小麦籽粒胚乳的主要组成成分,约占籽粒干重的65%-70%是决定小麦品质重要指标。为此,本研究选择不同淀粉含量的4个小麦品种为材料,借助于十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、双向凝胶电泳(2-DE)、飞行时间质谱仪(MALDI-TOF-MS)、扫描电镜(SEM)、布拉本德粘度分析仪、差示扫描量热仪(DSC)、激光粒度分析仪、X-射线衍射仪(X-RD)等现代测试技术与手段对其籽粒胚乳A、B淀粉淀粉粒的形成机理和理化特性进行了系统细致的研究,旨在为小麦淀粉品质育种目标的制定提供理论依据,为培育优质专用型小麦品种增加了研究基础。本文的主要研究内容与结果如下：1.用常量法和微量法对4个小麦品种淀粉粒提取、分离纯化以及后续的测定分析,发现常量法得到的A型淀粉粒纯度在79.46%-85.23%之间,B型淀粉粒纯度在90.27%-96.43%之间；微量法分离的A型淀粉粒纯度在92.68%-96.70%之间,B淀粉粒纯度在96.48%-99.00%之间。A、B淀粉粒分离与纯化效果在常量中均达到显著水平,在微量中达到极显著水平,即微量法提取、分离及纯化的A、B淀粉粒纯度高于常量法的,但常量法最终得到的A、B淀粉粒产物远大于微量法的。2.通过用扫描电镜SEM对小麦花后籽粒胚乳淀粉粒发育的持续观察发现,小麦淀粉体和A型淀粉粒出现于花后3d左右,B型淀粉粒出现于花后15d左右,淀粉体从9d开始逐渐消失,而A、B淀粉粒一直生长发育到籽粒成熟。运用聚丙烯酰胺凝胶电泳(SDS-PAGE)、双向凝胶电泳(2-DE)、飞行时间质谱(MALDI-TOF-MS)分析仪器与鉴定手段对小麦籽粒胚乳A、B型淀粉粒结合蛋白进行鉴定分析,结果表明A型淀粉粒形成的原因是它具有3种结合蛋白SGP-145, SGP-150和SGP-152,它们三者与支链淀粉,尤其与支链淀粉分支链聚合度>40(DP>40)的长分支链的支链淀粉合成有关,从而最终影响到了A型淀粉粒的形成,而B型淀粉粒因缺乏这3种结合蛋白而不能使其粒径发育到大于10μm。3.用扫描电镜(SEM)对小麦发育籽粒自然断面和分离纯化后的A、B型淀粉粒观察发现,小麦胚乳淀粉粒大小可以10μm为界限划分为A型和B型淀粉粒；形态上,淀粉粒可分为规则形状和反常形状。分离纯化后的小麦A型淀粉粒形态表现为蚌壳状；B型淀粉粒大多数表现为球状或椭球状。用直链、支链淀粉试剂盒(Megazyme International Ireland 2006)对发育小麦籽粒胚乳淀粉粒中直、支链和总淀粉测定,结果表明小麦籽粒胚乳中直链淀粉形成早于支链淀粉,但最终积累量支链淀粉大于直链淀粉,且A、B型淀粉粒中支链淀粉含量与小麦总淀粉含量成正相关趋势。4.对4小麦品种小麦籽粒胚乳A、B型淀粉粒膨胀势测定发现,B型淀粉粒膨胀势均大于各自A型淀粉粒的。用布拉本德粘度分析仪和差示扫描量热仪(DSC)对小麦籽粒胚乳A、B型淀粉粒粘度糊化和热力学特性测定结果显示,小麦A、B型淀粉粒糊化后均为天蓝色,淀粉峰值粘度和热力学糊化焓变表现为B型淀粉粒>A型淀粉粒。5.通过用激光粒度分析仪、X-射线衍射仪(XRD)分别对小麦籽粒胚乳淀粉粒的粒度分布和A、B型淀粉粒晶体特性的测定分析。结果显示,小麦籽粒胚乳淀粉粒的体积和表面积分布都是以10μm为界限呈双峰分布,数目分布呈单峰。小麦A、B型淀粉粒晶体结构均呈现典型的“A型”特征。A型粉粒相对结晶度均显著高于B型淀粉粒的,4个尖峰强度也呈现A型淀粉粒>B型淀粉粒。A型淀粉粒的相对结晶度与其体积分布呈极显著正相关,与B型淀粉粒的表面积和数量分布依次呈显著和极显著负相关；B型淀粉粒的相对结晶度分别与其表面积和数量分布依次呈显著和极显著正相关,与A型淀粉粒的体积分布呈极显著负相关。更多还原

【Abstract】 Wheat is one of most important food crops in the world, yield and quality of which are suggested to have a role in international food supplies security and regional condition tranquilization. In China, wheat is the second large food crop just less than rice. With ever increasing standard of living of pepole and development of food industry, the quality requirement level for production of wheat is more and more strict. However, more scholars mainly concentrated on the research of wheat protein and amino acid in past a long period, and finally ignored the study of wheat starch. Starch accounted for 65%-70% weight in dry wheat grain, thus it was the main composition of wheat grain and was a more important quality character. Therefor, four wheat cultivars with different starch content were chosen for this research work and a series of modem detecting techniques were used to systemicly study the formation mechanism and physico-chemical characteristics of A, B type starch granules in wheat endosperm. These techniques included Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Two-dimensional Gel Electrophoresis (2-DE), and Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) for the research of formation mechanism, whereas Scanning Electronic Microscope (SEM), Viscosity Amylograph (Brabender, Germany), Differential Scanning Calorimeter (DSC), Laser Particle Analyzer and X-ray Diffractometer (X-RD) etc for the research of physico-chemical characteristics to A, B type starch granules in wheat endosperm respectively. The major contents and results of this paper were as follows:1. Starch granule were isolated and purified from four wheat cultivars grain using macromethod and micromethod, and were tested by SEM. The results showed that the purity of A type starch granules ranged from 79.46% to 85.23%, which of B type starch granules ranged from 90.27% to 96.43% by using macromethod; if using micromethod, the purity of A type starch granules ranged from 92.68% to 96.70%, which of B type starch granules ranged from 96.48% to 99.00%.The effects of isolation and purification for A, B-type starch granule indicated significant level in macromethod, and distinct significant level in micromethod. In other words, the purity of A, B type starch granules with micromethod was higher than that of macromethod, but the ratio of A, B type starch granules with micromethod was more less than that of macromethod.2. The developing condition of starch granules in wheat endosperm were continuously observed with Scanning Electronic Microscope (SEM), and found that amyloplast and A type starch granules emerged at approximately 3 day post anthesis (DPA) and B type starch granules emerged at approximately 15 DPA. The amyloplast began to disappear from 9 DPA, but A and B type starch granules continuously grew till.wheat grain matured. Starch granule band protein (SGP) of wheat A, B type starch granules were separated and identified by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Two-dimensional Gel Electrophoresis (2-DE), and Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The results showed that the reason why A type starch granules formation was that it had 3 sort of SGPs (SGP-145, SGP-150 and SGP-152), which could promote the biosynthesis of amylopectin, especially the degree of polymerization of chain length of amylopectin> 40(DP>40), and finally caused the formation of A type starch granules. The diameter of B type starch granules was less than 10μm, due to they lacked of such 3 SGPs.3. The natural ruptured side of kernel of different deveoping periods and the separated & purified A, B type starch granules of wheat endosperm in wheat were observed by Scanning Electron Micrograph (SEM). The results showed that wheat starch granules could be divided into A and B type by its diameter of 10μm, and could be divided into normal and abnormal by its shape. The shape of separated & purified A type starch granules was shell structure but the shape of most B type starch granules indicated spherical structure. The contents of amylose and amylopectin in A, B type starch granules of wheat endosperm of different developing periods were determined by AMYLOSE/AMYLOPECTIN ASSAY PROCEDURE, and the results indicated that the formation of amylose in starch granule of wheat endosperm was earlier than that of amylopectin, while the final content of amylopectin was much higher than that of amylose, and the amylopectin contents in A, B type starch granules had a positive correlation trend to total starch content.4. The swellings power of A, B type starch granules in matured wheat endosperm were measured according to method of McCormick, and the results showed that the swelling power of B-type starch granule was higher than that of A-type starch granule. Viscosity and thermal characteristics of A, B type starch granules of wheat were determined by Viscosity Amylograph (Brabender, Germany) and Differential Scanning Calorimeter (DSC). The results showed that the color of A, B type starch granules after cataplasm were sky blue, both the peak value of paste (PV) and the value of enthalpy change (△H) of B-type starch granule were higher than that of A-type starch granule.5. The size distribution of starch granules were determined using Laser Particle Analyzer and the crystalline properties of A, B type starch granules were determined by X-ray Diffractometer (X-RD) The results of size distribution showed that the volume and surface area in wheat endosperm displayed a bimodal distribution by its diameter of 10μm, while the number displayed a single peak. The results of crystalline properties showed that the crystalline structure of A, B type starch granules were typical "A" type and the crystalline value of A type starch granule is totally notable larger than that of B type, the same trends as the value of four shape peaks.The analysis of correlation relationship study between size distribution and crystalline properties showed that the crystallinity of A type starch granules have extremely positive significant associated with the volume of A type, but have negative or extremely negative significant with surface area and number distribution of B type starch granule, thus the crystallinity of B type starch granules have extremely negative significant with the volume of A type, whereas have positive significant or extremely significant with surface area and number distribution of B type starch granule in wheat.更多还原