【作者】 赵法茂；

【导师】 王宪泽；

【作者基本信息】 山东农业大学 ， 作物品质改良与质量检测控制， 2008， 博士

【摘要】 淀粉是成熟小麦籽粒的主要组成成分,约占小麦籽粒重量的65%左右,由分支很少的直链淀粉和高度分支的支链淀粉两种葡萄糖多聚体组成。直链淀粉与支链淀粉的比例(直/支比)影响淀粉的理化性质和黏滞性谱(RVA谱)特性,进而影响小麦的加工品质、食用品质和工业用途。然而直链淀粉含量相同或相近的品种之间,淀粉品质却表现出明显差异,说明除直/支比外,支链淀粉的精细结构如聚合度、分支化度、链长分布和平均链长等结构参数也显著影响小麦品质。淀粉分支酶(SBE)是唯一引入α–1,6糖苷分支键的淀粉合成酶,因此SBE的性质和活性是影响支链淀粉精细结构和含量的重要因素。本研究选用种源地分布有代表性的90个小麦品种为材料,应用非变性聚丙烯酰胺凝胶电泳(Native–PAGE)和SDS–聚丙烯酰胺凝胶电泳(SDS–PAGE)检测了淀粉分支酶同工酶的等位基因分布特点和基因型组成,鉴定了SBE各同工酶的遗传多态性,研究了SBE等位基因位点和基因型与酶活性的关系,分析了SBE等位基因位点和基因型对支链淀粉含量的遗传效应,以期为阐明小麦淀粉品质形成的分子机制和小麦淀品质改良提供理论依据,主要研究结果如下。1.胚乳可溶性基质SBE遗传多样性鉴定应用Native–PAGE检测表明,SBE有SBEⅠ和SBEⅡ两种同种型,SBEⅠ同种型表现丰富多态性,具有A、Dⅰ、Dⅱ和B 4个基因位点,其出现频率分别为22.2%、98.9%、48.9%和75.6%。而SBEⅡ同种型只具有SBEⅡa单一基因位点,未显现多态性。在所有测试品种中,共检测到7种基因型, Dⅰ基因位点几乎出现在所有基因型中,对基因型组成的贡献率最大;D与B位点组成的基因型(DⅰB和DⅰDⅱB)的出现频率高于与A位点组成的基因型(ADⅰB和ADⅰDⅱ);每一个基因型(品种)只有4个基因位点的1～3个,未检测到具有4个基因位点的基因型。2. SBE两种同种型酶活性分析应用Nagamine等文献中的方法在Native–PAGE凝胶上对SBE进行酶活性染色,并通过降低酶活性染色的协同酶–磷酸化酶的浓度,对SBE两种同种型酶活性进行分析,结果在凝胶上只显现SBEⅡ酶带,但SBEⅠ酶带未显现,表明SBEⅡ活性大于SBEⅠ。3. SBEⅡb亚细胞定位及表达分析SBEⅡb在小麦籽粒发育的早期开始表达,但表达量较低,从花后14d至28d其表达量几乎恒定不变。不同发育时期SBEⅡb的活性有差异,其酶活性表达高峰出现在花后第21d左右。在籽粒发育后期,SBEⅡb被淀粉粒所包裹,成为淀粉粒结合蛋白,暂时丧失活性,一旦淀粉粒结构遭到破坏释放SBEⅡb,其活性立即恢复。SDS–PAGE检测表明,所有被试品种都具有单一SBEⅡb位点,表明SBEⅡb不具有遗传多样性。4. SBE基因型与酶活性的关系统计分析SBE各个基因位点和基因型的酶活性并进行差异显著性测验和多重比较。单一基因位点分析表明:A、Dⅰ、Dⅱ和B 4个基因位点的酶活性之间差异达极显著水平(p<0.01),其中A基因位点所表达的酶活性最高,而其它3个基因位点之间差异未达到显著水平;基因型分析表明:由A位点组成的基因型酶活性最高,与不含A位点的基因型相比差异达显著水平(p<0.05),而缺乏A位点的基因型之间酶活性无显著差异(p<0.05)。由于SBEⅡ同种型不具有多态性,因此SBE酶活性与SBEⅠ具有显著相关性,与SBEⅡ相关性不显著,其中A基因位点或含有A位点的基因型对酶活性的影响最大。5. SBE基因型对支链淀粉含量遗传效应分析单一基因位点的分析结果表明:A基因位点的支链淀粉含量最高,Dⅰ位点最低,在0.01水平上差异显著;多个等基因位点(基因型)的联合效应分析表明,组成基因型的基因位点越多,支链淀粉含量越高,与酶活性分析结果呈完全一致的趋势。其中,含有A基因位点的基因型(ADⅰDⅱ和ADⅰB)所对应的支链淀粉含量较高,与不含A位点的基因型(DⅰB、DⅰDⅱ和Dⅰ)的支链淀粉含量差异达显著水平(P<0.05)。SBE不同基因型对支链淀粉生物合成具有不同的遗传效应,显著影响支链淀粉含量;而且A基因位点和含有A基因位点的基因型遗传效应最大,可为筛选不同支链淀粉含量的小麦品种及淀粉品质改良提供参考。更多还原

【Abstract】 Strach is the major component of wheat grain, accounting for about 65% of the final dry weight of grain, and is composed of two distinct types of glucose polymer; amylose, which makes up 22～26% of normal starch, is an essentially linear molecule in which the glucose units are joined byα–1, 4 linkages, amylopectin is a much larger branched glucan polymer typically constituting 74～78% of the starch mass, produced by the formation ofα–1, 6 linkages between adjoining straight glucan chains. It is generally held that the ratio of amylose and amylopectin is a major determinant for physiochemical characteristics and Rapid Viso–analyzer(RVA) profile of starch, further influencing processing, cooking and eating qualities of wheat. However, previous studies also showed that there were significant differences in wheat quality among the varieties with similar amylose content. It is infered that fine structure of amylopectin, e.g. degree of polymerization, degree of branching, average chain length and chains distribution, also played an important role in wheat quality. Starch branching enzyme(SBE) is a sole enzyme to introduce branching points in the amylopectin molecules. Therefore the properties and activities of SBE are responsible for amylopectin structure.In order to elucidate the molecular mechanism of wheat quality and provide theoretical base for improving the quality of wheat starch, Ninety wheat cultivars from different provenances were used in the present study, including some typical hexaploid wheat from various province of China and some high–yielding modern varieties as well as exotic ones. The gene loci, genotypes and genetic diversity of SBE isozymes were determined by using native and SDS polyacrylamide gel electrophoresis (Native–PAGE and SDS–PAGE). The correlation between isozyme genotypes and enzyme activities of SBE was investigated. The genetic effects analysis were made for isozyme genotypes of SBE on amylopectin content. The main results were as follows.1. Genetic diversity of SBE in soluble stroma of endospermThe genetic diversity of SBE was characterized through Native–PAGE. The result showed that there were two SBE isoforms, ie. SBEⅠand SBEⅡ. SBEⅡisoform failed to exhibit genetic polymorphism but SBEⅠmanifested genetic diversity. Four gene loci, A, B, Dⅰand Dⅱ, were identified at SBEⅠlocus, with frequency 22.2%, 98.9%, 48.9% and 75.6%, respectively. Seven genotypes were determined, and Dⅰlocus was included almost in all genotypes, which was the most contribution to genotypes formation. The frequency of genotypes comprised by D and B locus was higher than that by D and A. Each genotype was composed of the one to three gene locus among four ones. The genotype constituted by four gene locus was not observed.2. Activity analysis of SBE isoformsIn this study, we conducted activity staining of SBE using Nagamine’s literature method in Native–PAGE gel. The result demonstrated that if phosphorylase concentration, which acted as co–working enzyme SBE activity staining, was lowered, the sole SBEⅡa but no SBEⅠzymogram was observed, indicating that SBEⅡactivity is higher than that of SBEⅠ.3. Subcellular localization and expression level of starch granule–bound SBEⅡbSBEⅡb expressed at the early stage during wheat grain development, and kept constant from 14th to 28th day post anthesis. There was different expression level of SBEⅡb in term of protein amount in different period of developing wheat grain, and its activity reached the maximum at the 21th day after anthesis. SBEⅡb was entraped into granule in the late period of developing grain, temporarily lossing enzyme activity. Once SBEⅡb was released from within granule by mechanical and chemical methods, the activity was recovered immediately. SDS–PAGE was conducted to examine SBEⅡb diversity. The result indicated that all cultivars tested had SBEⅡb locus, and there was no differences of the electrophoresis profile among cultivars. Therefore, similar to SBEⅡa, SBEⅡb was not of genetic polymorphism. 4. Correlation between isozyme genotypes and activity of SBEIn order to explore the relationship between the isozyme genotypes and enzyme activity of SBE, enzyme activity statistics corresponding to gene loci and genotypes was made, and multiple comparison analysis conducted. The result indicated that from single gene locus analysis, there were significant differences of the enzyme activity among A, B, Dⅰand Dⅱlocus (p<0.01), which the activity of A locus was the highest, and the one of remaining three locus had no significant differences. From genotype analysis, the activity of genotypes constituted by A and B or by A and D was the highest and exhibited a statistically significant level compared to other genotypes (p<0.05). But there was no significant differences between genotypes without A locus (p<0.05). Consequently, SBE activity correlated to SBEⅠisoform, but no significant correlation to SBEⅡwas observed. Furthermore, the effects of A gene locus and genotypes comprised by A locus on SBE activity was the most significant.5. Genetic effects of isozyme genotypes of SBE on amylopectin contentFrom single gene locus analysis, amylopectin content of A locus was the highest, and the lowest for Dⅰ, which showed a statistically significant differences at 0.01 level. From joint effects analysis of multiple locus, the more gene loci constituting SBE genotypes, the more significant effects of the genotypes on amylopectin content, representing same scenario as enzyme activity analysis. Amylopectin content of genotypes containing A locus ( A DⅰDⅱand ADⅰB ) were the highest, which there was significant differences at 0.05 level compared to that of genotypes of DⅰB and DⅰDⅱ, especially to Dⅰ. There is different genetic effects for different genotypes of SBE on amylopectin content, and SBEⅠisoform plays a major role in determining amylopectin content. The effects of genotypes containing A locus is the most significant, which can provide theoretical base for the breeding and improving of wheat quality.更多还原