Genetic Diversity Of Isozyme Of Starch Branching Enzyme And The Effects On The Activity And Amylopectin Content In Wheat Grain

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.