| Objective: This study was carried out in order to elucidate the reasons for the difference in the contents of resistant starch in Wheat Varieties(lines),and provide the theoretical basis for wheat resistant starch content of marker assisted selection.Method: In this study, the SBE IIa, SBE IIb gene and SBE IIa promoter sequence were cloned from wheat varieties(lines) with different contents of resistant starch. Were among different wheat varieties(lines) gene sequence alignment and polymorphism analysis, explore the influence of wheat resistant starch synthesis gene loci, and based on allelic loci design specific AS-PCR primer. The functional markers(FMs) of the marker assisted selection of the resistant starch were developed by using the wheat varieties(lines) with different contents of the resistant starch。The main results and conclusions:1. The relative expression of SBE II a and SBE II b gene in Wheat Cultivars with high and low resistance starch contents were significantly different from the grain. SBE II A and SBE II B gene relative expression quantity in High resistant starch content SD06-5(RS=3.86%) was only 50 percent of the genes expression of resistant starch content low M190(RS=0.60%),which also showed that it have a significant negative correlation relationship between the contents of the resistant starch in wheat grains and the relative expression of SBE A and SBE II B gene in wheat kernel.2. SBEⅡa,SBEⅡb gene and SBE II a promoter sequence were cloned by RT-PCR technology The result of sequencing showed that the open reading frame(ORF) of SBEⅡa and SBEⅡb gene was 2471 bp and 2510 bp respectively, and SBE IIa promoter sequence length was 2896 bp.Sequence analysis displayed sequence of SBEⅡa(AF286319.1),SBEⅡb( AY740401.1) and the promoter region of SBEⅡa genes(AY357072.1) published sequences in genebank wheat corresponding gene sequence blast comparability of results were 98.64% and 99.07% and 99.92%.3. Polymorphism analysis of SBEⅡa gene promoter, SBEⅡa and SBEⅡb originated from difference spring wheat cultivars.The result of blasting ORF sequences of SBEⅡa showed, there is a mutation in the Alpha amylase-C-terminal all-beta and Alpha starch catalytic respectively. And in the leading sequences of N-terminal Early set domain,Xinchun12,M150,M190,SD06-5,Annong90202 and Xinchun19 showed six mutation loci comparison with the other wheat cultivars.But in the promoter of SBEⅡa gene,only in a certain sequence from wheat cultivars showed single-base mutation, missing or insert. The result of blasting ORF sequences of SBEⅡb showed, M190, Fuchun1,Xinchun12 and Ningchun4 show twelve mutation loci in the leading sequences of N- terminal Early set domain and three mutation loci in the Alpha starch catalytic domain comparison with the other wheat cultivars.M65,SD06-5,Botamu and Yemao show two mutation loci compare with the other wheat cultivars and M190 Fuchun1 and Xinchun19 compared with the other wheat cultivars show one mutation loci in the N-terminal Early set domain.4.Biological information analysis of SBEⅡa, SBEⅡb and determine the important alleles sites effecton the synthesis of RS.The results of biological information analysis of SBEⅡa,SBEⅡb showed that, SBEⅡa and SBEⅡb gene contain three domains: N-terminal early set, Alpha amylase-C-terminal all-beta and Alpha starch catalytic. Meanwhile two different candidate sites(2248and 2302) showed in the C-terminal Early set domain in the SBEⅡb gene, which determined the important alleles sites(A?G) effect on the synthesis of RS.5. We design and screening the specific primers of AS-PCR. The AS-PCR results showed that the primers could be used in wheat RS content of marker-assisted selection, effectively.In this study, it failed to grasping any possible candidate loci could effect on the synthesis of starch in the ORF of SBEⅡa gene.While in SBE II B gene ORF sequence of 2248, 2302 alleles on A G mutation is the effective influence of wheat resistant starch synthesis gene deviation of ectopic. |
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