抗性淀粉含量不同的小麦品种(系)淀粉分支酶SBEⅡa和SBEⅡb基因多态性分析

Polymorphism Analysis of SBEⅡa and SBEⅡb Originated from Different Spring Wheat Cultivars with Starch Resistant Contents

【目的】克隆抗性淀粉含量不同的小麦品种(系)中淀粉合成酶关键基因SBEⅡa和SBEⅡb进行多态性分析,从分子水平阐述不同小麦品种(系)间抗性淀粉含量差异的原因。【方法】根据Gene Bank中公布的小麦SBEⅡa(AF286319.1)和SBEⅡb(AY740401.1)基因序列设计特异性引物,利用RT-PCR技术克隆SBEⅡa和SBEⅡb基因序列,利用Clustal.W进行测序结果的比对分析,NCBI Conserved domain和inte Pro在线网站对SBEⅡa和SBEⅡb氨基酸序列进行结构域预测,寻找影响抗性淀粉含量的主效基因位点。【结果】克隆了SBEⅡa(2 471 bp)、SBEⅡb(2 510 bp)基因ORF(open reading frame,ORF)全长。序列分析表明:SBEⅡa、SBEⅡb基因与公布的序列同源性分别为98.64%和99.07%。【结论】SBEⅡa基因ORF的三个结构域中未发现影响籽粒抗性淀粉含量的候选差异位点。在SBEⅡb基因编码区羧基C-末端的3个候选差异位点和α-淀粉催化酶结构域的1个候选差异位点可能是造成小麦品种(系)间抗性淀粉含量差异的原因。SBEⅡa、SBEⅡb氨基N-末端前面序列中共计10个单碱基变异作为潜在差异位点,也可能参与SBE基因的表达。

【Objective】We aim to clone sequences of SBEⅡa and SBEⅡb genes for polymorphism analysis in different spring wheat cultivars in order to explain the reason why different spring wheat cultivars show great significant difference in RS（ resistant starch,RS） content at the molecular level. 【Method】According to the sequence of SBEⅡa（ AF286319. 1）,SBEⅡb（ AY740401. 1） gene in wheat published in Gene Bank,special primers were designed and SBE Ⅱa,SBE Ⅱb gene partial sequence were cloned using PT- PCR. Afterwards,we blasted the nucleotide sequencing result with Clustal. W software,predicting the conserved domainthough web of NCBI conserved domain and inter Pro. The important allele＇s sites effect on the content of resistant starch were searched. 【Result】The results about sequencing showed that open reading frame of SBE Ⅱa and SBEⅡb genes were 2,471 bp and 2,510 bp,sequence analysis displayed sequence between SBEⅡa and SBEⅡb from different spring wheat cultivars and SBE Ⅱa（ AF286319. 1）,SBE Ⅱb（ AY740401. 1） were homologous 98. 64% and 99. 07%,respectively.【Conclusion】Much single nucleotide mutation（ SNP） appeared in different spring wheat cultivars,but it failed to grabble any possible candidate loci on the synthesis of starch in the three conserved domain of SBEⅡa gene. While three different candidate sites were showed in the N- terminal early set domain and the only one candidate site in the Alpha amylase catalytic domain in the SBEⅡb gene,that may be the reason for different contents of resistant starch in different spring wheat cultivars. Then the ten SNPs that were found in the front of N- terminal Early set domain of SBEⅡa and SBEⅡb may be regarded as the potential candidate sites that take an active part in controlling the expression of SBE gene.