小麦LMW-GS基因PCR初步研究

Primary study on PCR of LMW-GS genes from wheat

用CTAB法提取小麦材料基因组DNA,根据基因库中公布的已知LMW-GS基因序列,设计并合成染色体位点特异的PCR引物1～7;探索出优化的PCR反应体系,即20μL反应体积中,Mg2+浓度为2.5mmol/L,dNTP浓度为200μmol/L,模板DNA30～60ng,每种引物50ng,Taq酶0.5U。利用特殊小麦材料——六倍体普通小麦(染色体组为AABBDD)、四倍体小麦(AABB)及二倍体一粒小麦(AA)和节节麦(DD)等的基因组DNA为模版,在优化的PCR反应体系下进行特异性扩增和引物验证。结果表明,引物3和引物4为小麦谷蛋白Glu-D3位点LMW-GS基因的特异引物,用其进行扩增时,循环反应条件为94℃变性1min,62℃退火1min,72℃延伸2min;扩增产物大小约为1.63kb,包括启动子和整个编码区。引物5和7为小麦谷蛋白Glu-B3位点LMW-GS基因的特异引物,用其进行扩增时,循环反应条件为94℃变性1min,64℃退火1min,72℃延伸2min;扩增产物大小约为1.45kb,包括启动子和整个编码区。

The genomic DNA was extracted from wheat cultivars Suneca and Cook using CTAB method.Based on the known LMW-GS gene sequences reported in genebank,the primer 1-7 for specific chromosome locus genes was designed and synthesized.An optimal reaction system suitable for LMW-GS PCR was established.By using the genomic DNA from special wheat material-hexaploid (AABBDD),tetraploid (AABB) and diploid (AA or DD) as templates,specific LMW-GS genes in an optimal reaction system was amplified.The result showed that,primer3 and 4 were specific for LMW-GS genes at Glu-D3 locus in wheat.The size of the PCR products was about 1.63 kb,including promoter and the whole CDS.Primer5 and 7 were specific for the LMW-GS genes at Glu-B3 locus in wheat.The size of the PCR products was about 1.45 kb,including promoter and the whole CDS.These results will provide some important information for wheat high-quality LMW-GS gene and its promoter cloning.