甘蓝S位点受体激酶基因(SRK)编码区的甲基化分析  

Methylation Analysis of S-locus Receptor Kinase Gene (SRK) Coding Region of Brassica oleracea

作　　者：张莉[1] 朱利泉[1] 贾华[1] 李明[1] 吴玮铷[1] 唐章林[1] 王小佳[1] 

机构地区：[1]西南大学农学与生物科技学院,重庆400716

出　　处：《农业生物技术学报》2010年第3期482-488,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(30671429;30971849)资助

摘　　要：克隆了位于自交不亲和型甘蓝(Brassic aoleracea)S位点受体激酶基因(SRK)上第一编码区中包含两个CCGG位点的目的片段,通过甲基化敏感限制性内切酶-PCR法,利用对甲基化敏感性不同的MspⅠ和HpaⅡ分别对柱头乳突和花药基因组DNA及其PCR产物进行交叉组合式的酶切与电泳,首次对SRK基因编码区的特定DNA区段进行了甲基化分析。使用相同的SRK基因特异性引物时,柱头乳突和花药基因组DNA作为模板均可以扩增出清晰目标谱带,且目标谱带经MspⅠ/HpaⅡ完全酶切后可产生预期的谱带类型;而经MspⅠ/HpaⅡ完全酶切后的此二基因组DNA再作为模板进行PCR,均无特异性的目标谱带扩出。这些结果初步表明,自交不亲和型甘蓝花粉的SRK基因可能不存在甲基化封闭。A target fragment which contains two CCGG sites was cloned from the first coding region of S-locus receptor kinase gene （SRK） of Brassica oleracea with typical self-incompatibility （SI）. Then the method combining methylation-sensitive restriction endonucleases with PCR （MS-RE-PCR） was used to analysis primarily the methylation of the specific DNA fragment of SRK gene coding region, that is, genomic DNA （gDNA） extracted from stigma papilla and anther and their PCR amplification products were digested and electrophored interlacedly and combindly. The restriction endonucleases were MspⅠand HpaⅡ,whose methylation-sensitivity were different. When using the same specific primer, the target bands could be got by using gDNA as template, and all target bands could also be digested completely by MspⅠ/HpaⅡ producing the expected smaller bands, but there was no specific PCR amplification products by using gDNA digested by MspⅠ/HpaⅡas template. These results indicated that SRK gene in anther of SI B. oleracea may be not blocked by DNA methylation.

关 键 词：S位点受体激酶基因(SRK) DNA甲基化 甲基化敏感限制性内切酶-PCR法 MspⅠ/HpaⅡ 

分 类 号：Q523.8[生物学—生物化学]