红麻野败型CMS胞质SNP分子标签的发掘

Discovery of An SNP Marker Associated with CMS Cytoplasm in Kenaf(Hibiscus cannabinus)

利用同源克隆的方法,克隆了红麻野败型CMS胞质SRAP标记位点Me14上游的侧翼序列,并采用RT-PCR方法研究该位点的表达。结果表明:(1)红麻不育系P3A和保持系P3B的mtDNA在Me14结合位点处存在1个G/A SNP位点;HpaⅡ内切酶可特异性酶切以保持系P3B扩增获得的E1纯化片段,而不育系P3A的E1纯化片段不能被酶切。(2)对红麻的9对不育系/保持系、5个恢复系和F1杂交种在该SNP位点的分析发现,以保持系和恢复系总DNA为模板扩增获得的E1纯化片段均可为HpaⅡ内切酶特异性酶切,而不育系和F1杂交种的E1纯化片段不能被酶切。(3)RT-PCR结果表明,E1片段对应基因在不育系P3A和保持系P3B中的时空表达模式无差异,在GenBank中也没有与E1相匹配的蛋白序列。研究表明,该研究发掘的红麻CMS胞质SNP标记位点处,不育系的mtDNA存在点突变,该标签并非具有嵌合阅读框的不育基因。

Analysis was carried out to explore the nature of Me14/Em8 marker for kenaf WA-CMS cyto- plasm and develop the more credible markers linked to kenaf CMS cytoplasm. The upstream flanking se- quence of Mel4 binding site was amplified by homologue cloning. Semi-quantitative RT-PCR was used to analyze the expression levels of the loci in different tissues. （1）The sequences analysis showed a G/A SNP in the Mel4 binding site of the CMS line （P3A） and its maintainer line （P3B） mtDNA. Purified E1 PCR product amplified using the total DNA of P3B as template can be digested by Hpa Ⅱ because of the “CCGG” sequence in the Mel4 binding site,in contrast with that of P3A because of the “CCGA” sequence in the corresponding locus. （2）The characteristics of the SNP loci showed that the E1 fragments that were amplified using the total DNA of the nine maintainer and the five restorer lines as template can be digested by Hpa Ⅱ ,in contrast with that of the nine CMS lines and the F1 hybrids. （3）RT-PCR results also showed the existence of E1 transcripts. However, no differences were found in the expression patterns between P3A and P3B. Blastx searches of publicly available gene and protein databases did not produce any statisti- cally significant “hit” to known genes. The SNP marker associated with the CMS cytoplasm can be regar- ded as a point mutation in mtDNA of male sterile lines,which is not related to chimeric gene.