甘蓝细胞质雄性不育相关基因orf138的分子特性分析

The Molecular Characteristics of Ogura Cytoplasmic Male Sterility Related Gene orf138 in Cabbage(Brassica oleracea var.capitata)

以甘蓝Ogura细胞质雄性不育系为材料,找出能够有效鉴定由orf138导致的甘蓝CMS的标记,并明确此基因在甘蓝OguCMS基因组中的位置。根据萝卜CMS相关基因orf138的序列信息,设计特异引物,并在甘蓝不同类型不育系和保持系中鉴定PCR产物的稳定性。随后利用Tail-PCR技术,扩增获得此基因的侧翼序列并进行了生物信息学分析。根据orf138的全长序列设计引物Bo138300BF/R,在甘蓝不育型材料中,能够稳定扩增出300bp左右的单一条带,而在其他细胞质不育类型和可育材料中均未扩出条带,经多次验证结果稳定可靠。甘蓝中orf138的上下游侧翼序列有效碱基1789bp,通过生物信息学分析,获得包括起始密码和终止密码的orf138的完整序列共417bp。同源性比对结果显示:与甘蓝型油菜、白菜和萝卜的orf138片段具有高度保守性。分析侧翼序列表明甘蓝orf138的3’端是由ORF83、trnfM、ORF125等基因片段构成的一个复杂序列。获得了甘蓝OguCMS特异的分子鉴定标记,明确了orf138在甘蓝线粒体中的位置,以上结果为甘蓝雄性不育的进一步研究奠定良好的基础。

The objective was to develop effective specific markers of Ogura cytoplasmic male sterility （Ogu CMS） line in Brassica oleracea and illustrate the upstream and downstream genome sequence arrangement of the CMS gene orf138. The orf138 gene fragment in cabbage CMS line was amplified based on specific primers designed according to radish orf138 gene sequence information. The upstream and downstream mitochondrion sequences of orf138 sterile line were identified by use of TaiI-PCR technology. Ogu CMS specific primer Bo138300 BF/R was designed according to the full-length sequence of 05138. It could stably amplify a single PCR product of about 300 bp in OguCMS cabbage line, while never in fertile maintainer lines and other types of cytoplasmic male sterility lines. Upstream and downstream flanking sequence of orf138 were aligned and resulted in a 1789 bp sequence. Bioinformatic analysis showned that this sequence included to obtain the complete sequence of 417 bp of the oriel38. Homology comparison showed that orf138 gene of cabbage. Brassica napus and radish was highly conserved but the downstream. The 3＇ flanking mitochondrion sequenceof the orf138 in cabbage showed a complex sequence included ORF83 trnfM, ORF125. The research here developed an effectice specific DNA marker Bo138300BF/R and illustrated the genome location of orf138 in cabbage mitochondrion. These results lay a good foundation for further study of cabbage Ogu CMS.