大白菜细胞质雄性不育系RAPD特异扩增片段的克隆及其序列分析

Cloning and sequencing of a specific DNA fragment related to cytoplasmic male sterility line of chinese cabbage(Brassica campestris ssp.pekinensis)

利用RAPD技术从大白菜细胞质雄性不育系CMS3411-7的DNA中得到1个特异扩增片段CMSO-PL01670,进一步以该片段为探针进行Dot和Southern杂交证实了该片段为不育系所特有。回收该特异扩增片段并将其克隆到pGEM-TEasy载体上进行序列测定。测序结果表明该片段全长673bp,其碱基组成为A+T=67.16%。通过与GenBank+EMBL+DDBJ+PDB中的455972个序列进行比较,同源性均小于30%,表明该片段为一新发现序列。并且该序列的+4～+216的区域内含有1个可编码70个氨基酸的开放阅读框架。上述结果为今后从分子水平进一步研究大白菜细胞质雄性不育打下了坚实的基础。

A specific amplified DNA fragment was obtained from cytoplasmic male sterility line 'CMS34117' of Chinese cabbage using RAPD technique.Verification of Southern blotting and Dot blotting using this fragment as probe showed that the DNA fragment was peculiar to CMS34117.After cloning this fragment into pGEMT easy vector and sequencing it,we found that the full length of the fragment was 673 bp and its A+T content was 67.16%.Sequences of comparison of CMSOPL01670 with 455 972 sequences from GenBank+EMBL+DDBJ+PPB revealed that their homology were low (less than 30%),which implied that CMSOPL01670 maybe a newly found DNA fragment.The nucleotide sequence of CMSOPL01670 contains an open reading frame encoded 70 amino acid residues,which locates at the positions from +4 to +216.The ORF may play an important role in CMS of Chinese cabbage,because this similar facts has been found and proved in CMS of maize.