甘蓝型油菜WRI1基因cDNA的克隆与序列分析

Cloning and sequence analysis of cDNA WRI1 gene from Brassica napus

通过RT-PCR方法克隆了甘蓝型油菜湘油15号5个WRI1基因cDNA。测序结果显示,BnWRI1-1大小为1 248 bp,BnWRI1-2和BnWRI1-3大小为1 254 bp,BnWRI1-4、BnWRI1-5大小为1 242 bp;聚类分析结果显示,所克隆WRI1基因cDNA在进化关系上属于AP2/ERF转录因子家族,同拟南芥AtWRI1(At3G54320)同处1个分支,并且所克隆基因cDNA来自甘蓝型油菜不同的基因组;通过核苷酸序列比对分析,得到所克隆WRI1基因cDNA单核苷酸变异位点40个;特异性酶切位点分析显示,来源于A基因组的BnWRI1-1、BnWRI1-2、BnWRI1-3第892位与来源于C基因组的BnWRI1-4和BnWRI1-5第880位的核苷酸发生变异,导致Bcl I识别位点的变化;酶切试验结果表明,采用BclI酶可区分甘蓝型油菜的AtWRI1-like WRI1基因的A或C基因组来源。

Five WRI1-Like WRI1 cDNA samples were isolated frorn Brassica napus （Xiangyou No. 15 cultivar） by RT-PCR. Among the five cDNAs, BnWRI1-1 is 1 248 bp, BnWRI1-2 and BnWRI1-3 are 1 254 bp, BnWRI1-4 and BnWRI1-5 are 1 242 bp in length. Cluster Analysis showed WRI1 genes belong to transcription factor AP2/ERF gene family, which clustered in the same group with AtWRIl（At3G54320） gene ofArabidopsis thaliana. Alignment of the coding regions of WRI1 genes indicated 40 single nucleotide polymorphic sites. A nucleotide transition from A to G at position 892 in the coding sequence ofBnWRI1-1, BnWRI1-2, BnWRI1-3 and at position 880 in the coding sequence ofBnWRI1-4, BnWRI1-5 produced a specific restriction endonuclease cleavage site which was original identified by Bcl I. Further digestion test showed that Bcl I cleavage could differentiate the genome origin ofBrassica napus At WRII-like WRII.