大白菜CMS雄性不育发生相关基因的克隆与分析

Clone and analysis of CMS related gene of heading Chinese cabbage

【目的】筛选和克隆与大白菜CMS雄性不育相关的应答基因。【方法】以大白菜不育系06J45和恢复系01S325-1为材料,利用cDNA-AFLP方法从F2S5群体不育株和可育株花蕾中筛选分离差异表达基因,通过RACE技术对该差异表达基因进行克隆,对基因序列进行生物信息学分析,并利用qRT-PCR定量分析不育株和可育株花蕾发育过程中基因表达的变化。【结果】在F2S5可育株花蕾cDNA混合池中扩增出1条特异表达条带,通过3′RACE和5′RACE克隆得到相应的BrUP基因,全长1 407bp,编码403个氨基酸残基。生物信息学分析表明,BrUP基因与大白菜Bra007268基因序列相近,由6个外显子和5个内含子组成,与拟南芥未知功能基因AT3G56750同源,一致性达90%。保守结构域分析表明,BrUP基因可能是1个具有跨膜结构的O-型岩澡糖基转移酶基因,主要功能是进行糖基化修饰。qRT-PCR定量发现,BrUP只在花蕾发育早中期表达,且在大白菜不育株花蕾发育早期其表达量显著性下调。【结论】成功筛选克隆获得1个与大白菜雄性不育发生相关的功能未知基因BrUP。

【Objective】This study aimed to screen and clone cytoplasmic male sterility(CMS)related gene of heading Chinese cabbage.【Method】The cDNA-AFLP technology was used to screen and identify differentially expressed gene from male sterile flower bud and male fertile flower bud deprived from the hybrids of male sterile line 06J45 and its restorer line 01S325-1.Then RACE was used to clone the selected gene,the bioinformatics analysis was done to analyze its sequence and qRT-PCR was used to detect its expression.【Result】A differentially expressed cDNA fragment was selected from flower bud of fertile plants and sterile plants of the F2S5 generation.A full-length cDNA of 1 407 bp encoding 403 amino acids,named as BrUP,was amplified using RACE.Blast analysis showed that the BrUPgene was homologous to an unknown protein AT3G56750 with similarity of 90%.Conserved domain analysis showed that the BrUPgene belonged to the O-FucT like superfamily and may involve in the O-fucosylation of protein substrates.qRT-PCR analysis showed that BrUP mainly expressed at early stages of flower bud development,and the expression was significantly lower in the development of male fertile flower bud.【Conclusion】An unknown function gene BrUPrelating to CMS of heading Chinese cabbage was screened and cloned.