普通白菜1,5-二磷酸核酮糖羧化/加氧酶小亚基基因Bcrbc S的克隆及表达分析

Cloning and Expression Analysis of Ribulose-1,5-bisphosphate carboxylase/oxygenase Small Subunit Gene Bcrbc S in Pakchoi

利用RACE技术,从普通白菜抗霜霉病品种苏州青叶片克隆到1,5-二磷酸核酮糖羧化/加氧酶小亚基(ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit,Bcrbc S)基因的全长c DNA序列。采用q RT-PCR分析该基因在普通白菜不同组织的表达模式。利用SDS-PAGE技术分析了该基因的原核表达特征。序列分析结果表明,Bcrbc S基因的c DNA序列全长为733 bp,其中开放阅读框长度为543 bp,共编码181个氨基酸,分子质量为20.3×10~3 Da,理论等电点为8.23。氨基酸同源系统进化分析表明,普通白菜Bcrbc S基因与同科植物的进化关系相近。实时定量分析结果表明,Bcrbc S基因在普通白菜叶中表达最强;在SA和Na Cl处理下,Bcrbc S基因表达量均在处理24 h后达到峰值。原核表达载体经IPTG诱导表达出分子质量约为20×10~3 Da的融合蛋白。

The full-length cDNA sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit(BcrbcS)gene was cloned from the leaves of pakchoi cultivar‘Suzhouqing’with resistance to downy mildew by RACE technique.This paper analyzed the expression pattern of this gene under different tissues by qRT-PCR and also prokaryotic expression characteristics of this gene by SDS-PAGE technology.Results of sequence analysis showed that the full-length cDNA of BcrbcS gene was 733 bp,among which the length of open reading frame was 543 bp.The total encodings were 181 amino acids.The molecular mass was 20.3×10^3 Da,and theoretical isoelectric point was 8.23.The phylogenetic analysis of amino acid homology showed that the pakchoi BcrbcS gene had similar evolutionary relationship with the other plants in the same family.Results of quantitative real-time analysis showed that the expression of BcrbcS gene was the strongest in pakchoi leaves.The expression of BcrbcS gene in pakchoi peaked at 24 hours after infection with SA and NaCl.Prokaryotic expression vector was induced by IPTG to express a fusion protein with a molecular weight of about 20×10^3 Da.