甘蓝型油菜BnPPR636及其启动子的克隆与表达分析

Cloning and Expression Analysis of BnPPR636 and Its Promoter in Rapeseed(Brassica napus)

PPR蛋白对植物的生长发育、逆境抗性以及育性具有重要的调节作用。本研究根据氨基酸序列的保守性,利用CODEHOP方法,通过简并引物扩增甘蓝型油菜基因组DNA,得到PPR基因片段。结合RACE技术,在甘蓝型油菜品系120中扩增得到了PPR基因cDNA的全长序列。分析发现该PPR基因编码一个含636个氨基酸的蛋白质,具有11个PPR基序,不含有内含子,具有典型的PPR基因特征,命名为BnPPR636。Blast比对发现,BnPPR636与白菜P2克隆的一个未知蛋白的氨基酸序列一致性最高,达到75%。RT-PCR分析表明,BnPPR636在花中的表达量最高,在根与角果中的表达量次之,叶和茎中较低。进化树分析表明,BnPPR636蛋白与其他植物中和CMS育性恢复有关的PPR蛋白序列的一致性较高。以基因组DNA为模板,利用染色体步移技术,得到了BnPPR636基因起始密码子上游1145bp的DNA片段。经生物信息学软件分析表明,该序列具有典型的启动子序列特征,并含有许多相关的顺式作用元件,可能参与调节花和根的发育。

PPR protein plays an important role in plant development,especially for characters involved in fertility restoration and resistance to biotic and abiotic stresses.Based on the conserved amino acid sequence,a fragment of PPR gene was cloned in Brassica napus by CODEHOP method.The full length cDNA of the gene was amplified by RACE which contains an ORF of 1 908 bp in length encoding a protein with 636 amino acids including 11 PPR motifs,named BnPPR636.Genomic DNA sequence of BnPPR636 was completely identical to the cDNA sequence,indicating no intron in the gene.The highest homology was found with an unknown protein of P2 clone of B.rapa,which had 75% identity to BnPPR636.RT-PCR with BnPPR636 gene specific primers showed it was highly expressed in flower,moderately expressed in root and developing silique,with lowest expression in leaf and stem.Phylogenetic analysis showed that BnPPR636 had high consistency with the PPR protein sequence of other plants related to CMS Restoration.Using chromosome walking techniques,a 1 145 bp DNA fragment located upstream of the start codon of BnPPR636 gene was obtained based on genomic DNA as template.Analysis of bioinformatics software showed the sequence had the typical characteristics of the promoter sequence,and contains many cis-acting elements,which may be involved in the regulation of flower and root development.