甘蓝型油菜PEP转运子BnPPT1基因的克隆、序列分析和表达模式

Molecular cloning and characterization of BnPPT1 gene from Brassica napus

磷酸烯醇式丙酮酸/磷酸转运子基因(Phosphoenolpyruvate/phosphate translocator1,PPT1)是细胞质体碳水化合物转运的一个关键基因,在植物莽草酸代谢和脂肪酸生物合成途径中起着重要作用。根据十字花科拟南芥PEP转运子AtPPT1基因序列与油菜数据库BBSRC Brassica DB中相应ESTs设计全长引物,以甘蓝型油菜(Brassica napus)幼嫩叶片基因组DNA和种子cDNA为模板,克隆到了甘蓝型油菜PEP转运子基因全长序列,命名为BnPPT1。该基因基因组gDNA全长为2 075 bp,含有8个内含子,编码区长度为1 224 bp,编码407个氨基酸。序列比对结果表明,BnPPT1与同属于十字花科拟南芥AtPPT1转运子同源性很高,仅有个别氨基酸的差异。进一步采用半定量RT-PCR分析表明,BnPPT1基因在油菜各器官中表达差异很大,在幼嫩的子叶和茎中有较高丰度的表达,在中早期发育的种子中表达丰度逐步升高,种子成熟后期没有表达,暗示该基因在油菜籽粒中油份等物质积累中有重要功能。

Phosphoenolpyruvate（PEP）/phosphate translocator 1（PPT1） gene is best known for its role as the translocator in importing the PEP into plastids as substrates for fatty acid biosynthesis and more important for the shikimate pathway.In order to investigate the function of PPT1 from Brassica napus,we genetically isolated a full-length cDNA encoding PPT1 with total RNA of developing seeds from Brassica napus,and the corresponding genomic DNA region from DNA of young leaves with specific primers designed according to sequences of AtPPT1 and ESTs from Brassica napus by searching the BBSRC BrassicaDB.This gene,designated BnPPT1,is 2 075 bp in length of the genomic DNA with eight introns and is 1 224 bp in length of the complete coding sequences encoding 407 amino acids.Multiple sequence alignment of protein sequences showed BnPPT1 shared high homology with AtPPT1 of Arabidopsis thaliana.Furthermore,semi-quantitative RT-PCR analysis carried out with RNA isolated from tissues and various stages of developmental seeds from Brassica napus demonstrated that BnPPT1 was preferentially expressed in seeds at middle developmental stages,which indicated that BnPPT1 gene might play an important role in carbohydrate metabolism of developing seeds in Brassica napus.