玳玳花瓣脂氢过氧化物裂解酶基因cDNA的克隆及原核表达

以玳玳花瓣为材料,采用RT-PCR和RACE技术,克隆了玳玳HPL基因cDNA全长,全长为1 776 bp,其中包含52 bp的5′非编码区,224 bp的3′非编码区,1 500 bp的编码区(编码499个氨基酸,分子量为55.7 ku)。将该HPL基因cDNA编码区的核苷酸序列及所推导的氨基酸序列与其他植物的HPL基因cDNA序列进行比较,推断玳玳花瓣HPL属于13-HPL类。通过PCR扩增得到了玳玳HPL基因组全长。测序结果显示玳玳HPL基因中包含1个长2 374 bp的内含子。将分离出来的玳玳HPL基因cDNA的编码区序列插入pET-15b载体上构建原核表达载体,在细菌BL21细胞中进行原核表达。结果表明:该编码区片段可在原核细胞中大量表达,其表达产物分子量大小约为55 ku,与13-HPL蛋白的分子量55.7 ku相符。本研究为下一步利用HPL基因cDNA进行遗传转化研究奠定基础,将为花卉香味的遗传改良提供一条新途径。

cDNA Cloning and Prokaryotic Expression of Hydroperoxide Lyase in Daidai (Citrus aurantium)

In this research,the full length cDNA of Hydroperoxide lyase (HPL) from Daidai (Citrus aurantium) petals was cloned by RT-PCR and RACE and tried to express this gene in E.coli.The cDNA sequence of Daidai HPL was 1 776 bp long.It contained an ORF encoding 499 amino acid residues (Mw=55.7 ku),a 5'untranslated region of 52 bp and a 3'untranslated region of 224 bp.Daidai HPL was a hydrophilic protcin.Compared with HPLs of other plants,this phylogenetic analysis suggested that Daidai HPL was a member of the 13-HPL family.Genomic fragment of Daidai HPL gene was also amplified by using PCR,and subsequent sequencing analysis showed that the Daidai HPL gene contained one intron of 2 374 bp.The Daidai HPL gene cDNA was inserted into the vector pET-15b.The expression construct was transformed into BL21 and was induced.Daidai HPL was expressed at high level in E.coli.SDS-PAGE analysis showed that Daidai HPL was 55 ku,which is approximately the same with the calculated molecular weight 55.7 ku.The research aimed to control the expression of HPL gene and regulate he production of scents in plants,and provide a new approach to the genetic improvement of flower scents.