桂花OfCCD1基因启动子克隆与表达特性

Cloning and transient expression assay of OfCCD1 gene promoters from Osmanthus fragrans

类胡萝卜素裂解双加氧酶(CCDs)参与植物中多种类胡萝卜素的代谢,在桂花Osmanthus fragrans中类胡萝卜素裂解双加氧酶基因1(OfCCD1)对花香物质的合成具有重要作用。根据前期获得的桂花‘堰虹桂’O.fragrans‘Yanhong Gui’转录组数据库中OfCCD1的序列和前人已发表的OfCCD1序列设计引物,利用染色体步移技术从桂花基因组DNA中扩增获得OfCCD1基因2个拷贝的启动子序列OfCCD1P-L和OfCCD1P-S,其长度分别为2 747 bp和981 bp。2个启动子序列中均含有参与光响应的元件,热激响应元件(HSE),脱落酸(ABA)响应元件(ABRE)和乙烯响应元件;此外,2个启动子中均含有4个ACGT序列。将2个启动子序列分别替代p BI121上的35 S启动子,将构建的载体通过农杆菌Agrobacterium tumefaciens介导转化烟草Nicotiana tabacum叶片进行瞬时表达,发现2个启动子都具有驱动GUS报告基因表达的功能。

Charge-coupled devices（CCDs） are involved in the degradation and biosynthesis pathways of carotenoid metabolism, and in Osmanthus fragrans, OfCCD1 plays an important role in the biosynthesis of scent compounds. In this study, primers were designed based on the transcriptome database of O. fragrans ‘Yanhong Gui＇ and another reported OfCCD1 gene sequence. Two promoter sequences of OfCCD1, named OfCCD1 P-L and OfCCD1 P-S, were cloned by genome walking. Results showed that the two promoter sequences in length were 981 bp for OfCCD1 P-L and 2 747 bp for OfCCD1 P-S. Also, the light responsive element, heat stress element, abscisic acid（ABA） responsive element, and ethylene responsive element were found in the two promoter sequences which including four sequences： ACGT Adenine（A）, cytosine（C）, guanine（G）, and thymine（T）.The 35 S promoter in p BI121 was replaced by OfCCD1 P-L and OfCCD1 P-S. Afterward, the new vectors were transformed into Nicotiana tabacum leaves by Agrobacterium tumefaciens. The transient expression showed that the two cloned promoters could drive the expression of downstream reporter genes.