Cloning, Expresssion And Function Analysis Of CCD1 Gene Critical To Cleavage Of Tobacco Carotenoids

Tobacco carotenoids and their apocarotenoid derivatives play essential physiological and developmental roles and provide plants tolerance to a variety of stresses. Carotenoid cleavage dioxygenases(CCDs) is one of the key genes involved in the metabolic pathways of carotenoid. The CCD gene family contained nine members. Among them, the function of CCD1 is most complicated. In this research, CCD1 was cloned. Their sequences were analysed using Bioinformatics softwares. The gene expression patterns were analysed in different issues of tobacco and in different ecological zones as well as in different varieties. Meanwhile, the overexpression and RNA interferencing vectors were constructed, and then transplanted to tobacco. At the same time, prokaryotic expression vectors were also constructed and expressed in E.coli. These results provide some basis for the function analysis to CCD1. The main results were as follows:1. Using RACE method, three copies(named CCD1-1, CCD1-2 and CCD1-3) of the full-length c DNA sequence of tobacco carotenoid cleavage oxygenases1 were cloned from K326. Sequences analysis showed that they contained 1635-bp, 1647-bp and 1641-bp open reading frame(ORF) encoding the deduced protein of 544, 548 and 546 amino acid residues with the theoretical isoelectric point(p I) of 6.51, 6.55 and 6.25, respectively. The calculated molecular masses were all 61 k Da. The similarity of CCD1 with tomato, pepper and other plants reached to more than 80%. Protein hydrophobicity and transmembrane region’s prediction analyses indicated that they all inclued one transmembrane helical region The secondary structure prediction results showed that they were mainly made up by beta turns and random coil. The tertiary structure prediction results displayed that they showed high similarity except in random coil.2. Expression analysis showed that the expression level of CCD1 was higher in flower, and the least in root. And, the gene expression patterns in different ecological zones displayed that the expression of CCD1 in K326 showed the highest level in Henan province. After topping, among the three sub-ecological zones including Nanxiong in Guangdong province, Xuancheng in Anhui province and Pingdingshan in Henan province, the expression of CCD1 in K326 presented the highest level in Nanxiong. Accordingly, the amout of carotenoids were the most in Nanxiong. In addition, expression level of CCD1 and CCD3 in Yuyan 10 was higher than in K326 in Henan province and in K326 higher than in Anyan 240 in Anhui province, while in NX232 higher than in K326 in Guangdong province.3. The overexpression and RNA interferencing vectors were constructed using in-fusion and gateway methods. Then the vectors were transplanted to tobacco using agrobacterium mediated method. Fluorescence quantitative analysis showed that the expression level of CCD1 increased in overexpression transgenic positive plants and reduced in RNAi transgenic positive plants. At the same time, prokaryotic expression vectors of CCD1 were constructed and expressed in BL21-Codon Plus(DE3)-RIPL. The results showed that the soluble fusion protein with the molecular weight of 64 k D could be induced by IPTG. These results provided the fundamental bases for the function analysis of CCD1.