Cloning,Function Analysis Of NCED3 Gene Critical To Cleavage Of Tobacco Carotenoids

Carotenoid and its degradation products are an important precursor for aromatic compounds.And the kind and content of degradation products is one of main indices to measure the quality of tobacco.Doing an in-depth study about key genes in the degradation pathway of tobacco carotenoid,identifying the function of important genes,can lay the foundation for improving the quality of tobacco leaf.Carotenoid cleavage dioxygenase(CCDs)is a key gene in plant carotenoid degradation pathway,the CCDs family has 9 members,including NCED2,NCED3,NCED5,NCED6,NCED9 and so on.The study obtains two c DNA sequence of the tobacco variety K326 carotenoid degradation genes NCED3 by homologous cloning method,and carry out bioinformatics analysis.Use the drought stress gene expression,while using in-fusion and gateway methods to build carriers of over expression and inhibition expression respectively,and transform tobacco plants by an Agrobacterium-mediated method.The results of the study provide a foundation for further analysis of NCED3 in the aspect of function research of drought resistance of tobacco.The main results are as follows:1 The flue-cured tobacco varieties K326 carotenoid degradation key gene NCED3 2 copies NCED3-2,NCED3-1 by means of cloning,and submit to the Genbank NCBI,the accession number are KM605434 and KM605435.Sequence analysis showed that NCED3-1 K326 and NCED3-2 genes contained an open reading frame(ORF)of 1842 bp and 1830 bp,each encodes 613 and 609 amino acids.Forecast the protein are 68177.8 Da and 67754.2Da respectively,the isoelectric point(PI)were 7.66 and 7.28.Transmembrane regions predicting?hydrophilic and signal peptide analysis showed that it is likely to be hydrophilic membrane proteins confined to mitochondria.Two secondary structure prediction model shows that the protein structure is main beta sheet and coil in it.Three protein secondary structure prediction analysis showed that the spatial structure of NCED3-1 and NCED3-2 is very similar.2 PEG6000 drought stress analysis showed that drought stress can induce the expression of NCED3 gene and the accumulation of endogenous ABA.And in the process before 12 h,the expression of NCED3 and ABA accumulation rate increased,then decreased gradually.3 Construct the over expression vector of K326 NCED3 gene and RNAi vector,and transform tobacco plants by an Agrobacterium-mediated method.Obtain over expression of NCED3-1 and positive plants of inhibiting expression.