Cloning And Functional Studies Of CCD1,a Key Gene For Carotenoid Oxidative Cleavage In Osmanthus Fragrans

Carotenoids and their derivatives are not only precursors of biosynthesis of many stress-resistant substances,but also regulate plant color,aroma and flavor.They are widely used in medicine,health,food,health and other industries.The degradation of carotenoids is regulated by carotenoid cleavage dioxygenase.CCDs are the key enzymes in the pathway of carotenoid degradation and metabolism in plants.There are nine members,of which the regulation mechanism of CCD1 is the most complex.In order to further study the physiological function of Osmanthus fragrans OfCCDl gene in aroma biosynthesis,RACE-PCR technology was used to clone the OfCCDl gene of Osmanthus fragrans,and the sequence of nucleic acid and protein was analyzed to understand its structural characteristics.Semi-quantitative PCR method was used to study the expression pattern and content changes of OfCCD1 gene,and to clarify its expression pattern.Prokaryotic expression method was used to induce and purify the CCD1 gene.The protein of CCD1 was transformed into tobacco and Arabidopsis thaliana by leaf disc method and inflorescence dipping method,and its physiological function was revealed.The following experimental results were obtained:1.Nucleic acid sequence analysis showed that the full open reading frame(ORF)length of OfCCD1 gene was 1632 bp.The encoded protein sequence contained 543 amino acids.The molecular formula was C2785H4294N720O791S24.The relative molecular weight was about 61288.52,the theoretical isoelectric point was 6.27,the lipid solubility index was 80.77,and the total average hydrophobic index was-0.262.Its secondary structure is mainly composed of alpha-helix(11.60%),irregular curl(58.38%),elongation chain(23.76%)and beta-rotation angle(6.26%).Its three-dimensional structure of protein is the most similar to VP14 in maize.2.The expression levels of OfCCD1 gene in Osmanthus fragrans var.thunbergii,Osmanthus fragranscv.Latifoliu,Osmanthus fragrans(Thunb.)Lour and Osmanthus fragrans var.semperflorens were the lowest at the budding stage,and reached the peak at the early blossom stage,middle blossom stage,middle blossom stage and early blossom stage respectively,which indicated that the expression abundance of OfCCD1 gene varied with the species of Osmanthus fragrans,and had temporal and spatial specificity.3.0.5 M IPTG could induce the expression of OfCCD1 protein.Purified by nickel column,the target protein was obtained with molecular weight of 64 KDa.At the same time,the target band was identified by His antibody,and the size of the target protein was consistent with that of the target protein.4.Tobacco plants were transformed by leaf disc method,and resistant transgenic plants were obtained through resistance screening.At the same time,OfCCD1 gene could be amplified by semi-quantitative PCR detection.Arabidopsis thaliana(Col-0)were transformed by inflorescence dipping method,and positive plants with over-expression of OfCCD1 were obtained,which laid a foundation for further study on the physiological function of OfCCD1.