Function Study And Expression Analysis Of Tobacco Carotenoid Cleaving Dioxygenase(CCD1)

Tobacco(Nicotiana tabacum)is a herbaceous plant of the Nicotiana genus Solanaceae,and it is an important leaf-use cash crop in the world.Carotenoids are important precursors for the formation of volatile aroma components of plants,and their enzymatic degradation products have an important impact on the aroma quality of plants.Carotenoid cleavage dioxygenase(CCDs)is the main functional enzyme that catalyzes the cleavage of carotenoids to produce ketones and aldehydes in plants.The cleavage products are necessary for the growth and development of plants,and the formation of plant color and aroma substances.However,its specific mechanism and products in the degradation of tobacco carotenoids are still unclear.Therefore,this study used common tobacco K326 as the experimental material,three key genes NtCCD1 for carotenoid degradation were cloned,and detailed bioinformatics analysis was performed;qPCR was used to analyze the expression pattern of NtCCD1 in different tobacco varieties at different developmental stages.The subcellular localization indicated that its protein was located in cytoplsam of tobacco;The one-step cloning method was used to construct the p MAL-NtCCD1a-His expression vector,and the NtCCD1 recombinant protein was isolated and purified;The bacterial color experiment combined with SPME-GC-MS was used to investigate its enzymatic activity,and it demonstrated that NtCCD1 a can cleavage ?-carotenoids to generate ?-ionone in vitro.Our study provides a theoretical basis for the industrial application of NtCCD1.The main findings are as follows:1.In this study,three NtCCD1 genes were cloned from common tobacco K326,named NtCCD1 a,NtCCD1 b and NtCCD1 c.The bioinformatics analysis revealed that the three NtCCD1 genes were 1632 bp,1644 bp and 1638 bp in length,encoding 543,547 and 545 amino acids,respectively.The protein sequence comparison revealed that they also contained the conservative structure of the CCDs family.NtCCD1 a and NtCCD1 b have a close homology relationship,while the homology relationship between NtCCD1 a and NtCCD1 b and NtCCD1 c is relatively far;Phylogenetic analysis shows that NtCCD1 has the closest genetic relationship with potato St CCD1 and tomato Sl CCD1,indicating that they are highly conserved in the process of evolution,and may have evolved from the same species.Analysis of the gene structure and conservative motifs of NtCCD1 shows that the CCD1 gene has the most complex structure in the CCDs family,suggesting their functions are diversity.2.qPCR indicated that there is no significant difference in the expression of NtCCD1 gene in tobacco K326 and Honghua Dajinyuan during the growth periods,but NtCCD1 m RNA level is significantly higher in normal tobacco K326 than Honghua Dajinyuan durig the mature period;Among the three NtCCD1 genes,the expression level of NtCCD1 c gene is significantly higher than that of NtCCD1 a and NtCCD1 b in all the tissues,including roots,roots,stems and leaves Moreover,the expression of NtCCD1 gene in leaves was significantly higher than the other tissues.3.The subcellular localization vector of NtCCD1 was constructed.Confocol images indicated that the proteins of tobacco GFP-NtCCD1 a,GFP-NtCCD1 b and GFP-NtCCD1 c were located in the cytoplasm of tobacco,which is consistent with the localization of CCD1 proteins in other higher plants that have been reported.4.The prokaryotic expression vector p MAL-NtCCD1c-His was constructed,and its expression was detected by SDS-PAGE electrophoresis.The induction conditions were optimized.The results showed that the best induction conditions are: 20?,1 mmol/L IPTG induces the highest expression of recombinant protein of p MAL-NtCCD1c-His.5.The enzymatic activity of NtCCD1 c was studied through the bacterial color complementation experiment.The study shows that the NtCCD1 c protein can cleave?-carotene,lycopene and zeaxanthin in vitro;SPME-GC-MS was used to analyze the enzyme digestion products,The results show that NtCCD1 c protein can produce ?-ionone by cleaving ?-carotene,but the products of lycopene and zeaxanthin cleavage by NtCCD1 c protein are still unclear.