Identification And Prokaryotic Expression Analysis Of CCD Genes Related To Carotenoid Metabolism In Cerasus Humilis

Chinese dwarf cherry[Cerasus humilis(Bge.)Sok],a small shrub of the genus Cerasus in the Rosaceae family,is a Carotenoid fruit tree.As a natural pigment,Carotenoid plays an important role in the production of medicines,cosmetics and food additives.The regulation of Carotenoid synthesis through bioengineering can contribute to the development and promotion of functional products,improve the effect of food safety management,and maintain human health.A variety of Carotenoid are produced by the catalysis of Carotenoid cleavage dioxygenase(CCD),which plays an important role in the formation of aroma,color,Carotenoid and stress response of plants.In this study,the Ch CCDs gene family(Ch CCD)was identified and analyzed by bioinformatics method,and the expression pattern of Ch CCDs gene in ’Nongda 4’ fruit was analyzed by q RT-PCR..In addition,the genes Ch CCD7,Ch NCED1 and Ch NCED5 were cloned from ’Nongda 4’,and the prokaryotic expression vectors of Ch CCD7 and Ch NCED1 were constructed,it is valuable for the function analysis of the Carotenoid engineering bacteria and the application of the Carotenoid of the lysate.It will lay a foundation for the breeding work in Chinese dwarf cherry.The main findings are as follows:1.A total of 10 CCD genes were identified based on the Chinese dwarf cherry genome data,including Ch CCD-like-a,Ch CCD-like-b,Ch CCD1 a,Ch CCD1 b,Ch CCD4,Ch CCD7,Ch CCD8,Ch NCED1,Ch NCED5 and Ch NCED6.The protein size,isoelectric point and other characteristic parameters of Ch CCD family members have little difference,and all of them belonged to hydrophilic proteins.Amino acid sequence alignment revealed that most Ch CCD proteins contained four conserved histidine active sites,while Ch CCD-like-a and Ch CCD-like-b lacked the first histidine active site,which may be the difference between the two genes and the others.Phylogenetic analysis showed that the 10 CCD families were divided into six subfamilies: CCD1,CCD4,CCD7,CCD8,NCED and CCD-like.The CCD protein of Chinese dwarf cherry was more closely related to peach and plum than to apple and pear.The genetic structure of each subtribe was found to be similar,in which NCED subtribe had only one exon and no intron.The results of promoter cis-element analysis showed that the genes of Ch NCED5 and Ch NCED6 were closely related to the regulation of Abscisic acid.2.Transcriptome analysis showed that the expression trends of 10 genes were not identical during the different growth stages of ’Nongda 3’,’nongda 4’ and ’Nongda 5’ fruits in Chinese dwarf cherry,which could be divided into four types,for example,Ch CCD-like-a,Ch CCD-like-b,Ch CCD7,Ch CCD8 and Ch NCED6 were not expressed,Ch NCED1 and Ch NCED5 were on the rise,Ch CCD1 a and Ch CCD1 b had no significant difference,the expression of Ch CCD4 was low and on the decline.At the ripening stage of ’Nongda 4’ and ’Nongda 5’,the expression trends of 10 genes were basically the same,Ch CCD-like-a,Ch CCD-like-b,Ch CCD7,Ch CCD8 and Ch NCED6 were not expressed,the expression of Ch CCD4 was lower,and the expression of Ch NCED5 was the highest,the expression of Ch NCED1 was higher in ’Nongda 4’ fruit than in ’Nongda 5’ fruit.There were five expression patterns in different tissues of ’Nongda 4’,the expression of Ch NCED5 were high in fruit,the expression of Ch CCD1 a,Ch CCD1 b and Ch CCD4 were high in leaf,and the expression of Ch CCD1 a and Ch CCD1 b in Seed kernel,the expression of Ch CCD1 a was highest in rhizomes and true roots.It is suggested that the Ch NCED1 and Ch NCED5 genes may be closely related to fruit maturation.3.The expression patterns of 10 CCD genes in different growth stages of ’Nongda 4’ fruit were analyzed by q RT-PCR.There are roughly three types: Ch CCD-like-a,Ch CCD-like-b,Ch NCED1 and Ch NCED6 showed an ascending-descending-ascending-descending trend;Ch CCD1 a,Ch CCD1 b,Ch CCD4,Ch CCD7 and Ch CCD8 showed a descending-ascending-descending trend;and Ch NCED5 showed an ascending trend,which was consistent with the expression pattern of previous transcriptome data analysis.The results suggested that there might be a certain relationship between Ch NCED5 and fruit development.4.The CDS sequences of Ch CCD7,Ch NCED1 and Ch NCED5 genes were obtained from the database obtained by the whole genome sequencing,and the primers were designed,the CDS sequence of Ch CCD7 gene with restriction site was amplified from tender stem c DNA of ’Nongda 4’.The CDS sequences of Ch NCED1 and Ch NCED5 genes with restriction endonuclease sites were amplified from the mature fruit c DNA of ’Nongda 4’.The analysis of Ch CCD7,Ch NCED1 and Ch NCED5 proteins showed that Ch CCD7 encodes 615 amino acids,the relative molecular mass is about 68499.76 Da,the theoretical isoelectric point is 5.67,the liposolubility index is 83.51;Ch NCED1 encodes 632 amino acids,the molecular weight is about 70288.74 Da,the theoretical isoelectric point is 6.73,the liposolubility index is 73.61;Ch NCED5 encodes 617 amino acids,the molecular weight is about 68027.19 Da,the theoretical isoelectric point is 6.45,the liposolubility index is 79.00;Ch CCD7,CHNCED1 and CHNCED5 are hydrophilic proteins without signal peptide,which have a high proportion of extended chain and random coil in secondary structure,and have typical structure domain of CCD family.5.The prokaryotic expression vectors p ET-Ch CCD7 and p ET-Ch NCED1 were successfully constructed.After IPTG induction at 37 °C,the two genes were able to express the target protein,and the size of the target protein was about 69 and 70 KDa,which were consistent with the predicted protein size.