Cloning And Functional Analysis Of CCDs Gene From Cerasus Humilis

Chinese dwarf cherry(Cerasus humilis Bge.)is a unique shrub fruit tree of China.Its fruit is of bright color,unique flavor,rich aroma and rich nutrition.It is important to study the metabolism of color and flavor The CCDs gene can be catalyzed by catalytic cleavage at different sites of carotenoids to generate a variety of apo-carotenoids.The apo-carotenoids have many biological functions,such as CCD1 and CCD4,which mainly regulate the color and flavor of plant fruits.,CCD7 and CCD8 are involved in the synthesis of solokinase.NCED is the main rate-limiting enzyme in the synthesis of abscisic acid.In this study,the Cerasus humilis variety 'Nongda 4' was used as the material to clone the ChCCDl gene and the ChCCD4 gene.The prokaryotic expression vector was constructed and introduced into the engineering bacteria for the synthesis of carotenoids.The color change and GC-MS of the bacterial liquid were observed.Lysates were detected and the function of the ChCCDs gene was analyzed.The main findings are as follows:(1)Using the RT-PCR cloning method,the CDS sequences of the two ChCCDs genes of Cerasus humilis.sinensis were obtained from the analysis of Cerasus humilis.sibiricus transcriptome data,which were ChCCD1 and ChCCD4,respectively.The CDS sequence length of ChCCD1 gene is 1644bp,which encodes 547 amino acids.The CDS length of ChCCD4 gene is 1794bp and encodes 597 amino acids.The bioinformatics analysis revealed that the two ChCCDs genes belong to the CCDs gene family,and the encoded proteins all contain RPE65 conserved domains.Phylogenetic tree analysis showed that the CCDs proteins of Eurasian and Lie were closely related to the CCDs of Prunus mume,Prunus persica and pear.(2)Extracting the total RNA of fruits from different varieties at different stages of fruit production of Cerasus humilis varieties 'Nongda 4','Nongda 5','Nongda 6' and 'Nongda 7'.The cDNA was transcribed into cDNA and the expression levels of CCDs in different cultivars were analyzed by fluorescent quantitative PCR.The results showed that the total expression of ChCCD1 in the four cultivars was higher in the young fruit period and lower in the maturity period;the expression level of ChCCD4 in the young fruit period of 'Nongda 4' was higher and the maturity period was lower,among other three cultivars.The expression level in young fruit is relatively low,and the maturity is relatively high.(3)Connect the ChCCDs gene to the prokaryotic expression vector pet-28a to construct prokaryotic expression vectors pet-ChCCD1 and pet-ChCCD4.The two prokaryotic expression vectors were respectively introduced into engineering bacteria capable of synthesizing carotenoids.As a result,it was found that ChCCD1 can degrade lycopene ?-carotene and zeaxanthin,and ChCCD4 can also degrade lycopene and P-carotene,but there is no ChCCDl is significant,and ChCCD4 hardly degrades zeaxanthin.GC-MS analysis of lysates of ChCCDs gene revealed that ChCCDl lycopene produced 6-methyl-5-hepten-2-one,and ?-carotene was cleaved to produce ?-ionone.