Study On The Enzyme Activity Characteristics And Functional Differentiation Of Carotenoid Cleavage Dioxygenase 4(CCD4) In Brassica Napus L.

Carotenoids are a natural isoprene like pigment composed of 40 carbon atoms,typically appearing in red,yellow,or orange.Our previous research results showed that the loss of function of the carotenoid lyase dioxygenase 4 gene(BnaC3.CCD4)in Brassica napus significantly increases the content of violaxanthin in the petals,resulting in a change in flower color from white to yellow.However,it is currently unclear whether the variation in flower color is caused by the action of BnaC3.CCD4 on violaxanthin.In Brassica napus,the Bna.CCD4 gene has four copies: BnaA1.CCD4,BnaA8.CCD4,BnaC1.CCD4,and BnaC3.CCD4,with differentiated expression patterns.When the four copies were expressed separately in yellow flower Brassica napus,the flower colors of the transgenic materials were significantly different;When using CRISPR-Cas9 technology to knock out the Bna.CCD4 gene in Brassica napus,there were significant differences in carotenoid content among different mutant materials.Therefore,the enzyme activity characteristics of the four proteins have differentiated,but the specific differentiation characteristics need further research.Based on the above issues,this study investigated four CCD4 proteins in Brassica napus using methods such as bioinformatics prediction and in vitro enzyme activity,further revealing the action mode of Bna.CCD4 at the molecular level and the possible reasons for functional differentiation.The main research findings are as follows:1.Bioinformatics analysis: The results of DNA sequence and amino acid sequence analysis showed that the four CCD4 genes in B.napus could be divided into two groups,BnaA1.CCD4 and BnaC1.CCD4 were the first group,BnaA8.CCD4 and BnaC3.CCD4 were the second group,and the sequence similarities within and between groups were97.0 % and 88.0 %,respectively.Phylogenetic analysis showed that AtCCD4 had the closest homology with Bna.CCD4.Domain analysis showed that all four Bna.CCD4 proteins contained the RPE65 domain.Conserved motif analysis showed that there was no difference in motif composition among the four proteins.Comparing the conserved motifs of Bna.CCD4 with the conserved motifs of AtCCD4,CitCCD4,GjCCD4,etc.,it was found that it has the same motif composition as AtCCD4.2.The three-dimensional structures of BnaA1.CCD4,BnaA8.CCD4,BnaC1.CCD4 and BnaC3.CCD4 proteins were established by sequence homology modeling,and molecular docking was performed with 11 carotenoid substrates such as All-trans-violaxanthin,β-carotene and Lutein.Molecular docking results showed that BnaA8.CCD4 and BnaC3.CCD4 had similar substrate affinity.The three substances with the highest affinity were 9-cis-violaxanthin,β-cryptoxanthin and Zeaxanthin,respectively.BnaA1.CCD4 and BnaC1.CCD4 have similar substrate affinity.The three substances with the highest affinity are All-trans-neoxanthin,9-cis-neoxanthin and Lutein,respectively,while the four CCD4 proteins all show low affinity for ζ-carotene,Lycopene and Phytoene.3.The prokaryotic expression vector pET-28a-Bna.CCD4 of Bna.CCD4 gene was constructed.The optimal induction conditions were determined to be 16 °C,20 h,0.2mmol/L IPTG,and an in vitro enzyme activity reaction system was established.The results of in vitro enzymatic activity showed that four Bna.CCD4 proteins could cleave Lutein,Zeaxanthin,β-carotene,α-carotene(C40)and 8-apo-β-carotenal(C30)in vitro.The results of GC-MS and LC-MS analysis showed that Bna.CCD4 could cleave β-carotene and 8-apo-β-carotenal at 9 and 10 carbon double bonds to produce volatiles of C13 and dialdehydes of C14 and C17.4.The affinity and catalytic efficiency of BnaA1.CCD4,BnaA8.CCD4,BnaC1.CCD4 and BnaC3.CCD4 proteins for β-carotene and Lutein were determined by Lineweaver-Burk equation.The results showed that in terms of substrate affinity,the order of affinity for β-carotene and Lutein was BnaA1.CCD4 > BnaA8.CCD4 >BnaC1.CCD4 > BnaC3.CCD4 and BnaC3.CCD4 > BnaC1.CCD4 > BnaA8.CCD4 >BnaA1.CCD4.In terms of substrate catalytic efficiency,the order of catalytic efficiency for β-carotene and Lutein was BnaA1.CCD4 > BnaC1.CCD4 > BnaA8.CCD4 >BnaC3.CCD4 and BnaC3.CCD4 > BnaC1.CCD4 > BnaA1.CCD4 > BnaA8.CCD4.5.Nine point mutations were generated in BnaC3.CCD4 protein by site-directed mutagenesis,which were the same as BnaA8.CCD4.Then the enzyme activity of the mutant protein was studied in vitro.The results showed that the mutation of amino acids173 and 389 could significantly improve the catalytic efficiency of BnaC3.CCD4 forβ-carotene,indicating that the mutation of these two sites led to the difference in the catalytic efficiency of BnaA8.CCD4 and BnaC3.CCD4 for β-carotene.