The Function Of Carotenoid Cleavage Dioxygenase Gene Family In Gardenia Jasminoides

Objective:The mature fruit of Gardenia jasminoides is rich in carotenoids and secondary metabolites of carotenoids,which is an ideal plant to study the biosynthesis pathway of carotenoids.Under the action of carotenoid cleavage dioxygenase（CCD）,carotenoids in plants will remove their prosthetic groups and produce a variety of apo-carotenoids,which can affect plant color,aroma,fruit ripening,and stress resistance.To study the function of CCD in Gardenia jasminoides,we sequenced the transcriptome by a high-throughput sequencing platform.The GjCCD gene sequence was screened from the sequencing results and the prokaryotic expression vector was constructed,and then its function was verified by catalytic reaction in vitro and in vivo.Methods:（1）The total RNA of the stem,leaf,flower,and fruit of Gardenia jasminoides was sequenced by Illumina Hi Seq X-ten high-throughput Next-generation sequencing platform separately,and the mixed RNA of the stem,leaf,flower,and fruit of Gardenia jasminoides was sequenced by the 3^rd generation sequencing platform Pacific Bioscience Sequel Platform.Through the joint analysis and functional annotation,the non-repetitive GjCCD gene sequences with complete open reading frame were screened.（2）The screened GjCCD genes were amplified and cloned into plasmid p ET-32a（+）expression vector and p SYNO-1 expression vector,respectively.After induction,the recombinant plastid which can express the target protein was screened,and the induction conditions were optimized to make sure to express enough soluble target protein for the follow-up experiment.The target protein was purified by Ni-NTA affinity chromatography for subsequent experiments.（3）The carotenoid-producing plasmids and the expression plasmids containing CCD genes were transformed into BL21（DE3）Escherichia coli and extracted the fat-soluble ingredient in the E.coli after induction.The carotenoid content and its shear products were detected by HPLC and UPLC-MS.Results:（1）Through the joint analysis of Gardenia jasminoides sequence data,11 GjCCD gene sequences were obtained.Through bioinformatics analysis,8 non-repetitive GjCCD gene sequences with complete open reading frames were obtained finally,which were tentatively named FF1RH5,FA1RM4,Gj5136,Gj3912,Gj9691,Gj9158,Gj8785,and Gj6843.Organelle localization prediction showed that proteins Gj8785,Gj5136,Gj3912,FA1RM4,and FF1RH5 were located in the cytoplasm,proteins Gj9691 and Gj6843 were located in the chloroplast,cytoplasm or nucleus,and protein Gj9158 was located in the chloroplast or cytoplasm.The results of the three-dimensional protein structure simulation show that FF1RH5,FA1RM4,Gj5136,Gj3912,Gj9158,and Gj8785 proteins have typical CCD protein structure,with a Fe²⁺and an oxygen ligand in the center,respectively.There are no ligands and metal ions in the Gj9691 protein center,and there is an extra OH-ligand in the Gj6843 protein center,which suggests that Gj9691 and Gj6843 proteins are structurally unstable.Phylogenetic analysis showed that FF1RH5 and Gj8785 proteins were clustered with At CCD1proteins and predicted to be CCD1 subfamily proteins;FA1RM4,Gj5136,Gj9691,Gj9158,and Gj3912 proteins were clustered with At CCD4 proteins and predicted to be CCD4 subfamily proteins;Gj6843 proteins clustered with Ce NCED1 proteins and predicted to be NCED1 subfamily proteins.（2）The GjCCD genes were cloned into plasmid p ET-32a（+）expression vector and p SYNO-1 expression vector.The recombinant plasmid was induced by IPTG,the fusion protein containing the solubilizing tag and six histidine tags can be expressed in BL21（DE3）E.coli.The expected size of the fusion protein produced by the p ET-32a（+）expression vector was88～105 KD,and the expected size of the fusion protein produced by the p SYNO-1expression vector was 102～119KD.The results of SDS-PAGE electrophoresis showed that Gj5136,Gj3912,Gj8785,FA1RM4,and FF1RH5 genes could be expressed on the p SYNO-1 vector,and Gj5136,Gj9691,Gj6843 genes could be expressed on p ET-32a（+）vector,which could be used in follow-up experiments.After the optimization of expression conditions,it was determined that the best concentration of inducer IPTG was 0.5mmol/L.After co-expression with molecular chaperone plasmid p G-KJE8,the expression of soluble target protein was significantly increased.The target protein can be purified by Ni-NTA affinity chromatography,and the purified protein was enough to be used for the follow-up catalytic test in vitro.The splicing product of CCD protein was not detected in vitro.（3）The plasmids which can synthesize zeaxanthin,lycopene,orβ-carotene were cotransformed into E.coli BL21（DE3）with the expression plasmids inserted GjCCD genes,respectively.The transformed plasmids were co-expressed,and the fat-soluble products were screened by HPLC and verified by UPLC-MS.Finally,it was found that FA1RM4 and Gj5136 proteins of the CCD4 subfamily could cleave zeaxanthin to produce crocetin dialdehyde,while Gj3912 and Gj5136 proteins could cleave lycopene to crocetin dialdehyde.Conclusion:Through gene screening,codon optimization,construction of expression vector,optimization of expression conditions,transformation,and induction of co-expression,catalysis in vitro,catalytic in vivo,and detection of HPLC and UPLC-MS,this study successfully verified the function of three CCD4 subfamily genes of FA1RM4,Gj5136 and Gj3912 from Gardenia jasminoides in E.coli.The results showed that FA1RM4 and Gj5136 proteins could cleave zeaxanthin in 7,8（7′,8′）site,Gj3912 and Gj5136 proteins could cut lycopene in 7,8（7′,8′）site,resulting in the formation of crocetin dialdehyde.