Full-length CDNA Cloning And Quantitative Expression Analysis Of The Key Enzyme Gene In The Synthesis Pathway Of Chlorogenic Acid In Lonicera Macranthoides Hand-Mazz

Lonicera macranthoides Hand-Mazz is an important medicinal plant,and widespread planting in Chian.Chlorogenic acid(CGA)is an important secondary metabolite of plants and has important medicinal value of clearing heat and detoxification,antibacterial and antiviral.However,the molecular mechanism of chlorogenic acid synthesis and the key enzyme genes in L.macaroni have not been studied thoroughly.therefore,this paper used the gene cloning,determination of chlorogenic acid and expression pattern analysis,prokaryotic expression,as well as the carrier construction and other aspects to cloning full-length cDNA cloning and quantitative expression analysis of key enzyme genes in chlorogenic acid synthesis pathway.This study will provide the molecular mechanism of chlorogenic acid biosynthesis of L.macrocephala.The main research contents of this paper are as follows:(1)We cloned The full-length cDNA sequences of key enzyme genes Lm4CL,LmHCT and LmCCoAOMT in the chlorogenic acid synthesis pathway of L.macrocephala were cloned by RACE.The results showed that the full length of Lm4CL gene was 1960 bp(GenBank:MN 103349),the CDS sequence is 1617 bp and encodes 539 amino acids.The part sequence of LmHCT gene is 840 bp,the full length of LmCCoAOMT gene was 1096 bp(GenBank:MN103348),the full length of CDS was 735 bp,encoding 245 amino acids.Through cluster analysis,it was found that the Lm4CL gene is the most closely related Lonicera japonica,the furthest related Tsuga chinensis pritz,The LmCCoAOMT gene is most closely related to Nicotiana sylvestris and Petunia hybrid,and furthest related to Prunus mume and Eriobtrya japonica.The results showed that the target genes were distantly related between woody plants and herbaceous plants,which fully reflected the differences between woody plants and herbaceous plants.Amino acid sequence analysis shows that both Lm4Cl and LmCCoAOMT were hydrophobic amino acids without transmembrane structure,and the tertiary structure of the protein exists in the form of a-helix and ?-fold.(2)Using RT-qPCR to analyze the expression patterns of these three genes.The results of RT-qPCR showed that the expression level of Lm4CL gene was the lowest in middle flower and the highest in late stem.The expression of LmHCT gene was the lowest in early flower and the highest in late stem.The expression of LmCCoAOMT gene was the lowest in the middle stage of flower and the highest in the middle stage of stem.The gene expression in the stem and leaf tissues of L.macranthoides macronioides increased significantly during the growth process,which also shows that these genes are not only the key enzyme genes in the synthesis pathway of chlorogenic acid in L.macranthoides macronioides,but also may play an important role in the synthesis of lignin.(3)HPLC was used to determine the chlorogenic acid content of L.macrocephala in different stages,and the chlorogenic acid content of L.macranthoides and Lonicera japonica were compared.The results showed that the chlorogenic acid content of L.macranthoides was 2.88%in early stage,2.56%in middle stage and 2.81%in late stage.The chlorogenic acid content of Lonicera japonica was 2.59%in early stage,2.22%in middle stage and 1.37%in late stage.Correlation was used to analyze the correlation between gene expression and chlorogenic acid content.The results showed that the expression level of Lm4CL gene was positively correlated with the content of chlorogenic acid,while the expression level of Lj4CL gene was negatively correlated with the content of chlorogenic acid.The expression level of LmHCT gene was negatively correlated with the content of chlorogenic acid,while the expression level of LjHCT gene was strongly positively correlated with the content of chlorogenic acid.The expression level of LmCCoAOMT gene was weakly positively correlated with the content of chlorogenic acid in anthesis,while the expression level of LjCCoAOMT gene was strongly positively correlated with the content of chlorogenic acid.(4)We constructed prokaryotic expression vectors of Lm4CL-pcold-TF and LmCCoAOMT-pcold-TF gene,and the effects of different concentrations of 0.0,0.4,0.6 and 0.8 mmol/L of IPTG on protein expression were analyzed.The results showed that the two proteins mainly existed in the form of inclusion bodies.Compared with pcold-TF,Lm4CL had a large amount of protein enrichment at 100 kD-150 kD,and the IPTG induction was the best at the concentration of 0.6 mmol/L.Compared with pcold-TF,LmCCoAOMT protein had a large amount of protein enrichment at about 70 kD-100 kD,and IPTG induction was the best at 0.8 mmol/L concentration.In addition,the above proteins were purified and a good purification effect was obtained.(5)We constructed the eukaryotic expression vectors of Lm4CL-pCAMBIA1301 and LmCCoAOMT-pCAMBIA1301 gene,and subsequent studies were carried out to lay a foundation for exploring the molecular mechanism of chlorogenic acid synthesis and further improving chlorogenic acid content.