Study On The Cloning, Expression And Functional Of Boehmeria Nivea 4CL And CCR Gene Family Members

Ramie(Boehmeria nivea)is a widely planted textile staple crop in southern China.Ramie stem generates natural bast fibers.Whereas lignin content in the stem influences the fiber quality remarkably.In recent years,most of the research on reducing lignin content by gene engineering methods used to regulate key enzyme gene activity and to change lignin biosynthesis pathway were focused on model plants,less research on the phloem harvest plants.The functional characteristics of 4CLs and CCRs in ramie are still not clear.It is worth to study whether their functional characteristics in the lignin metabolic pathway of ramie is the same or different from other plant species.Ramie variety ‘Xiangzhu 3’ was used as the material in this study.According to the transcriptome information,the family members of 4CL and CCR were cloned from ramie,and the bioinformatics,expression differences,lignin and general flavone,and relevance of these genes were analyzed.The prokaryotic expression vector and the eukaryotic expression vector were constructed.The inducible expression,purification in vitro and the enzymatic characteristics of these recombinant proteins were analyzed.To explore and analysis the effects of two gene family members on their upstream and downstream material flow relations,it is helpful for understanding the mechanism of lignin biosynthesis in ramie stem.The main results were showed as follows:1.Three 4CL genes were cloned from ramie,named as Bn4CL1,Bn4CL2 and Bn4CL3;and four CCR genes were cloned from the ramie,named as BnCCR,BnCCR-1,BnCCR-2 and BnCCR-3.2.There were high similarity among the three Bn4 CLs and the 4CL from other plant.All of them contain two conserved motifs that called Box I and Box II,while 2 bases were altered in Bn4CL3.Bn4CL1 and Bn4CL2 belong to the stable proteins and have no transmembrane region,while Bn4CL3 belongs to unstable protein and has two transmembrane regions.Bn4CL1 and Bn4CL2 belong to class I,and Bn4CL3 belongs to class II.The three-dimensional structure homologous models were constructed for the three Bn4 CLs proteins.3.The four BnCCRs shared high identity with other plant CCR homologous sequences that acquired in public databases.BnCCRs contained conserved motifs of CCR.BnCCR has two transmembrane regions and 1 extra transmembrane helices,while other BnCCRs have no transmembrane region.BnCCR belonged to Group I,while other BnCCRs belonged to Group II.4.The highest expression levels of Bn4CL1 and Bn4CL2 were at the maturity stage,and the highest expression level of Bn4CL3 was at the late maturity.The contents of lignin and total flavonoids were positively correlated with the expression of Bn4CL3,but not significant correlated with the expression of Bn4CL1 and Bn4CL2.5.The highest expression levels of BnCCR and BnCCR-2 were at the maturity stage,and the highest expression levels of BnCCR-1 and BnCCR-3 were at the rapid growth period.The lignin content was negatively correlated with the expression of BnCCR,but not significant correlated with the expression of other BnCCRs.6.The recombinant prokaryotic expression vector of pQE30-4CL3 was constructed successfully.The recombinant protein of Bn4CL3 was induced and purified in vitro.The optimum pH and temperature were 8 and 40℃ respectively.The recombinant protein could catalyze p-coumaric acid,cinnamic acid and caffeic acid to produce corresponding coenzyme A ester,especially cinnamic acid,but it did not catalyze ferulic acid and sinapic acid.7.The recombinant prokaryotic expression vector of pQE30-CCR,pQE30-CCR-1,pQE30-CCR-2,pQE30-CCR-3 were constructed successfully.The recombinant protein of BnCCR,BnCCR-2 and BnCCR-3 were induced and purified in vitro.The optimum pH and temperature were 6 and 30℃ respectively for the enzymic catalytic reaction of the recombinant protein BnCCR.The recombinant protein of BnCCR could catalyze the p-coumaryl CoA to produce coumaraldehyde,whlie the recombinant protein of BnCCR-2 and BnCCR-3 may catalyze p-coumaryl CoA.8.The recombinant eukaryotic expression vector of pCAMBIA1302-4CL3 and pCAMBIA1302-CCR were constructed successfully,which could lay the foundation for the transient expression and the genetic transformation of ramie and the verification of gene function in plants.