Cloning And Functional Analysis Of Chalcone Synthase Gene And Cinnamoyl-CoA Reductase Gene In Pinus Koraiensis

Chalcone synthase(CHS) is a key enzyme during biosynthesis of flavonoids in plants.Itcatalyzes the condensation of a4-coumarate CoA with three malonyl-CoAs to produce chalconeand further generates flavonoids.Flavonoids play an important role in plants color and defensesystem.Cinnamoyl-CoA reductase(CCR) is the first key enzyme in lignin biosynthesis pathway.Itcould catalyze three coenzyme A esters of hydroxycinnamic acid to the corresponding cinnamicaldehyde.Lignin also plays an important role in plants defense system.Pinus koraiensis is pinekossen genus,evergreen coniferous trees.It is one of the endemic species in NortheastChina.Study on CHS gene and CCR gene in Pinus koraiensis is of great significance inunderstanding the biosynthesis of flavonoid and lignin.This research cloned CHS and CCR genefrom P. koraiensis.It lays the foundation for further study on CHS and CCR protein structure andfunction of P. koraiensis. The results are as follows:1.CHS-F and CHS-R were taken as primers for PCR amplification of CHS gene.Sequencingresult showed that the fragment is1191bp.The sequence was submitted to GenBank and theaccession number is KJ796482. P. koraiensis CHS gene contains an open reading frame of1191bp,the gene encodes396amino acids,molecular weight is43.37kD,theoretical isoelectricpoint is5.88.CCR-F and CCR-R were taken as primers for PCR amplification of CCRgene.Sequencing result showed that the fragment is975bp.The sequence was submitted toGenBank and the accession number is KJ796481. P. koraiensis CCR gene contains an openreading frame of975bp,the gene encodes324amino acids,molecular weight is35.63kD,theoretical isoelectric point is6.19.2.BLASTP results showed that,P.koraiensis CHS gene encoding protein has high homologywith A. thaliana,Capsicum annuum,Vaccinium ashei,Populus trichocarpa,83%,86%,84%,83%respectively.P.koraiensis CCR gene encoding protein has high homology withEucalyptus saligna,Picea abies, Pinus radiate,77%,93%,100%respectively.It has low homologywith Arabidopsis thaliana, Oryza sativa,49%,57%respectively.3.Multiple sequence alignment results showed that the amino acid sequence of P. koraiensisCHS gene is highly conservative in system evolution with other plants.The amino acid sequenceof P. koraiensis CCR gene is highly conservative in system evolution with other plants.4.The phylogenetic tree of P. koraiensis CHS gene and P. koraiensis CCR gene wereconstructed by using MEGA5.0with Neighbor-Joining method among P. koraiensis and other plants respectively.The result showed:Pinus massoniana and Picea abies have the closestphylogenetic relationship with P. koraiensis CHS gene.Pinus radiate and Picea abies have theclosest phylogenetic relationship with P. koraiensis CCR gene.Eucalyptus saligna and Ginkgobiloba also have a closer genetic relationship with P. koraiensis CCR gene.