Cloning Of Bp4CLs Promoter And Preliminary Identification Of Gene Functions In Betula Platyphylla Suk.

Betula platyphylla Suk.is one of the important tree species in the north of China.It has important characteristics such as fast growth,cold-tolerance and drought tolerance,so it has important research value.4-Coumaric acid:Coenzyme A ligase is the last enzyme of the phenylpropanoid pathway and plays a key role in the synthesis of lignin.The 4CL gene can catalyze the formation of corresponding CoA esters from caffeic acid,p-coumaric acid,sinapic acid and ferulic acid,and provide a material basis for the synthesis of plant secondary metabolites.In this experiment,three white birch 4CL gene sequences,namely Bp4CLl,Bp4CL2,and Bp4CL3 genes,were found by database search,and their overexpression vectors and silencing vectors were constructed.The promoter sequences of the Bp4CL1 and Bp4CL3 genes were obtained by aligning the known gene sequences in the Betula genomics database,and the plant expression vectors containing the promoter of the gene were named as Bp4CL1::GUS and Bp4CL3::GUS.Subcellular localization results showed that Bp4CL1 gene was localized on the cell membrane;Bp4CL2 gene was expressed in the nucleus;Bp4CL3 gene was expressed in whole cells.The promoter transient expression and GUS staining analysis showed that Bp4CLl gene was expressed in stems,leaves and roots of birch.It was expressed in Arabidopsis rosette leaf stage and was expressed in roots,leaves and flowers at flowering stage;Bp4CL3 gene was The expression of Betula platyphylla in roots and stems was expressed in Arabidopsis rosette leaves,and was expressed in roots,stems,leaves,and flowers during flowering.The above results suggest that Bp4CL1 gene may be involved in the synthesis of lignin.Bp4CL3 gene may have other functions in addition to its involvement in the synthesis of lignin.It also indicates that the Bp4CL1 and Bp4CL3 promoters have transcriptional activity.In this experiment,a positive transgenic plant that overexpressed Bp4CL2 was obtained,and histological observation of stem sections revealed that the xylem staining of transgenic positive plants was deeper than that of wild type plants,indicating that the lignin content increased.The results of qRT-PCR showed that the expression level of Bp4CL2 gene was increased in stems and petioles of transgenic plants.Phenotypic statistics showed that the plant height of transgenic plants became shorter and the number of internodes decreased.Therefore,the above results indicate that the Bp4CL2 gene plays an important role in the growth and development stages of plants.