Cloning And Functional Identification Of Cinnamoyl CoA Reductase Genes From Caragana Intermedia

Lignin is a kind of aromatic polymer.It is the second most abundant natural biopolymer in the earth,after cellulose.They have been widely studied recently by scientists all over the world.In this study,two cinnamoyl-CoA reductase encoding genes,CiCCR2 and CiCCR3,of lignin biosynthetic pathway were cloned from Caragana intermedia through PCR.We constructed the binary vector of these two genes and obtained the homozygous transgenic Arabidopsis thaliana plants overexpression these two genes.The results are as follows.(1)The open reading frame(ORF)of CiCCR2 is 897bp,the gDNA consists of 4 introns(with a length of 131 bp,1351 bp,176bp and 222bp respectively).The deduced protein comprised 299 amino acids with a calculated molecular weight of 33.696KD,and isoelectric point at 5.11,as well as the grand average of hydropathicity(GRAVY)is-0.288.The ORF of CiCCR3 is 966bp,the gDNA consists of 5 introns(with a length of 829bp,856 bp,197bp,522bp and 1191bp respectively).The deduced protein comprised 322 amino acids with a calculated molecular weight of 35.91KD,and isoelectric point at 6.01,and GRAVY of-0.173.(2)Binary vectors p35S::CiCCR2 and p35S::CiCCR3 were constructed,8 lines.9 lines homozygous transgenic Arabidopsis thaliana overexpression these two genes were obtained respectively.(3)Quantification assay and Histochemical staining showed that the lignin content increased by overexpressing of CiCCR2 and CiCCR3 in Arabidopsis compared with the wild type during both seedling and mature stages.At the same time,The fresh and dry weight of overexpressing plants were higher than that of the wild type.