Cloning and bioinformation analysis of 4CL gene in Neosinocalamus affinis.

Abstract

【Objective】 4-coumarate:CoA ligase(4CL) plays an important role in ligin biosythesis pathways.Up to now,there are not reports about the gene coding 4-coumarate:CoA ligase of Neosinocalamus affinis.The cloning of 4CL gene of Neosinocalamus affinis would be useful for improving the quality of Neosinocalamus affinis as raw materials for paper making.【Method】 The degenerate primers were designed according to 4CL known function of other plants.The conserved sequence of 4CL gene in Neosinocalamus affinis was cloned using cDNA of the young shoot of Neosinocalamus affinis and degenerate primers as a template and primers respectively.Then,the complete 4CL gene sequence was cloned by 5′RACE and 3′RACE using the conserved cDNA sequence cloned as a template.【Result】 The full-length sequence of 4CL in Neosinocalamus affinis(Na4CL) was successfully cloned in this study.The bioinformational analysis showed that the CDS region of the nucleotide sequence was 1 674 bp,coding 558 predicted amino acids with the conserved amino acid blocks SSGTTGMPKGV and GEICIRG.An active M residue embedded in the SSGTTGMPKGV motif of Na4CL,which is different from L residue in SSGTTGLPKGV motif of 4CL of other plants.Phylogenetic analysis showed that Na4CL was most similar to Os4CL4 in rice and Lp4CL2 in Lolium perenne.【Conclusion】 The 4CL gene named Na4CL was first cloned in Neosinocalamus affinis,which would play an important role in ligin biosythesis pathways of Neosinocalamus affinis.And then the CDS sequence of Na4CL has been submitted to GenBank with the accession number EU327341.