Cloning And Characterization Of KNOX2 Gene From Larix Gmelinii

Larix gmelinii(Rupr.)Kuzen is a deciduous tree in the north of China,that has a straight trunk,few nodes and strong mechanical properties,and is used in the construction,wood processing and paper-making industry.Therefore,it is very important to isolate key genes related to wood formation and to improve wood quality by regulating the expression of the genes by using molecular biological methods.In the study,the key gene in regulating the differentiation of apical meristem and timber formation,KNOX gene(named LgKNOX2),was isolated from L.gmelinii,and analyzed the expression and function of the gene.The full length of LgKNOX2 cDNA contains 1484 bp,including an open reading frame of 1320bp(ORF),a 5'UTR of 27 bp and a 3UTR of 137 bp,encoding 439 amino acids with a molecular weight of 49.3kdDa and the theoretical isoelectric point of 5.46.Bioinformatics analysis showed that the protein of LgKNOX2 has no signal peptide sequence and transmembrane domain.Phylogenetic analysis showed that the LgKNOX2 is close to PpKNOX2 of Pinus pinaster,PmKNOX2 of Picea mariana,PaKNOX2 of Picea abies,LgKNOX1 of L.gmelinii.The results of real-time fluorescence quantitative PCR showed that LgKNOX2 gene was maory expressed in roots,stems and leaves of L.gmelinii seedlings with the highest expression in stems.The promoter cloning and sequence analysis showed that the expression abundance of the gene was regulated by auxin,methyl jasmonate,gibberellin,abscisic acid light and other factors.Using Agrobacterium tumefaciens-mediated transformation method,two transgenic Arabidopsis thaliana lines were obtained.The phenotypic analysis showed that the transgenic plants over-expressing LgKNOX2 gene had longer and well-developed roots,while smaller leaves,surface shrinks,the bendy petiole,and albefaction in base blade were observed in the two transgenic lines.These results indicate that the important transcriptional regulator,the LgKNOX2,gene can affect plant development and control plant morphogenesis.The results of the expression characteristics and functional analysis of the LgKNOX2 gene laid a foundation for understanding the molecular mechanism of wood formation in L.gmelinii.