Isolation And Characterization Of Cellmlose Synthase Gene From Larix Gmelinii

Dahurian larch,Larix gmelinii(Rupr.),is a deciduous tree which is mainly grows in northeastern China and the major forest tree species for afforestation in Great Khingan.L.gmelinii has extensive application in building,timber processing,wood fiber industry and so on owing to its high-quality timber,and shows great economic value.Celll?lose Synthase(CesA)is the key enzyme in cell?lose synthase pathway,which expression abundance influences biosynthesis of cell?lose and reg?lates wood formation.Therefore,the study on expression reg?lation mechanism and function of the CesA genes has important significance.In the study,the ortholog of CesA was isolated from L.gmelinii,referred to as LgCesA3,using degenerate-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE),and function and expression characteristics of the gene was analyzed.The res?lts showed that the 4220 bp of the LgCesA3 cDNA contains 3297bp of open reading frame(ORF),531 bp of 5'-untranslated region(UTR)and 392 bp of 3'-UTR,which encodes a protein of 1099 amino acids.The 7255 bp of the LgCesA3 DNA contains 14 exons and 13 introns.The subcell?lar localization analysis showed that of LgCesA3::GFP fusion protein signal was detected periphery of onion epidermal cells suggesting that LgCesA3 was located in the cell membrane.For investigating the expression mechanism of the LgCesA3,1180bp of LgCesA3 promoter sequence was cloned using genome walking technique,sequence analysis revealed that the LgCesA3 promoter region contains some elements responding to plant hormones including salicylicacid and Methyl Jasmonate,photoinducible elements and abiotic-stress response elements such as drought and mechanical pressure.Real-time fluorescent quantitative-PCR(real time Q-PCR)analysis showed that the LgCesA3 expressed in all organs of the larch plants with higher expression in stems than in leaves or roots.The expression of LgCesA3 gene was up-reg?lated by treatment with exogenous hormonal,including methyl jasmonate and salicylic acid,while the expression of the gene appeared after rising downward trend under 15%PEG and exogenous hormonal GA3.In order to further analyze functions and heterologous expression features of the LgCesA3,pBI101-LgCesA3 was constructed,and transgenic Arabidopsis were obtained via agrobacteri?M-mediated genetic transformation.The res?lts of RT-PCR and enzyme activity detection confirmed the stable expression of LgCesA3 in transgenic A.thaliana.Phenotypic analysis showed that the excessive expression of LgCesA3 promoted the accpM?lation of cell?lose in transgenic plants.These research res?lts showed that growth and development of plants,as well as timber formation,can be reg?lated through the genetic modification of CesA,the key enzyme in the synthesis pathways of cell?lose,which provided basis for further understanding the expression and reg?lation mechanism of LgCesA3 gene,and will likely be usef?l for timber improvement of forestry trees.