Study On The Gene Involved In The Cellulose Biosynthesis Of Moso Bamboo(Phyllostachys Edulis)

Bamboo(Phyllostachys edulis ) is not only one of the important forest resources in the world but also a non-lignification resource which was characterized by rapid growth and strong reproducible ability. With the change of the global environment and the drastic shrinkage of forest reserves, bamboo would play a pronounced role in relieving contradiction of supply and demand on woods resource, and so the exploitation of bamboo has received great attention around the world.The cellulose content of bamboo affects its practical and economic value directly. The cellulose synthase is the key enzyme for cellulose biosynthesis, and the transcription and expression of the gene of cellulose synthetase influence the content and quality of cellulose directly.Three full-length cDNA of CesA genes (PeCesA1: FJ495287, PeCesA2: AAY43217 and PeCesA4: FJ475350) and two partial cDNA (PeCesA6 and PeCesA7) were cloned from Moso bamboo ,which is one native and economic useful bamboo of China, by using RT-PCR and RACE method. Their nucleotide sequences and encoded amino acid sequences were analyzed and the structure of proteins was also predicted with bioinformatics approaches.PeCesA1 and PeCesA4 have been ligated into the prokaryotic expression vector and Pichia pastor vector, its expression in different strainswere carried out respectively. The expression vector of PeCesA1 was constructed using Gateway technology and then transformed into the tobacco leaf and the cell suspension of BY-2. The transgenic resistant buds and the resistant cell lines were obtained. The genetic transformation system of Dendrocalamus latiflorus was established. The callus regeneration system from embryo of Moso bamboo was studied.Those researches provided technical support for bamboo genetic engineering. Those main results are as follows.1.Cloning and analysis of CesA gene(1)A gene of cellulose synthetase named PeCesA1 was cloned from Moso bamboo in length of 3244 bp with an ORF of 3237bp which encode a protein of 1078 amino acids. The Mw and pI of PeCesA1 were predicted to be 121 586 02 Da and 7.12 respectively. The similarity of PeCesA1 with those of Oryza sativa, Zea mays, Populus tremuloides and Arabidopsis thaliana arrived more than 80%. The homology with BoCesA1 was the highest (98%).The secondary structure of PeCesA1 was abundant in helex, sheet regions and L-loop regions, PHD fingers and eight transmembrane domains. Spacial structure prediction showed that PeCesA1 belonged to compact globular protein. (2)PeCesA2 was of 3215 bp with an ORF of 3 213 bp which encode a protein of 1070 amino acids. The Mw and pI of PeCesA1 were predicted to be 120604.86 Da and 6.55 respectively. The results of protein prediction showed that the amino acid sequences of PeCesA2 consisted of 2 N-myristoylation sites, 3 anamidation sites, 9 Casein kinase II phosphorylation sites, 12 N muscade acylation sites and an amidation sites, 14 protein kinase C phosphorylation sites, 1 casein kinaseⅡphosphorylation site, 1 Cysteine-rich region profile,Bipartite nuclear localization signal and 2 zinc finger motifs.(3)PeCesA4 was of 3257 bp with an ORF of 3246bp which encode a protein of 1081 amino acids. The Mw and pI of PeCesA1 were predicted to be 120928.68 kDa and 7.57 respectively. The results of protein prediction showed that the amino acid sequences of PeCesA4 consisted of 3 anamidation sites, 7 N-myristoylation sites, 2 cGMP- or cAMP-dependent protein kinases, 10 Casein kinase II phosphorylation sites, 16 N muscade acylation sites , 14 protein kinase C phosphorylation sites, 1 casein kinaseⅡphosphorylation site, 1 Cysteine-rich region profile, Bipartite nuclear localization signal and 2 zinc finger motifs.(4)The length of PeCesA6 was 1462 bp with a 621 bp-untraslated region at the 3'end. A conservative region of 990 bp was found in the cDNA of PeCesA7.(5)Phylogenetic trees of cellulose synthetase gene was generated by MEGA4. Based on the information of CesA gene in model plant, it was presumed that PeCesA1, PeCesA2 and PeCesA4, were related with cellulose synthesis in the primary wall. PeCesA6 and PeCesA7 were related with cellulose synthesis in the secondary wall.(6)The expression analysis of gene showed that the expressions of PeCesA1 were different obviously in the shoots, root, stem, leaf and sheath of Moso bamboo The highest expression was examined in the stem, and then were shoots, root, stem and leaf. There is no expression in the sheath.The highest expression of PeCesA6 was examined in root and sheath, and then were stem, shoots and leaf. The lowest expression of PeCesA7 was also examined in leaf and no difference among other organs.2.The prokaryotic expression of PeCesA1 and PeCesA4The PCR products of PeCesA1 was ligated into the vector pEASY-E1 by TA Cloning, expression vector pEASY-E1-PeCesA1 was formed. The recombinant vector transformed the E.coli host strain BL(DE3) and single clone was induced to yieldrecombinant PeCesA1 by 1mM IPTG. The recombinantion of PeCesA1 with fusionHis-Tag migrated at 120 KDa in SDS-PAGE. Though the comparisons of among His-PeCesA1 in BL (DE3), BL(DE3) plysS and Rossata (DE3) strains, the result showed that the expression quantity of BL(DE3)plysS strain was the highest, and moreover it was higher markedly at 30℃than at 37℃.Meanwhile a pGEX-4T-1- PeCesA4 recombinant plasmid was constructed byinserting PeCesA1 gene into pGEX-4T-1 vector, and was transformed into E. Coli. BL-21. After induction by 1mM IPTG. The result of SDS-PAGE electrophoresis showed that the expression of fusion protein GST-PeCesA4 was higher than that of His-PeCesA1 obviously. Furthermore, the expression increased with the increasing of temperature ranging from 25℃to 40℃and reached the highest at 40℃. But no expression was detected at 43℃.3. The expression of PeCesA1 in Pichia pastorisThe expression vector of pPIC9K-PeCesA1 was constructed by sub-cloningtechnology and then transformed into P. pastoris KM71 through electroporation afterlinearization by PmeⅠdigestion. The recombinant P. pastoris KM71/ pPIC9K-aglu were screened in MD and YPD/Amp plates and identified using PCR. In shaking culturecondition, methanol was added to reach a final concentration of 0.5% to induce thesecretion of PeCesA1. Finally condon preference was used to analyze the cause of thelow level of PeCesA1 expression.4.Plant expression vector construction of PeCesA1 gene and its transformation of tobacco leaves and suspension cells (BY-2)GatewayTM cloning technology was used to construct the plant expression vector.An entry clone was performed by a BP recombination reaction with attB-PCR productsand donor vector pDONR207, and an expression clone was processed for selecting LR recombination reaction with the entry clone and destination vector pH7WG2D or pH7WGF2 with GFP gene. The plant expression vector of pH7WGF2-PeCesA1 and pH7WG2D-PeCesA1 regulated by 35S promoter were successfully constructed. PeCesA1 gene was transformed into tobacco leaves and suspension cells (BY-2) via Agrobacterium invadation. The resistant buds and aerosol cell line were obtained. The fluorescence protein was found in the buds and cells under confocal microscope. This result indicated that GFP-PeCesA1 was expressed stably in the buds and cells.5.Construction of Callus Transformation SystemStems and stem tips of tube seedlings (Dendrocalamus latiflorus) were selected as explants, and factors that influenced the callus development were studied. The results showed that the best medium for callus induction was MS+3 mg·L-1 2,4-D+0.02 mg·L-1KT, for differentiation was MS+3 mg·L-1 6-BA+0.02 mg·L-1 IBA, for root induction was 1/3MS+3 mg·L-1 IBA, and for the growth of roots was 1/3MS+1 mg·L-1 IBA+0.05mg·L-1 6-BA. The regeneration activity of the embryo cells lasted more than 1 year.The mature seeds of Moso were also used as explants to induce callus. The results showed that callus could be induced by 2,4-D at concentrations between 4 and 8 mg·L-1, and the best medium was M4(HBmacro + 1/2MSmicro + 1/2 MSCalcium +1/2 MS Iron + 8 mg·L-1VB1 + 5 mg·L-1 2,4-D. Combination of a low concentration ofKT with 2,4-D could enhance the inducing rate.