【作者】 袁丽钗；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2009， 博士

【摘要】 竹子是世界重要的森林资源之一,随着全球环境的变化和森林资源的锐减,竹子作为一种生长迅速、再生性强的非木质植物资源,在缓解木材供需矛盾方面发挥着重要作用,其开发利用受到了国际社会的高度关注。竹子纤维素含量直接影响到其应用价值,而纤维素合成酶是纤维素生物合成的关键酶和特征酶,纤维素合成酶基因的转录与表达水平直接影响到纤维素的含量和品质,从而影响到竹子的经济价值。本论文以中国重要经济竹种毛竹(Phyllostachys edulis)为材料,利用RT-PCR和RACE方法克隆到5个维素合成酶基因PeCesA1(登录号FJ495287)、PeCesA2(登录号FJ475350)、PeCesA4(登录号FJ475351)、PeCesA6及PeCesA7。运用分子生物信息学方法对核苷酸序列及其编码的氨基酸序列了进行分析。将PeCesA1和PeCesA4构建到原核及毕赤酵母表达载体上,进行了异源表达研究。利用GatewayTM克隆技术构建了PeCesA1的植物表达载体,通过转化烟草叶片,获得了转基因抗性芽。建立了麻竹愈伤再生体系,并对毛竹种胚愈伤再生体系进行了探讨。主要结果如下:1.毛竹纤维素合成酶基因克隆与分析(1) PeCesA1基因cDNA长3 244 bp,开放阅读框为3 237 bp,编码1 078个氨基酸,理论分子量为121 586.02 Da,等电点7.12。聚类分析表明,PeCesA1蛋白序列与绿竹、水稻、玉米、大麦、杨树等的一致性在80%以上,其中与绿竹BoCesA1的一致性最高,达98%;其次是水稻,为97.5%;二级结构富含а-螺旋,β-折叠及L-环状区域,具有8个跨膜区及锌指结构域;球状结构预测显示PeCesA1属于结构致密的球状蛋白。(2)PeCesA2基因cDNA长3 215 bp,开放阅读框为3 213 bp,编码1 070个氨基酸,理论分子量为120 604.86 kDa,等电点6.55;蛋白结构预测表明,PeCesA2包含一个纤维素合成酶保守域的同时,还包含2个氨基化位点、3个N-糖基化位点、9个酪蛋白激酶II磷酸化位点、12个N-肉豆蔻酰化位点、14个蛋白激酶C磷酸化位点、1个酪氨酸激酶磷酸化位点;1个赖氨酸富集区、2个双向定位信号和2个锌指结构域。(3)PeCesA4基因cDNA长3 257 bp,开放阅读框为3 246 bp,编码1081个氨基酸,理论分子量为120 928.68 kDa,等电点7.57;蛋白结构预测表明,PeCesA4包含一个纤维素合成酶保守域的同时,还包含2个氨基化位点、7个N-糖基化位点、2依赖于cAMP和cGMP的蛋白激酶磷酸化位点、10个酪蛋白激酶II磷酸化位点、16个N-肉豆蔻酰化位点、14个蛋白激酶C磷酸化位点、1个酪氨酸激酶磷酸化位点;1个赖氨酸富集区、2个双向和定位信号、2个锌指结构域。(4)PeCesA6基因片段长1 462 bp,包括3’端621 bp非编码区。PeCesA7含有990bp的保守区。(5)通过构建系统进化树分析比较,推测PeCesA1、PeCesA2、PeCesA4可能与毛竹细胞初生壁纤维素合成有关,PeCesA6、PeCesA7可能与毛竹细胞次生壁纤维素合成有关。(6)基因表达分析表明,PeCesA1、PeCesA6、PeCesA7的表达谱存在着差异。PeCesA1茎部表达量最高,芽、根、叶次之,在箨片中未检测到;PeCesA6在根、茎、叶及箨片中均有表达,以根和箨片中表达量最高,其次是茎、芽,在叶中表达量最低;PeCesA7芽、根、茎、叶及箨片中均有表达,其中以叶片中表达量最低,其余器官无明显差异。2.PeCesA1和PeCesA4基因在大肠杆菌中的表达采用TA克隆的方法直接将PeCesA1基因的PCR产物链接到pEASY-E1表达载体上,重组质粒转化大肠杆菌,经1 mmolL-1M IPTG诱导在120 KD处有一特征带,实现了其在大肠杆菌中表达。通过比较重组蛋白His-PeCesA1在BL(DE3)、BL(DE3)plysS及Rossata(DE3)菌株中表达情况发现,在BL(DE3)plysS菌株中表达量最高,其中30℃表达量明显高于37℃。同时又以pGEX-4T-1为载体,将PeCesA4基因序列构建到高pGEX-4T-1表达载体上。SDS-PAGE电泳结果发现,融合蛋白GST-PeCesA4的表达量明显高于His-PeCesA1,在25℃~43℃温度范围内,重组蛋白的表达量随着温度增高而提高,40℃表达量达到最高峰,43℃重组蛋白不表达。3. PeCesA1在毕赤酵母中的表达利用亚克隆技术将毛竹PeCesA1构建到毕赤酵母表达载体pPIC9K上,成功地构建了PeCesA1基因的真核表达载体pPIC9K- PeCesA1;重组载体经PmeI线性化后电转化毕赤酵母KM71,PCR检测获得了7个转化子,诱导表达后SDS-PAGE结果未发现特征带。4.PeCesA1基因植物表达载体构建及烟草叶片、悬浮细胞BY-2的转化应用GatewayTM技术将PeCesA1通过BP反应克隆到pDONR207中,构建了入门载体pDONR207- PeCesA1,然后通过LR反应将入门克隆上的目的基因平行构建以绿色荧光蛋白(GFP)为选择标记的pH7WG2D或pH7WGF2植物表达载体上,构建了35 S启动子调控的组成性二元植物表达载体pH7WGF2-PeCesA1及pH7WG2D-PeCesA1。通过叶盘法转化烟草叶片,获得了转PeCesA1基因抗性芽。利用农杆菌侵染烟草悬浮细胞BY-2,获得抗性细胞团。通过荧光共聚焦显微镜观察,GFP-PeCesA1在抗性芽及悬浮细胞中都得到表达。5.愈伤受体转化系统的建立与探讨以麻竹试管苗茎段及茎尖为外植体,对影响麻竹愈伤再生体系的因子进行了研究。筛选出最适愈伤诱导培养基为:MS+3 mg·L-1 2,4-D+0.02 mg·L-1KT;最适不定芽分化培养基为:MS+3 mg·L-1 6-BA+0.5 mg·L-1 IBA;不定根诱导培养基为:1/3MS+3 mg·L-1 IBA,不定根生长培养基为1/3MS+1 mg·L-1 IBA+0.05 mg·L-1 6-BA。通过脱分化所建立的麻竹胚体细胞系的再生特性已保持了一年以上。以毛竹成熟种子为外植体进行了愈伤诱导,结果发现:2,4-D浓度在4～8 mg·L-1之间均能诱导愈伤组织发生,以5 mg·L-1 2,4-D效果最好;在所使用的四种基本培养基中,M4(HB大量+ 1/2MS微量+ 1/2MS钙盐+1/2 MS铁盐+ 8 mg·L-1VB1+ 200 mg·L-1肌醇)愈伤诱导率最高,配合使用低浓度的0.02 mg·L-1KT提高2,4-D的诱导效果。更多还原

【Abstract】 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.更多还原