铁皮石斛DoUAM基因克隆及转化拟南芥

Cloning of Do UAM from Dendrobium officinale and Transformation into Arabidopsis thaliana

为了探究铁皮石斛阿拉伯吡喃糖变位酶(UAM)基因的功能,本研究采用RT-PCR技术从铁皮石斛叶片中克隆出阿拉伯吡喃糖变位酶基因,获得1104 bp的ORF序列并命名为DoUAM,编码367个氨基酸,分子量大小为41.433 kD。生物信息学预测表明:DoUAM蛋白与模式植物拟南芥(AtRGP1和AtRGP2)和水稻(OsUAM1)的同源序列高度保守。Motif预测表明DoUAM含有DXD-Motif、KKXX-Like基序和一个RGP基序,三个糖基转移酶基序。系统发育分析结果显示,DoUAM与PeUAM1亲缘关系最近,并将其归类到具有自我糖基化功能的ClassⅠRGPs亚族中。蛋白三维结构预测显示,DoUAM蛋白的Ile27,Gly51,Asp88,Asp108,Ile239等残基可能与底物UDP-Sugars的糖基相互作用,Asp108、Asp109和Asp110残基可能为二价金属离子的结合位点,以提高酶的活性。通过DNA同源重组技术(Clone expressⅡ,Vazyme)将其连接到pBI121s载体上,成功构建植物表达载体pBI121s-DoUAM。利用农杆菌介导的浸花法转化到拟南芥(Col-0)中,经筛选获得6株具有卡那霉素(kan)抗性的T2代阳性转基因拟南芥植株,对6株再生植株进行PCR检测表明,目的基因已经成功整合到拟南芥基因组中。表型观察发现T2代转DoUAM基因拟南芥植株生长较对照组(WT)缓慢,尤其是根生长缓慢,叶片瘦小,分析表明DoUAM可能与植物的发育有关。对转基因拟南芥植株的总多糖含量进行检测发现,转DoUAM植株多糖含量明显高于对照组(WT),进一步分析表明铁皮石斛DoUAM基因与植物细胞多糖的合成有关。本研究结果为深入研究DoUAM基因在铁皮石斛发育过程以及多糖合成过程中可能发挥的功能和作用机制提供了帮助。

In order to explore the function of DoUAM gene in Dendrobium officinale,the Arabinopyranose mutase gene was cloned from Dendrobium officinale leaves by RT-PCR technology.The ORF sequence obtained from Dendrobium officinale is 1104 bp in length and named DoUAM,which encodes a putative protein about 367 amino acids with a molecular weight of 41.433 kD.Bioinformatics predictions showed that the DoUAM protein is highly conserved with the homologous sequences of the model plants Arabidopsis thaliana(AtRGP1 and AtRGP2)and rice(Os UAM1).The Motif predictions indicated that DoUAM contains a DXD-Motif,a KKXX-Like motif and an RGP motif,three glycosyltransferase motifs.The Phylogenetic analysis showed that DoUAM was closely related to PeUAM1 and was classified into ClassⅠRGPs subgroup with self-glycosylation function.Three-dimensional structure prediction showed that the residues of DoUAM protein such as Ile27,Gly51,Asp88,Asp108,Ile239 might interact with the glycosyl group of UDP-Sugars,and Asp108,Asp109 and Asp110 residues may be the binding sites of bivalent metal ions to improve the enzyme activity.The plant expression vector pBI121 s-DoUAM was successfully constructed by ligating DoUAM unto the pBI121 s vector by DNA homologous recombination technology(Clone expressⅡ,Vazyme biotech).We transformed pBI121 s-DoUAM into Arabidopsis thaliana(Col-0)by Agrobacterium-mediated Floral dip method.6 T2 generation positive transgenic Arabidopsis thaliana plants with kanamycin(kan)resistance were obtained by screening.PCR detection of the 6 regenerated plants indicated that the target gene had been successfully integrated into the genome of Arabidopsis thaliana.The phenotypic observation showed that the growth of T2 generation transgenic DoUAM gene Arabidopsis thaliana was slower than the control group(WT),especially as exhibited by the slow growth of the roots and the small leaves.The analysis indicated that DoUAM may be related to plant development.We detected the total polysaccharide content of transgenic Arabidopsis plants.The total polysaccharide content of the transgenic DoUAM plants was found to be significantly higher than that of the control group(WT).Further analysis showed that the DoUAM gene of Dendrobium officinale was related to the synthesis of plant cell polysaccharides.The results of this study can be helpful for further study of the function and mechanism of the DoUAM gene in the development of Dendrobium officinale and the process of polysaccharide synthesis.