Cloning, expression and purification of 3-Deoxy-D-manno-octulosonate 8-Phosphate Synthase from Phyll

来源 :第十届中国竹业学术大会 | 被引量 : 0次 | 上传用户:wangzhenyu86
下载到本地 , 更方便阅读
声明 : 本文档内容版权归属内容提供方 , 如果您对本文有版权争议 , 可与客服联系进行内容授权或下架
论文部分内容阅读
  The pectic polysaccharide rhamnogalacturonan Ⅱ (RG-Ⅱ) is present in all plant species.Even though the structures of the RG-Ⅱ are very complex,the sugar composition of the RG-Ⅱ is extremely evolutionary conserved and contain at least 12different glycosyl residues.Among its component sugar is the seldom-conserved eight-carbon monosaccharides 3-deoxy-D-manno-octulosonic acid (Kdo) which is required for pollen tube growth and elongation.Furthermore,the phosphorylated bioprecursor of the KDO is synthesized by 3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (AtkdsA1 and AtkdsA2 can code the synthase in Arabidopsis thaliana),and the biosynthetic pathway is also conserved between plants and Gram-negative bacteria.It can be seen that KDO-8P synthase plays a key role in structure stability.A full-length cDNA of encoding KDO-8P synthase gene was isolated from Phyllostachys heterocycla through RT-PCR,and named as PeAtkdsA (GenBank accession number: FP100420).The length of PeAtkdsA is 1207 bp,which contain an open reading frame encoding 291 amino acids.The result of amino acid sequence alignment analysis showed that PeKDO-8P synthase had high identities with other KDO-8P synthase in Oryza sativa,Zea mays,Brachypodium distachyon,Ricinus communis,Aquifex Aeolicus,Vibrio Cholerae,Physcomitrella,Haemophilus influenzae,Arabidopsis thaliana,E.Coli, Burkholderia Ambifaria, Selaginella moellendorffii, Coccomyxa subellipsoidea,especially with KDO-8P synthase from Oryza sativa up to 95%.Phylogenetic tree analysis showed that there were remarkably different branch sites.Tissue specific expression showed that PeKDSA expressed in root,stem,and leaf,much higher in root,which was similar with AtkdsA2 from Arabidopsis thaliana.In order to find out the difference and mechanism with different species,the structure of PeKDO-8P synthase should be analyzed.So in the research,PeKDSA was constructed into the expression vector pESAY-E1 and the fusion protein in expression was induced in E.coli strains:BL21(DE3) and RosettaTM(DE3).Then fusion protein was purified through steps chromatography (affinity chromatography and SEC).Native PAGE and SEC analysis shows that PeKDOPS protein mainly exists as tetramer,and SDS-PAGE shows the fusion protein has high degree of purity.Finally microcrystal was selected from the purity protein.These works provide the first step for its structure determination,or other,its function illumination from molecular lever.
其他文献
几年来一直是你的歌友。 其实自己也不明白是怎样喜欢上你的,为你的歌?你的人?还是你的笑?总之听了那首《三百六十五个祝福》,便认定了你是我心中最亮的星,最美的故事。 得
自2015年以来,西藏自然科学博物馆的建成与开放已经在科普工作领域取得了巨大成果,但从长期发展的角度来看,且对于科普平台的建设却仍然存在着很多有待提升之处.基于此,本文
通过计算机信息技术创造出各种各样实用的APP软件,给人们的生活带来了更大的方便快捷以及乐趣,其主要依靠的就是虚拟化技术.虚拟化技术在运用的过程中需要依靠可虚拟化的装置
设计完成热管回路的实验台架搭建,水浴加热的组装,以及热工、水力参数测量仪器的安装.并进行不同温度、不同充液率条件下热管回路的换热特性实验,通过对实验数据的分析,得到
本文针对该罕见路径和独特的强度变化特点,应用天气图、卫星云图等多种资料和空军T511数值预报系统,通过对涡度场、散度场、温湿场和强水汽对流等物理量场及环境引导气流和双
移动通信网络业务的辐射范围越来越广,企业应该加强传输电缆的维护工作,提升运维管理的智能化水平.本文首先对传输线路智慧运维管理的模式进行简单的介绍,其次总结智慧运维管
为摸清全市水泥制造业职业危害情况,以便制订相应的防治措施,1998年对全市6家大型水泥厂进行了劳动卫生学调查.1 对象和方法1998年对全市6家大型水泥厂的有关作业环境进行调
本文从目前集中告警系统局限性出发,介绍了知识图谱在通信线网集中告警应用和实践情况,就构建轨道通信资源与故障经验智能运维体系提出建议和思路.
一个名不见经传的乡镇煤炭企业。几年来,靠着他们勇于拚搏,不断开拓,艰苦创业,求实的精 神,从1987年起,公司就被大同市委、市政府命名为“文明单位”,曾获得山西省劳动竞赛
期刊