【作者】 王璐；

【导师】 冯彩平；

【作者基本信息】 山西大学 ， 生态学， 2008， 硕士

【摘要】 拟南芥具有基因组小、结构简单、形体小、生长周期短、繁殖系数高等优点,且其基因组全序列测定工作已于2000年12月完成。为此以模式植物拟南芥(Arabidopsis thaliana)为材料来研究MPK17基因的功能。MPK(Mitogen activatedprotein kinases MPKs)是一类广泛存在于各种真核生物体中的丝氨酸/苏氨酸型蛋白激酶。MPK与其他一系列激酶组成MPK级联信号通路,接受外界刺激信号,并将信号转入细胞内,直接调控特定基因的表达,进而控制细胞生物学反应,如细胞增殖、分化、转化及凋亡等。目前拟南芥中已知的有20个MPK基因,根据其中保守的TxY模体内氨基酸序列比较,将其分成TEY和TDY两个亚型,又可以将TEY类激酶分为A、B、C三组,TDY类激酶则单独列为D组。目前知道得研究最多的是A、B、C亚族的成员,如拟南芥中的MPK3,MPK4,MPK6、MPK7。D亚族成员在拟南芥中还没有见到报道,我们实验室前人从拟南芥转座子突变体库中,分离出一个MPK17的转座子插入突变株系MPK17,属于TDY族。因此通过研究,揭示含TDY这一保守结构的MPK17基因的功能以及在适应环境与植物激素等信号转导中的作用。首先利用NCBI(美国国立生物技术信息中心)提供的拟南芥MPK17基因的核酸序列和利用在线和本地的一些生物信息学软件对拟南芥MPK17基因的序列进行生物信息学的分析,主要内容包括:核酸序列开放阅读框分析、蛋白质结构分析、二级结构预测以及该序列的进化关系分析,初步结果它位于拟南芥2号染色体199510 bp—203 125 bp处,在芽、花的分裂组织、花、叶、根等多种组织和细胞中表达,在生长初期和末期基因的表达量最大。结合实验室前人工作,她们已经初步鉴定MPK17基因对环境因子,尤其是植物生长物质IAA及2,4-D有明显的应激反应。该转座子所携带的GUS报道基因在根系及幼叶组织中有特异表达。在此基础上我通过大量的实验在IAA、NAA、TIBA、2,4-D四种植物生长物质不同浓度条件下,观察拟南芥mpk17突变体与野生型之间主根长的差异,通过数据处理分析发现同一植物生长物质同一浓度处理下野生型与mpk17突变体之间主根长都没有明显的差异,表明MPK17基因功能缺陷植株在表型上与野生型没有差异,这就说明拟南芥MPK17基因的功能有可能是冗余的,与其他同源基因之间存在相互功能的替代的可能性,为了探明MPK17的功能,有必要进一步通过构建亲缘关系近基因的双突变体来研究。根据第二章的序列分析表明MPK9与MPK17亲缘关系最接近,通过PCR、抗性筛选、RT-PCR筛选出纯合的mpk17与mpk9单突变体,然后经过人工杂交,产生成功的F1代杂交种。近年来,在植物中分离到大量MPK,并在其作用机理研究方面取得了长足进展。植物中MPK的研究主要集中在双子叶模式植物(如拟南芥、烟草和紫花苜蓿等),关于玉米MPK方面的研究很少。因此利用生物信息学方法在玉米中发现和电子克隆了玉米的MPK17基因并对其核酸和蛋白序列进行了初步分析,表明单子叶植物玉米中也存在MPK17基因,对开展玉米中MPK种类和其作用机制的研究,对人为控制其生长发育和增强抗逆性进而增加产量具有重要的指导意义,为更好的研究MPK17提供了丰富的理论基础。更多还原

【Abstract】 Arabidopsis thaliana has good characteristic such as small genome, simple structure,small plant,short growth period,high propagating rate,and so on,and the whole genome sequencing has been completed in December 2000.So we make research for the function of MPK17 with Arabidopsis thaliana which is model plant,MPKs is a Ser/Thr-type protein kinase which is existed widely to each eukaryotes.MPKs is make up of MPK cascade signaling with other kinesis,accepting outside stimulate signaling,and changing signal over to cell to regulate directly the deliver of special gene,and then controlling cell biological reaction, such as proliferation of cells,differentiation,transform,apoptosis etc.At present,Arabidopsis thaliana known to have 20 MPK genes, according to the conservative TxY die body in the amino acid sequence comparison,divides into it TEY and the TDY two hypotypes,may also divide into a TEY kind of activating enzyme A,B,the C three groups, a TDY kind of activating enzyme alone lists as D group.At present what knows most studies are most is A,B,the C Asia race’s member,like MPK3,MPK4,MPK6,MPK7 of Arabidopsis thaliana.The D Asia race members in the Arabidopsis thaliana have not seen the report,our laboratory predecessor separates MPK17 transposable elements insertion mutation strain MPK17 from Arabidopsis thaliana transposable elements mutant storehouse,it belongs to the TDY race.Therefore with the research,the revelation contains TDY this conservative structure MPK17 gene function as well as in the adaptation environment and in the plant hormone equisignal transduction function.First,we make use of nucleic acid sequencing of Arabidopsis thaliana MPK17 which is provided with NCBI,and some on line and native bioinformatics software to analysis Arabidopsis thaliana MPK17 sequence with bioinformatics,the main contents contain:the analysis of nucleic acid sequence open reading frame,the analysis of protein structure,the forecast of the protein secondary structures and the evolution relationship analysis,the first fruit is that it is located to 199 510 bp—203 125 bp of second chromosome of Arabidopsis thaliana, and expressing with the division organizations of buds and flowers, many organizations and cells of flowers,leaves,root etc,and the most controlling is in the gene of preliminary stage and last stage.Unifies the laboratory predecessor to work,they already initially appraised the MPK17 gene to environment factor,particularly plant growth material IAA and,2,4-D have the obvious stress response.This transposable elements carries the GUS reporter gene has the special expression in the root system and the young leaf organization.Based on this,I through the massive experiments in IAA,NAA, TIBA,and 2,4-D four kind with different concentrations to examine the difference of main rooting length from MPK17 mutant and the wild on Arabidopsis thaliana,between wild and the MPK17 mutant the main rooting length does not have the obvious difference through data processing analysis discovery identical auxin identical concentration, indicated that the MPK17 gene function flaw,adult plant with wild does not have the difference on the phenotype,this explained that Arabidopsis thaliana MPK17 gene function to have the possibility is the redundancy, has the mutual function with other homologous gene between the substitution possibility,to verify the function of MPK17,it is necessary further studies through the construction the sibship near gene’s double mutant.According to sequential analysis of the second chapter indicated that MPK9 and the MPK17 sibship is closest,through PCR,the resistance screens and RT-PCR to screen the MPK17 and the MPK9 single mutant, then undergoes the hybridization,produces a successful F1 generation of hybrid.In recent years,a number of MPK have been isolated in the plant, and has made the considerable progress in its action mechanism research aspect.In the plant the MPK research mainly concentrates on the double seed leaf pattern plant(for example Arabidopsis thaliana,tobacco and alfalfa and so on),are very few about the corn MPK aspect’s research. Therefore we discovered and In silico cloning corn’s MPK17 gene using the biological information sciences method in the corn and carried on the preliminary analysis to its nucleic acid and the protein sequence, indicated that in the monocotyledon plant corn also has the MPK17 gene, to develops in the corn the MPK type and its function mechanism research,manual control its growth and strengthens the resistance then the gain in yield to have the important guiding sense,has provided the rich rationale for better research MPK17.更多还原