【作者】 王玫玫；

【导师】 郭兴启；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 促丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)是普遍存在于真核生物中的一类保守的丝氨酸/苏氨酸类蛋白激酶。MAPK级联途径(MAPKKK-MAPKK-MAPK)通过依次磷酸化传递环境信号,参与植物生长发育和多种生物、非生物胁迫信号转导。近年来许多研究发现MAPK与植物病原信号传递关系相当密切,并鉴定多种受病原侵染诱导的MAPK。棉花是一种重要的经济作物,利用基因工程方法培育棉花抗病新品种已经成为棉花育种的研究热点。本文以鲁棉22为材料,首次从棉花中克隆了MAPK基因(GhMAPK),并对其进行了序列比对,表达特性分析及功能鉴定。具体结果如下:1.根据同源序列设计简并引物,通过RT-PCR和RACE-PCR的方法从棉花(Gossypium hirsutum)中克隆到一个MAPK基因,命名为GhMAPK。GenBank注册号为DQ132852。其cDNA全长为1832bp,编码一个372氨基酸蛋白质。序列分析发现GhMAPK具有蛋白激酶所保守的11个亚区和TEY特征磷酸化位点。与C组MAPK同源性最高,同来自于拟南芥、杏、矮牵牛、豌豆、烟草中的几种MAPK同源性高达83-89%。cDNA序列与基因组序列比较后发现GhMAPK基因组序列内存在两个内含子。2.Southern杂交说明在棉花基因组中可能存在多个与GhMAPK具有一定同源性的其他MAPK基因,或者可以说存在一个小的MAPK基因家族。Northern杂交发现GhMAPK转录水平受机械损伤,高盐,冷害等非生物胁迫诱导而提高;植物抗病反应相关信号分子SA和H2O2也能诱导GhMAPK表达;棉花幼苗接种棉花枯萎病菌后,GhMAPK表达量也有明显提高,进一步证明了GhMAPK参与了病原侵染反应过程。3.将GhMAPK构建了正义植物表达载体pBI121-GhMAPK,采用农杆菌介导的方法,转化烟草(NC89),同时转空载体为对照。经卡那霉素筛选获得若干再生植株。经过PCR鉴定,并选取部分转基因植株进行了Northern和Western杂交分析,结果证明GhMAPK在转基因烟草中已成功得到表达。4.选取两个T0代转基因株系的自交种子扩繁,得到T1代转基因植株。在分子鉴定基础上,对T1代转基因植株进行了生物学功能分析。抗真菌实验中,接种了黑胫病原菌(Phytophtora parasitica var.nicotianae Tucker)的转基因植株的抗病能力明显高于对照植株。烟草花叶病毒(tobacco mosaic virus,TMV)侵染后,ELISA检测的结果表明,转基因植株中有部分株系表现出了明显的抗病性,但数量很少,大部分表现为感病,病毒抗性植株仅占接病总株数的约10%。5.构建原核表达载体pET- GhMAPK,并在大肠杆菌BL21(DE3)中表达融合蛋白,将特异诱导带切下,溶于PBS中获得抗原,免疫小鼠,其抗血清效价为1﹕1000。更多还原

【Abstract】 The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays pivotal role in the regulation of stress and developmental signals in plants. The MAPK cascades transfer signals through phosphorylations of MAPKKK- MAPKK- MAPK in turn.Numerous MAPK have been isolated and characterized from different species, and many of them had close relations with plant disease resistance. Cotton (Gossypium hirsutum) is an economically important species, its MAPKs, however, have not been reported before. In this thesis, a series of studies have been conducted on the isolation, sequence and expression analysis, function identification of cotton MAPK (GhMAPK). The main results are as follows:1. A novel gene, termed Gossypium hirsutum MAPK (GhMAPK), was isolated from cotton. The full-length cDNA of GhMAPK is 1832bp long, and encodes for a 372 amino acid protein that contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. Amino acid sequence alignment revealed that GhMAPK shared high identity with group-C MAPK in plants and showed 83%~89% similarities with MAPKs from Arabidopsis, apricot, pea, Petunia, and tobacco.2. Southern blot analysis indicated that the GhMAPK belonged to a multygene family in cotton. Two introns were found within the region of genomic sequence. Northern blot analysis revealed that the transcripts of GhMAPK accumulated markedly when the cotton seedlings were subjected to various abiotic stimuli such as wounding, cold (4℃), or salinity stress; Furthermore, GhMAPK was upregulated by the exogenous signaling molecules, such as salicylic acid (SA) and hydrogen peroxide (H2O2), as well as pathogen attacks. These results indicate that the GhMAPK, which has a high degree of identity with group-C plant MAPKs, may also play an important role in response to environmental stresses.3. Construct a sense expression vector pBI-GhMAPK, and transformed it in to the tobacco plant (NC89). We also transformed the empty vector into tobacco at the same time as control. We carried out Northern blot and Western blot on some transgenic plants, and found that GhMAPK has been expressed successfully in the tobacco plants.4. On the basis of the molecular identification, we analyzed the biological function of progeny T1 generation plants. The transgenic plants exhibited much milder symptoms than the nontransgenic plants upon challenge with Phytophtora parasitica var. nicotianae Tucker, but only 10% transgenenic plants showed resistance to TMV virions.5. Constructed an E.coli expression vector pET-GhMAPK and induce it to express in E.coli strain BL21(DE3). The strong induced fusion protein bands were collected into PBS solution and immuned little mouse to obtain antiserum. The value of antibody reaches 1:1000.更多还原