【作者】 徐慧妮；

【导师】 王秀峰；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2009， 博士

【摘要】 土壤次生盐渍化是温室蔬菜生长的主要障碍之一,严重影响栽培设施的利用效率和设施蔬菜栽培的可持续高效发展。温室土壤的盐分组成特点和滨海、内陆盐土不同,其阴离子主要是NO₃^-。目前前人对盐胁迫对植物的伤害机理方面的研究已取得了长足进展,但多以NaCl处理作为研究手段,而温室土壤中NO₃^-积累造成蔬菜伤害的分子机理缺乏深入系统的研究。黄瓜是我国日光温室主栽的蔬菜种类之一,黄瓜根系浅,吸肥能力差,易受盐害。因此,研究设施黄瓜在NO₃^-胁迫条件下的抗性机理具有重要的理论和实践意义。MAPK级联途径是真核生物中广泛存在的逆境胁迫信号转导途径。MAPK级联途径由三类蛋白激酶组成:MAPKKK-MAPKK-MAPK,通过依次磷酸化传递环境信号。植物中的MAPK途径与其他信号途径交织在一起,形成了一个网络系统,参与生长发育和各种生物、非生物胁迫信号转导。近年来,在植物中分离到大量MAPKs,并在其作用机理研究方面取得了很大进展。然而,前人对植物中MAPK的研究主要集中在双子叶模式植物（拟南芥、烟草和紫花苜蓿等）,关于黄瓜MAPK方面的研究很少。为此,本试验以黄瓜为试材,对黄瓜MAPK进行了基因克隆、表达特性和功能的研究,主要结果如下:1.利用同源序列设计兼并引物,通过RT-PCR的方法,在黄瓜根中分离到黄瓜MAPK基因的中间片段,再通过5′RACE和3′RACE分别克隆到黄瓜MAPK基因的5′片段和3′片段,拼接后设计特异引物扩增到黄瓜MAPK基因全长cDNA,命名为CsNMAPK（基因注册号:DQ812086）。该基因全长1636 bp,有开放阅读框1113 bp,编码370个aa。2.同源序列比较发现,黄瓜MAPK基因与已知的棉花、豌豆、苜蓿、马铃薯、番茄、辣椒的同源性分别为85.18 %、84.91 %、84.64 %、83.65 %、83.11%、81.60 %。对来自番茄、马铃薯、棉花等MAPK蛋白进行分子聚类分析,构建了MAPK蛋白的系统进化树,分析结果表明,CsNMAPK蛋白与棉花GmMAPK1、豌豆的PsMAPK3蛋白亲缘关系最近。聚类分析还表明CsNMAPK蛋白属于MAPK的Ⅰ类,预示可能在环境胁迫和激素信号转导中起着重要作用。3.Northern blot分析表明CsNMAPK受NO₃^-胁迫强烈诱导表达。56、98、140 mM NO₃^-处理后,该基因的表达量上升,182 mM NO₃^-处理后该基因的表达量下降。182 mM NO₃^-处理后该基因在黄瓜叶、茎和根中均有表达,且表达量差异不大。182 mM NO₃^-处理耐盐品种‘新泰密刺’10 min后该基因的表达量开始上升,处理30 min后表达量达到最大,随后开始下降;而盐敏感品种‘神农春五’的表达量在60 min达到最大。CsNMAPK也能被NaCl、ABA、H2O2、PEG和SA等诱导。4.利用农杆菌侵染法将pBI-CsNMAPK转入烟草中,通过PCR和Northern blot检测表明CsNMAPK已经插入烟草基因组中并且已经表达。通过种子萌发期NO₃^-抗性试验表明,转基因烟草种子的抗性提高。通过苗期NO₃^-胁迫试验表明,转基因烟草抗性提高。为了检测转CsNMAPK基因烟草抗性提高的原因,我们测定了一系列和植物逆境胁迫有密切关系的生理指标,包括MDA含量、膜透性、H2O2含量、抗氧化物酶活性（SOD、POD、CAT、APX）、净光合速率、游离脯氨酸的积累等。结果表明,未处理的转基因烟草和野生型烟草的这些指标没有明显差别。但是胁迫处理后,转基因植株中电解质相对渗漏率、MDA和H2O2含量都显著低于野生型植株;转基因植株中SOD、CAT、POD和APX酶活性均高于野生型植株,具有更高的活性氧清除能力;脯氨酸含量增加的更多,具有较强的渗透调节能力。5.构建了原核表达载体pET-CsNMAPK,并在大肠杆菌BL21中表达融合蛋白,免疫小白鼠,制备抗体。Western杂交表明,转正义植株中CsNMAPK已在蛋白水平过量表达。6.用子房注射法将携带CsNMAPK基因的正义和反义表达载体的质粒导入六个黄瓜品种中,T1代幼苗叶片用2500 mg/L的卡那霉素涂抹筛选,对未变黄的植株进行PCR和Real-time PCR检测,获得了8株转基因黄瓜植株,其中3株反义转基因植株,5株正义转基因植株。更多还原

【Abstract】 Excess application of chemical fertilizer has caused secondary salinization in Chinese greenhouse. Soil secondary salinization is one of the major limited factors affecting vegetable production in the greenhouse. Unlike the seashore, NO₃^- is the main accumualated anion in greenhouse soil. The mechanism of plant resistant to salt stress has been widely investigated, which mainly focused on the NaCl treatment and little was known about the molecular mechanism resisting to NO₃^- stress in plants. Cucumber is one of the most important vegetables cultivated in the Chinese greenhouse and was easily damaged by salt stress. Therefore, it is important to investigate the mechanism of cucumber tolerant to NO₃^- stress.Mitogen-activated protein kinase （MAPK） cascades are one of the major pathways in signal transduction in all eukaryotes. The MAPK cascades are composed of MAPK, MAPKK （MAPK kinase） and MAPKKK （MAPKK kinase）. They transfer signals through phosphorylations of MAPKKK-MAPKK-MAPK in turn. In plants, MAPK cascades interlace with other signal transduction pathways and form a molecular interaction network, and regulate various cellular functions, including growth, development, biotic and abiotic stress responses. To date, numerous MAPKs have been isolated from different plants, and considerable progress has been made in understanding plant MAPKs. The regulation of MAPK signaling has been discussed most in dicot plant species （Arabidopsis, tobacco and alfafa etc.） and the study on cucumber MAPKs was virtually lacking. In this research, cucumbers were used as experimental materials. Research about the gene clone, expression characteristics and functional analysis of CsNMAPK was done. The main results were summarized as follows:1. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from cucumber root according to the homologous sequences from other plants. The middle fragment of the cucumber cDNA was obtained by RT-PCR. The 5′and 3′fragment of the cDNA was isolated by 5′and 3′RACE. The clone, which named CsNMAPK （Acession Numeber: DQ812086）, contains 1636 bp nucleotides with an open reading frame （ORF） of 1113 bp comprising 370 amino acid residues.2. Comparing of the predicted protein sequence of the CsNMAPK with MAPKs from other plants showed that CsNMAPK was most homologous to the Glycine max （GmMAPK1） （85.18 %）, Pisum sativum （PsMAPK3） （84.91 %）, Medicago sativum （MsMAPK ） （84.64 %）, Solanum tuberosum （StWIPK） （83.65 %）, Lycopersicon esculentum （LeMAPK） （83.11 %）, Capsicum annuum （CaMAPK1） （81.60 %）. Construction of a dendrogram based on the homologous full-length amino acid sequences revealed that CsNMAPK belong to subgroupⅠMAPK in plants, which may play important roles in environmental stresses and hormone signal transduction.3. Northern blot analysis revealed that excess NO₃^- induced the expression of CsNMAPK. When treated with different concentrations of NO₃^-, the mRNA level of CsNMAPK was up-regulated by 56, 98, 140 mM NO₃^-, but down-regulated by 182 mM NO₃^-, compared to the normal NO₃^- concentration of 14 mM in the hydroponic systems. To determine the main location of cucumber CsNMAPK expression under excess NO₃^-, the expression pattern was examined in various plant organs, including roots, leaves and stems. Tissue specific expression showed that CsNMAPK expressed in all organs and little difference of the expression could be seen. When treated the salt tolerant cucumber cultivar （Xintaimici） and salt sensitive cucumber cultivar （Shennongchunwu） seedlings with 182 mM NO₃^- for different times, the two cultivars showed slight differences in expression kinetics. The CsNMAPK expression was a little up-regulated in Xintaimici within 30 min and then the expression decreased gradually, while Shennongchunwu maintained its expression until 1 h after down-regulation. The expression of CsNMAPK could also be induced by NaCl, ABA, H2O2, PEG and SA.4. The full-length CsNMAPK cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method. The transgenic tobacco plants were verified by PCR, Northern blot and Western blot. It indicated that the CsNMAPK gene had been recombined into tobacco genome and transgenic tobacco plants were obtained. Transgenic seeds were germinated on MS media with 150 mM NO₃^- to score their tolerance to NO₃^- stress. The results showed that the transgenic seeds exhibited enhanced resistance to NO₃^- stress during seed germination. Experiments were also done during the seedling stage with 98 mM and 182 mM NO₃^-. The results indicated that there was little difference of the transgenic and wild type tobacco plants under normal enviroment. When treated with 98 mM and 182 mM NO₃^-, transgenic plants showed more resistance. To investigate the effect of CsNMAPK over-expression in tobacco on the physiological response to NO₃^- stress, a panel of physiological parameters, including malondialdehyde （MDA） contents, relative electrolyte leakage, H2O2 content, antioxidant enzyemes, net photosynthesis rate and free proline content were investigated. The resultes indicated that relative electrolyte leakage, MDA content and H2O2 content in the transgenic plants were lower than that in the wild type tobacco plants. Under NO₃^- stress treatments, the enzyme activities of SOD, CAT, POD and APX of transgenic tobacco were higer than the wild type tobacco. Free proline accumulation increased in both transgenic and wild type plants, but the increase was higher in the transgenic lines, demonstrating that they have higher osmotic adjustment ability.5. A recombinant of prokaryotic expression vector pET-CsNMAPK was constructed and transformed into E.coli BL21. The strong induced fusion protein bands were collected into PBS solution and used to immunize white mice to obtain antiserum. Western hybridization revealed the presence of the strong positive protein signals corresponding to CsNMAPK in sense transgenic plants of tobacco.6. CsNMAPK was also introduced into 6 different cucumber cultivars by overy injection pathway with the sense-expression vector and antisense-expression vector. The T1 plants were selected by resistance to 2500 mg/L kanamycin. 8 of kanamycin-resistant transgenic cucumbers were identified by PCR and Real-time PCR including 3 of antisense transgenic plants and 5 of sense transgenic plants.更多还原