【作者】 李杰；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2003， 博士

【摘要】 干旱、盐碱及低温等环境条件，都会构成对植物的渗透胁迫，使植物缺水受伤害，甚至导致植物死亡。由此造成的作物严重减产，已成为全球性的问题。如何通过提高作物抗干旱、盐碱及低温的能力来增加粮食产量，以满足日益增长的粮食需求，无疑是关系到国计民生的重大问题。与传统的育种技术和分子标记辅助育种相比，利用基因工程手段提高植物的抗逆性是更具吸引力的途径。但是由于渗透胁迫反应在遗传上和生理上的复杂性，使得人们对渗透胁迫的耐受机制的认识还远远不够，限制了基因工程技术的应用。表现为单独导入某个功能基因难以获得理想的抗逆性。最新研究结果表明，在渗透胁迫条件下，信号传导过程中的关键基因，如DREB、CDPK等，能够调节信号传导和功能基因的表达。它们在转基因植物中的过量表达会激活许多内源抗逆功能基因协同表达，从而获得更强的抗逆性。 本研究针对上述问题，以水稻为材料，深入系统地进行了渗透胁迫信号传导过程中关键基因的克隆、植物表达载体构建以及再生体系和遗传转化体系建立等研究工作，为抗渗透胁迫转基因水稻品种(系)的培育以及水稻抗渗透胁迫机理研究奠定了基础。探索一条抗渗透胁迫分子育种的新途径。 本研究获得的主要研究结果如下： 1．利用RT-PCR方法从拟南芥中克隆了DREB2A基因。序列分析结果表明，该序列与GENBANK上的序列完全相同。 2．利用嵌套式RT-PCR方法，用水稻钙依赖的蛋白激酶基因osCDPK7特异引物从抗盐的水稻品种辽盐241中扩增到约1700bp的片段。经酶切鉴定，初步确定为osCDPK7基因。 3．利用RT-PCR方法，用水稻促分裂原活化蛋白激酶基因osMSRMK2特异引物从水稻品种辽盐241中扩增到约1200bp的片段。经嵌套式PCR和酶切鉴定，初步确定为osMSRMK2基因。 4．利用PCR方法，用水稻促分裂原活化蛋白激酶基因osMSRMK2特异引物从水稻品种辽盐241基因组DNA中扩增到约2500bp的片段。经嵌套式PCR鉴定，初步确定为osMSRMK2基因。 5．利用RT-PCR方法，用水稻促分裂原活化蛋白激酶基因osMAPK4特异引物从水稻品种辽盐241中扩增到约1500bp的片段。经酶切鉴定，初步确定为osMAPK4基因。 6．构建了8个由不同启动子调控的、带有不同克隆位点的、分别适合于在单子叶植物和双子叶植物中表达的植物表达载体卡盒。其中，pCU、pCE12适用于在单子叶植物中的组成型表达外源基因；pC29A、pCC29A适用于在单子叶植物中的渗透胁迫诱导型表达外源基因；pBE12、pBCE12适用于在双子叶植物中的组成型表达外源基因；pB29A、pBC29A适用于在双子叶植物中的渗透胁迫诱导型表达外源基因。 东北农业大学农学博士学位论文一7．构建了4个由不同启动子调控的、分别适合于在单子叶植物和双子叶植物中表达的 DREB基因植物表达载体。其中，pCDE12用于DREBIA基因在单子叶植物中的组 成型表达：水＊29A用于＊RE B IA基因在单子叶植物中的渗透胁迫诱导型表达；姆＊35s 用于DREB IA基因在双子叶植物中的组成型表达；pBD29A用于DREBIA基因在双子 叶植物中的渗透胁迫诱导型表达。8．构建了2个由不同启动于调控的适合于在单子叶植物中表达的DREBZA基因植物表达 载体。其中，pCDRE12用于DREBZA基因在单子叶植物中的组成型表达；pCDR29A 用于DREBZA基因在单子叶植物中的渗透胁迫诱导型表达。9．建立了水稻再生体系 门）愈伤组织诱导培养基为：MS＋3mg／L 2，4D＋30g／L蔗糖十sg／L琼脂。 （2）继代培养基为：＊以Zing／L 2，4o叶og／L蔗糖十10叭琼脂，提高蔗糖及琼脂浓度 有利干愈伤组织保持结构紧密的状态。 （3）不定芽诱导培养基为：MS＋Zmg／L6－BA＋30g／L蔗糖十gg／L琼脂。10．建立了农杆菌介导的遗传转化体系 门）确定了愈伤组织继代阶段双丙胺磷（PPT）的筛选压力为 10mg／L；愈伤组织分化 阶段双丙胺磷（PPT）的筛选压力为1刀mg／L。 （2）影响遗传转化的3个主要因素的正交试验较优组合为：农杆菌重悬液为WSB，共 培养pH值为5．2，添加阿魏酸100mglL。各影响因素的主次顺序是：酚类添加物 ＞重悬液组成＞pH值。11．用农杆菌介导法进行了DREBIA基因对5个水稻品种的遗传转化，现己获得抗性苗， 并有大量材料处于分化筛选阶段。12．建立了植物DNA的小量提取方法；确定了转基因植株PCR检测的反应条件。更多还原

【Abstract】 Drought, high salinity and low temperature are common stress condition that adversely affect plant growth and crop production, which is a global problem. How to promote the resist ability of crops to drought, high salinity and low temperature and then increase crop production is a important question related to the national economy and the people’s livelihood. Compared with traditional breeding technique and molecular marker aided selection, it is a more attractive approach to improve the resist ability of crops by plant engineering. But as a result of the complexity of osmotic stress reaction in heredity and physiology, the knowledge about molecular mechanism of osmotic stress is far from necessity, which restrict the application of genetic engineering technique. For example, it is difficult to obtain ideal resist ability when single functional gene was introduced into plants. The last research results showed that some key genes involved in osmotic stress signal transduction, such as DREB and CDPK, regulated the expression of functional genes. Overexpression of these genes in transgenic plants activated endogenesis stress resisted genes, therefore endowed higher resist ablity.According to above-mentioned problems, this study take rice as material to clone key genes during osmotic stress signal transduction, construct plant expression vectors and cultivate transgenic rice. These will provide the foundation for studying osmotic stress resist mechanism and cultivating transgenic rice varieties which resist to osmotic stress.The main results was summarized as follows.1. DREB2A gene was cloned from Arabidopsis thaliana by RT-PCR. The result of sequence analysis showed that the sequence we obtained was the same as that in GenBank.2. A 1700bp fragment was amplified from a salinity resist rice variety, liaoyan 241 by nested RT-PCR using osCDPK.7 gene special primer. It was identified as osCDPK7 gene by endonuclutidase analysis.3. A 1200bp fragment was amplified from a salinity resist rice variety, liaoyan 241 by RT-PCR using osMSRMK2 gene special primer. It was identified as osMSRMK2 gene by nested PCR and endonuclutidase analysis.4. A 2500bp fragment was amplified from a sality resist rice variety, liaoyan 241 genomic DNA by PCR using osMSRMK2 gene special primer. It was identified as osMSRMK2 gene bynested PCR.5. A 1500bp fragment was amplified from a salinity resist rice variety, liaoyan 241 by RT-PCR using osMAPK4 gene special primer. It was identified as osMAPK4 gene by endonuclutidase analysis.6. Eight plant expression vector cassettes were constructed, which were regulated by different promoters, contained different cloning sites and were fit for expression in monocotyledon or dicotyledon respectively. In these vector cassettes pCU and PCE12 were fit for constitutive expression of foreign genes in monocotyledon, pC29A and PCC29A fit for osmotic stress inducible expression of foreign genes in monocotyledon, pBE12 and pBCE12 fit for constitutive expression of foreign genes in dicotyledon, pB29A and pBC29A fit for osmotic stress inducible expression of foreign genes in dicotyledon.7. Four DREB 1A gene plant expression vectors were constructed, which were regulated by different promoters and were fit for expression in monocotyledon or dicotyledon respectively. In these vectors, pCDE12 is fit for constitutive expression of DREB 1A gene inmonocotyledon, pCD29A fit for osmotic stress inducible expression of DREB1A gene in monocotyledon, pBD35s fit for constitutive expression of foreign DREB1A gene in dicotyledon, pBD29A fit for osmotic stress inducible expression of DREB1A gene in dicotyledon.8. Twor DREB2A gene plant expression vectors were constructed, which were regulated by different promoters and were fit for expression in monocotyledon. In these vectors, pCDRE12 is fit for constitutive expression of DREB2A gene in monocotyledon, pCDR29A fit for osmotic stress inducible expression of DREB2A gene in monocotyledon.9. Rice更多还原