【作者】 王呈玉；

【导师】 李彦舫；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 本文采用cDNA末端快速克隆技术(RACE),以短芒大麦为试验材料,分离得到与非生物胁迫信号转导相关的两个基因,并对其基因特性进行了鉴定和分析。实验结果表明:转录因子HbDREB1基因cDNA全长899bp,其中包括660bp完整的开放阅读框(open reading frame,ORF),推测编码220个氨基酸的多肽;HbDREB1蛋白具有一个典型AP2/ERF DNA结合域;体外凝胶滞留实验表明全长HbDREB1蛋白能够与DRE元件特异性的相结合。同其作为转录因子的功能相一致,HbDREB1是一种细胞核内蛋白;Southern杂交表明HbDREB1基因在短芒大麦基因组中以单拷贝的形式存在,Northern杂交表明该基因主要受到低温强烈诱导,对高盐和干旱引起细胞渗透压发生的变化也有一定的响应,而对植物激素ABA不响应;转基因烟草的相对离子渗漏率和脯氨酸含量的测定结果表明,表达HbDREB1基因能够提高转基因烟草的抗性,但转基因植株表现出严重的生长迟滞现象。短芒大麦钙依赖蛋白激酶HbCDPK基因是植物钙依赖蛋白激酶家族的一个成员,且属丝氨酸/苏氨酸类蛋白激酶,是一个与膜相关的钙依赖蛋白激酶;cDNA全长2094bp,其中包括1596bp完整的开放阅读框; Northern-blot分析发现该基因受冷、干旱、高盐胁迫和激素诱导表达;转基因株系的生理生化指标测定结果表明,HbCDPK的表达降低了转基因植物的细胞膜在逆境条件下的损害程度。据此可以认为,短芒大麦DREB1转录因子和钙依赖蛋白激酶(HbCDPK)基因参与了非生物胁迫信号转导,并且具有提高转基因植物抗逆性的潜能。更多还原

【Abstract】 With the development of economy, environment living beings depend on has had a huge change. Drought, high salinity and low temperature have a serious adverse impact on growth and development of corps, economic plants and vegetation and adversely affect crop production, which is a global problem. One of the important restrictive factors to interfere growth of plant is changes temperature. Exploitation of novel botanical resources by bioengineering technology to promote the resist ability of crops to drought, high salinity and low temperature and then increase crop production not only is an important question related to the national economy and the people’s livelihood, but also become a hot point of study of modern bioengineering technology and area of plant improving. Macroscopical ecological principle combines to the microcosmic genetic engineering technology have been a new area of research. But as a result of the complexity of abiotic stress reaction in heredity and physiology, the knowledge about molecular mechanism of the resistance to abiotic 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.Many research results showed that some key genes encoded for the products responsive to abiotic stress, such as DREB and CDPK, participated in abiotic stress signal transduction and regulated expression of functional genes. Over-expression of these genes in transgenic plants activated the expression of stress resisted genes, therefore endowed higher resist ability. According to above-mentioned problems, this study take Hordeum brevisubulatum(Trin.)Link as material to clone key genes during abiotic stress signal transduction by RACE PCR method, DREB1 and CDPK, and analyses the characterization of two genes encoded for the products responsive to abiotic stress.DREBs transcription factors able to bind the DRE cis-acting element in the promoter of some genes concerned with cold, drought and high-salt stresses, resulting in initiating the expression of these genes at transcriptional level. Therefore, it would be a good strategy to improve tolerance of various kinds of agriculturally important plants to abiotic stresses by CBF/DREB genes transfer. Using RACE method, a DREB1 transcription factor cDNA gene was successfully isolated from Hordeum brevisubulatum(Trin.)Link and named as HbDREB1 with 899 bp (GenBank accession number: DQ250027), encoding a predicted proteins of 220 amino acid residues. It is shown from Protein Blast data that these proteins belong to a typical member of the AP2/EREBP transcription family with conserved AP2 DNA-binding domain (aa.49-aa.109). HbDREB1 had the PKK/RPAGRxKFxETRHP, DSAWR and LWSY“signature sequences”surrounding the EREBP/AP2 domain. These“signature sequences”clearly distinguish those proteins from other proteins that have the EREBP/AP2 domain. HbDREB1 is classified into the A-1 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. We found that HbDREB1 is most closely related to the barley HvCBF2 (GenBank accession number: AF442489), showing 91.4 % sequence identity. The DNA-binding properties of HbDREB1 were analyzed by electrophoretic mobility shift assay (EMSA), showing that HbDREB1 successfully binds to the DRE cis-element. Consistent with its role as a DNA-binding protein, HbDREB1 is preferentially localized to the nucleus of transgenic tobacco root cells. Northern blot analysis indicated that its expression induced by cold and weakly induced by drought and salt stress, but did not respond to ABA treatment. We further investigated the role of the HbDREB1 regulatory genes in cold acclimation in transgenic tobacco by gene transformation via Agrobacterium. PCR, PCR-Southern and Northern analyses of transgenic plants showed that the HbDREB1 gene was integrated into the tobacco genome and expressed at the transcriptional level. In addition, we show that over-expression of HbDREB1 results in elevated levels of Pro compared with the wild-type plants. Taken together, our results indicate that HbDREB1 is a DRE-binding transcription factor, heterologous over-expression of HbDREB1 may confer cold resistance in transgenic tobacco plants. The HbDREB1-expression tobacco plants used in the experiments described here also display dwarf and a delay in flowering phenotypes.Calcium-dependent protein kinases (CDPKs), which have been mainly identified in plants, are important signal factors involved in multiple physiological processes. This critical Ser/Thr protein kinase family can act as the sensor of calcium and transduce external and internal signal by phosphorylating the downstream signal factors involving proteins and transcriptional factors. CDPKs are a multigene family and play important roles in plant calcium signal transduction. Many researches showed that CDPKs participate in C/N metabolism, transmembrane transport of ion and water, cytoskeletal regulation, stomatal movement regulation, growth and development regulation and biotic/abiotic stress responses in plants. Calcium-dependent protein kinases (CDPKs) play important roles in multiple signal transduction pathways but the precise role of individual CDPK is largely unknown. A novel calcium-dependent protein kinase gene (designated as HbCDPK) was isolated from Hordeum brevisubulatum (Trin.) Link. The full-length cDNA of HbCDPK was 2094 bp long with an open reading frame (ORF) of 1596 bp encoding a polypeptide of 532 amino acid residues. There are four domains of a variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain from N- to C-terminal end of HbCDPK. HbCDPK located to cytoplasm membrane was determined by expression of green fluorescent protein fusions in tobacco root cells. Northern-blot revealed that the expression of HbCDPK was in root, leaf and mature embryo. Further expression analysis revealed that cold, dehydration, high salt stress and ABA treatments could up-regulate the HbCDPK transcription. We further investigated the role of the HbCDPK regulatory genes in cold stress in transgenic tobacco by gene transformation via Agrobacterium. PCR, PCR-Southern and Northern analyses of transgenic plants showed that the HbCDPK gene was integrated into the tobacco genome and expressed at the transcriptional level. In addition, we further show that over-expression of HbCDPK results in elevated levels of Pro compared with the wild-type plants. Taken together, our results indicate that HbCDPK was a membrane-associated Serine/Threonine protein kinase and might play vital roles in plant development and responses to environment stimuli. Over-expression of HbCDPK might confer cold resistance in transgenic plants.The cloning and characterizations of the above two genes would try to improve the ability to resist low temperature stress of plants and slow the adverse effects of various kinds stresses to distribution, growth and economic returns of plants. These would offer the theoretical basis to further investigate the mechanism of regulating the gene expression in stesses.更多还原