【作者】 阴翠翠；

【导师】 尉亚辉； 吴燕民；

【作者基本信息】 西北大学 ， 细胞生物学， 2009， 硕士

【摘要】 转录调控是植物应答逆境等一系列基因表达的最主要调控形式,其中DREB和ERF类转录因子主要参与生物和(或)非生物胁迫的响应。本研究以新疆沙生植物铃铛刺为材料,从中分离克隆新的DREB、ERF转录因子基因,以期为利用基因工程改良作物的抗逆性提够优良候选基因,同时为豆科植物耐逆胁迫的分子机制研究和作物抗逆育种提供理论基础。本研究取得以下成果:1.据植物中AP2/EREBP类基因DNA结合域保守区设计简并引物,以铃铛刺叶片为材料,RT-PCR扩增出2个AP2/EREBP基因同源片段;据此设计基因特异引物,通过RACE-PCR分离克隆出1个DREB、1个ERF转录因子基因,分别命名为HhDREB2、HhERF2,登陆号为:EU872018和FJ032362。2.半定量RT-PCR对2个基因的组织特异性表达情况进行了检测,结果在铃铛刺的根、茎、叶中均有表达;胁迫应答检测结果显示,两个基因的表达均受干旱、高盐和低温诱导,HhERF2还受茉莉酸甲酯的诱导。3.以基因组DNA为模板对HhDREB2、HhERF2进行了PCR扩增,扩增产物经序列分析显示,HhDREB2不含内含子,HhERF2含有1个内含子。4.酵母单杂交分析结果显示,HhDREB2和HhERF2都具有转录激活功能,β-半乳糖苷酶活性分别为30.37U和49.26U,Whatman滤纸显色均能呈现明显蓝色。5.构建绿色荧光蛋白融合表达载体,利用基因枪法转化洋葱表皮细胞的瞬时表达结果显示,HhDREB2和HhERF2蛋白均定位于细胞核中。6.构建植物表达载体pC13rd29A-HhDREB2、pC13rd29A-HhERF2及pC1302-HhERF2,利用Floral-dip法转化野生型拟南芥Columbia-0;通过Hyg抗性、PCR及RT-PCR筛选鉴定阳性苗;耐胁迫检测结果表明,HhDREB2、HhERF2在拟南芥中的表达,增强了转基因拟南芥对干旱、高盐、低温及细菌的胁迫耐受性。更多还原

【Abstract】 Transcription control is the most vital regulation ways of the genes expressed in plants response to stress. Transcription factors DREB and ERF are involved in the response of organism to biotic and/or abiotic stress. In this study, two novel genes encoding DREB and ERF transcription factors were isolated from salttree cultivars. The purpose of this research was to provide candidate genes to enhance crop tolerance to adverse conditions, meanwhile reveal the mechanism of stress tolerance in leguminous plant and provide a theoretical basis for crop tolerance.The following results were achieved in this study:1. According to the conserved regions of the AP2/EREBP DNA binding domains a pair of degenerate primers was designed. Using RT-PCR two fragments were amplified from leaves of salttree cultivars. Two novel genes encoding AP2/EREBP transcription factors, HhDREB2 and HhERF2 were isolated by RACE-PCR. GeneBank numbers are EU872018 and FJ032362, respectively.2. The expression pattern of HhDREB2 and HhERF2 in different organs was studied using semi-quantitative RT-PCR. It was proved that they expressed in roots, stems, and leaves of salttree, and were greatly induced by drought, high salinity and low temperature. In addition, HhERF2 were greatly induced by methyl jasmonate.3. HhDREB2 and HhERF2 were amplified using genomic DNA as templates. It was proved that HhDREB2 lacked intron. However, HhERF2 contained one intron.4. HhDREB2 and HhERF2 proteins both had transcriptional activation confirmed by the yeast one-hybrird method, and theirβ-galactosidase activity were 30.37 U and 49.26 U, respectively. The the yeast transformants of HhDREB2 and HhERF2 showed a clear blue in the colony-lift filters assay.5. We constructed two green fluorescent protein fusion expression vectors . They were transformed into the epidermal cells of onion by particle bombardmental method. Both HhDREB2 and HhERF2 proteins were localized in cell nucleus.6. HhDREB2 and HhERF2 were transformed into Arabidopsis thaliana with Agrobacterium tumefaciens C58C1 containing the plant expression vectors pC13rd29A-HhDREB2, pC13rd29A-HhERF2 and pC1302-HhERF2, respectively. The positive Arabidopsis thaliana transformants were selected using MS medium containing hygromycin, PCR and RT-PCR. Results of stress tolerance experiments showed that the expression of HhDREB2 and HhERF2 could improve resistance to drought, high salinity, low temperature and bacteria of transgenic Arabidopsis thaliana obviously.更多还原