【作者】 东丽；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 植物学， 2005， 硕士

【摘要】 黄瓜在我国蔬菜周年供应上占有重要地位,但因真菌病害的危害每年都有不同程度的减产。目前采用最多的化学防治手段虽取得了一定功效,但却带来了严重的环境污染、生态失调和危害人类健康等弊端。实践证明,最经济有效的方法就是培育抗病品种,然而常规育种手段由于育种周期长、效率低、基因资源匮乏及随病源生理小种的变化抗病性消失等原因,迄今,没有获得理想的效果。随着分子生物学和基因工程技术的日趋成熟,利用基因工程手段进行抗病分子育种已成为可能。转录因子DREB1A 是在拟南芥中克隆并鉴定的,它能在干旱、低温及高盐胁迫条件下调控报道基因的表达,并在上述逆境胁迫信号传递中起重要作用。由于DREB1A 转录因子可以调控植物自身内源多个与植物干旱、低温及高盐耐性有关的功能基因的表达,因此,利用转录因子来改良植物抗逆性与单基因的遗传转化相比有明显优势。拟南芥rd29A 启动子的表达受到干旱、低温和高盐的诱导,因为在它的启动子区域中,有两个与上述环境胁迫应答有关的DRE 顺式作用元件,是植物抗逆基因工程研究中理想的逆境诱导型启动子。因此,本研究以黄瓜为试材,建立黄瓜植株再生体系和遗传转化体系;构建了分别由双35S 组成型启动子、E12 强组成型启动子和rd29A 诱导型启动子调控的核糖体失活蛋白基因、几丁质酶基因和DREB1A 基因的多价基因植物表达载体;利用农杆菌介导法将其导入黄瓜优良品种,获得了转pBDCR T0及T1代PCR 阳性植株。T1代转基因植株的抗逆性实验表明,DREB1A 基因的表达可以提高黄瓜的抗旱性。本研究为培育抗逆境及抗真菌病转基因植株探索途径,并为进一步揭示核糖体失活蛋白基因、几丁质酶基因和DREB1A 基因的分子机理提供了理论依据。主要研究结果如下: (1) 植物表达载体构建构建了分别由双35S 组成型启动子、E12 强组成型启动子和rd29A 诱导型启动子调控的核糖体失活蛋白基因、几丁质酶基因和DREB1A 基因的多价基因植物表达载体,并命名为pBDCR。(2) 黄瓜植株再生体系建立①确定了黄瓜“津研四号”不定芽诱导培养基为:MS+1mg/L BA, 30g/L Suc, 0.8%Agar, pH5.8; ②不同基因型的不定芽分化率、平均每块外植体芽数及分化效率不同:“长春密刺”、“津研四号”不定芽分化率分别为85.8%、69.7%,平均每块外植体芽数分别为3.9、3.4,分化效率分别为334.6%和236.9 %。(3) 农杆菌介导的遗传转化体系建立更多还原

【Abstract】 Cucumber plays an important role in vegetable supply, but the yield is declined every year because of its fungi disease. One of the common protecting methods is chemical medicine. Although it has some effect, it also brings some disadvantages, such as heavy polluted environment, maladjustment ecology and harm to human healthy. It is testified that culture the fungi-resistant cultivates is the most economic and available way. However, the conventional breeding can’t meet the need of cultivating the fungi-resistant varieties, because of its long period, short fungi-resistant genes and disappearing resistance with the changes of pathogen. So, using the genetic engineering to breed the fungi-resistant varieties is a available method. But there are some problems in fungi-resistant engineering of cucumber One is that a single gene only can restrain a few varieties of fungi, and the other is that it is difficult to obtain a large quantity of transgenic plants because of lower rate of transformation. Transcription factor DREB1A is cloned and identified from Arabdopsis thaliana. It can regulate the report gene under drought ,cold and high salt stress and plays an important role in the signal conduction under all above stress. Transcriptional factor DREB1A can control the expression of many genes involved in antibiotic stress. It has much more advantage transferring a transcriptional factor than a single gene in the stress tolerance genetic engineering. The expression of rd29A gene of Arabdopsis thaliana is induced by drought ,cold and high salt stress. There are two DRE cis–acting element involved in responding to these stress signals in its promoter region. So it is an effective inducible expression promoter that can be used in genetic engineering of improving stress tolerance. According to above-mentioned facts, this study took cucumber as material to establish regeneration system and genetic transformation system. One multivalent plant express vectors was constructed, including ribosome inactivating protein (rip) gene、chitinase (chi) gene and DREB1A gene,which were regulated respectively by constitutive expression promoter double CaMV35S、E12 and inducible expression promoter rd29A. Cucumber was transformed by Agrobacterium Tumefaciens with the multivalent plant express vectors and obtained PCR positive plants(T0 and T1). The stress tolerance experiments showed that the drought stress tolerance of T1 transgenic cucumber has been improved because of the expression of DREB1A gene. The research work will provide the function for cultivating transgenic variety that resist to osmotic stress and fungi disease and studying resist mechanism of rip gene、chi gene and DREB1A gene. The main results were summarized as follows: (1) Construction of plant expression vectors One multivalent plant express vectors pBDCR was constructed, including that ribosome inactivating protein (rip) gene、chitinase (chi) gene and DREB1A gene, which were regulated respectively by constitutive expression promoter double CaMV35S、E12 and inducible expression promoter rd29A. (2) Establishment of cucumber regeneration system ①Identified adventitious buds inducing medium of cucumber“JinYanSiHao”:MS+1mg/L 6-BA, 30g/L Suc, 0.8%Agar, pH5.8. ②Differentiation frenquency of adventitious buds、the number of buds per explant and efficiency of differentiation are different of different genotypes :“ChangChunMiCi”and“JinYanSiHao”.differentiation rate of adventitious buds are 85.8% 、69.7% respectively. The number of buds per explant are3.9 、3.4 respectively. Efficiency of differentiation are 334.6 %、236.9 % respectively. (3) Establishment of cucumber genetic transformation system mediated by Agrobacterium Tumefaciens ①Identified the concentration of Km on stage of adventitious buds differentiation: Km concentration of“JinYanSiHao”is 25 mg/L. ②Resistant buds of “ChangChunMiCi”were obtained, which were transformed with LBA4404 (pBDCR). The rate of resistant buds are 39.79%; Resistant buds of “JinYanSiHao”were obtained. which were transformed with LBA4404 (pBDCR). The rate of resistant buds are 25.39%; (4) Melecular biology indentification of transgenic plants ①PCR positive plants of “ChangChunMiCi”were obtained, which were transformed with LBA4404 (pBDCR). 41 plants out of 142 detected Kmr plants of “ChangChunMiCi”were positive, The rate of PCR positive are 28.87%. The PCR positive plants number of chi 、rip and DREB1A gene are 57 、50 、53 respectively.The rate of PCR positive are 40.14 %、35.21%、37.32% respectively. ②PCR positive plants of “JinYanSiHao”were obtained, which were transformed with LBA4404 (pBDCR). 46 plants out of 151 detected Kmr plants of “JinYanSiHao”were positive, The rate of PCR positive are 30.46%. The PCR positive plants number of chi 、rip and DREB1A gene are 57 、52 、65 respectively.The rate of PCR positive are 37.75%、34.44%、43.05% respectively. ③The efficiency of transformation is different of different genotypes. The efficiency of transformation of “ChangChunMiCi”which were transformed with LBA4404 (pBDCR) is 11.49%. The efficiency of transformation of “JinYanSiHao”which were transformed with LBA4404 (pBDCR) is 7.73%. ④PCR positive plants of “ChangChunMiCi”(T1)were obtained, which were transformed with LBA4404 (pBDCR). 75 plants out of 377 detected Kmr plants of “ChangChunMiCi”were positive of DREB1A gene, The rate of PCR positive are 19.89%. 45 plants out of 377 detected Kmr plants of “ChangChunMiCi”were positive of chi、rip gene, The rate of PCR positive are 11.94%. (5) Biology Identification of transgenic plants ①The stress tolerance experiments showed that the drought stress tolerance of T1 transgenic cucumber has been improved because of the expression of DREB1A gene. The result of Pro analysis showed that the Pro amount of T1 transgenic cucumber has been improved. ②The Manicol sensitivity experiment of T0 transgenic seed showed that resistant to drought of seed has been improved ③P.cubensis experiment is being.done.更多还原