【作者】 邓春婷；

【导师】 欧阳波；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2009， 硕士

【摘要】 非生物逆境（干旱、冷害、盐碱等）是农作物产量和品质的主要限制因素之一。在各种非生物逆境胁迫下,植物通常都会出现活性氧（ROS）胁迫。植物在进化过程中形成了一套有效的抗氧化体系来减轻ROS胁迫以维持植物的正常生理功能。抗坏血酸（AsA）是植物组织中主要的抗氧化物质。脱氢抗坏血酸还原酶（DHAR）和单脱氢抗坏血酸还原酶（MDHAR）是抗坏血酸代谢中再生的关键酶,对于维持抗坏血酸氧化还原状态起着重要作用。逆境诱导型启动子（如受干旱、高盐或冷胁迫诱导的拟南芥RD29A启动子）在避免组成型超表达抗逆有关基因的负面影响方面具有积极的作用。本研究以番茄这一重要蔬菜作物为材料,通过调控番茄抗坏血酸代谢过程中的DHAR1和MDHAR1的表达,利用超表达、诱导表达和RNAi技术分析转基因番茄的抗非生物逆境的特性,探讨这两个基因在抗逆中的具体功能,并期望能获得具有应用价值的抗多种逆境的番茄材料。本研究获得的主要结果如下:1.从拟南芥中克隆了RD29A基因（AT5G52310）上游823bp的启动子序列IpeD29A。构建了一个含pRD29A的通用诱导表达载体pK2GW7I;2.通过Gateway重组技术构建了番茄DHAR1、MDHAR1的超表达载体pKOED、pKOEMD和诱导表达载体pKDI、pKMDI;3.通过农杆菌介导的基因转化技术,将有关载体导入番茄,获得转基因番茄苗44株。通过PCR验证T-DNA已经整合到番茄基因组;4.通过RT-PCR对抗坏血酸代谢相关基因在番茄不同组织中的表达谱进行了分析,发现DHAR1和MDHAR1均为组成型表达,但在各组织中的表达量有差异;5.对DHAR1和MDHAR1有关载体的转基因T₀代植株进行了初步抗逆性分析。并根据结果从每个载体转化的转基因后代中挑选出5个系,在T₁代中作了进一步的分析。初步发现超表达DHAR1和MDHAR1以及诱导表达DHAR1的转基因植株对提高番茄耐盐、耐旱、耐寒均有作用。初步结果表明盐胁迫下RD29A诱导型启动子在番茄中可以有效驱动下游基因的表达,对提高番茄的抗逆性具有重要意义。本研究获得了部分耐盐、耐寒、耐旱的转基因株系,为进一步的理论和育种研究提供了材料。更多还原

【Abstract】 Abiotic stress including drought, chilling and salinity etc. is one of the major threats to crop production and quality. Reactive oxygen stress generally accompanies with various abotic stresses. Plants have developed their own antioxidant defense systems to alleviate ROS stress and maintain normal physiological process. Ascorbate acid （AsA） is a major antioxidant in plants. Dehydroascorbate reductase （DHAR） and Monodehydroascorbate reductase （MDHAR） are key enzymes in AsA regeneration and essential for maintaining the redox state of AsA. The stress-inducible promoter, such as RD29A promoter which is induced upon drought, salt or cold stress, may be useful to minimize side effects caused by constitutively expressing of stress tolerance-related genes in plant. This study aims to decipher the functions of DHAR1 and MDHAR1 by analyzing of their roles in abiotic stress using strategies of over expression, inducible expression and RNA inference, which may also lead to creation of tomato breeding materials with multiple stress tolerance. The main results were as follows:1. The 823 bp upstream regulatory region（ pRD29A） of RD29A gene（AT5G52310） from Arabidopsis thaliana genome was amplified using PCR technique. pK2GW7I, a binary vector with pRD29A was constructed.2. The tomato inducible expression vectors- pKDI、pKMDI and over expression vectors- pKOED, pKOEMD were constructed for tomato DHAR1 and MDHAR1, respectively.3. A total of 44 putative transgenic tomato plants were obtained. PCR analysis results indicated that T-DNA was integrated into the tomato genome.4. RT-PCR analysis showed that DHAR1, MDHAR1 were constitutively expressed in different tissue of tomato, whereas the expression levels were different.5. Tolerance to oxidative stress for transgenic plants was investigated in T₀ generation. Five lines for each construct from T₁ transgenic plants were further chosen for function analysis of the DHAR1 and MDHAR1. It was found that that overexpression of DHAR1, MDHAR1 or inducible expression of DHAR1 could improve salt, drought and chilling stress tolerance on tomato. The preliminary results indicated pRD29A functions in tomato. Several transgenic tomato lines with tolerance to salt, drought and chilling stress were obtained and could serve as materials for future research on abiotic stress tolerance.更多还原