【作者】 李耀文；

【导师】 柳参奎；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2009， 硕士

【摘要】 植物在生活史中常常遭受到各种逆境胁迫,如盐碱、干旱、重金属、低温、微生物侵染、紫外线等,严重影响了植物的生长、发育、繁殖。植物在忍受各种胁迫,保持体内氧化还原内环境平衡上与体内的活性氧解毒系统密切相关。过氧化氢酶（EC 1.11.1.6,CAT）是生物体内主要的抗氧化酶之一,它催化H₂O₂生成水和氧气。CAT与SOD、POD一起,能有效的清除植物组织中的活性氧（ROS）,防止细胞的损伤。作为分子生物学研究中的模式植物,水稻的完整基因组序列信息已经被人们掌握,水稻CAT基因也已被克隆登录。在水稻中,有三种过氧化氢酶同工酶被发现。每种CAT的表达模式都是是独特和复杂的。本研究对水稻的过氧化氢酶基因同工酶CAT1、CAT2、CAT3三个基因进行了的研究。为了研究过氧化氢酶基因表达与环境应答之间的关系,利用半定量PCR和Northern杂交分析了NaCl, NaHCO₃, Na₂CO₃,PEG6000,H₂O₂,4℃冷处理逆境下,水稻中CAT基因在转录水平上的表达情况。研究结果表明:CAT基因对于这几种环境胁迫有一定的应答关系,CAT基因受到胁迫诱导而表达量增加,但是增加的趋势和达到最大表达量的时间,三个同工酶之间、在不同的逆境处理上有不同的表达特性,在叶中,CAT1基因表达增加量在NaHCO₃、Na₂CO₃逆境处理时较CAT2和CAT3基因表达量增加趋势明显,在根中,CAT2基因表达增加量在NaCl、NaHCO₃、Na₂CO₃逆境处理时增加趋势明显,而不论叶和根,PEG6000和冷处理时,CAT3基因表达增加量较为明显。这个研究结果表明,同一个CAT基因,对几个不同逆境的处理产生的胁迫都较为敏感,而对其他的逆境处理不如其他CAT敏感,不同的组织组织中,CAT的类型也会不同。为了研究CAT基因的生理生化特性,将CAT1、CAT3基因构建到原核表达载体中。IPTG诱导后,融合蛋白His-CAT1、His-CAT3表达;大量表达后的包涵体经变性,经过Ni⁺凝胶亲和层析获得了高纯度的His-CAT1、His-CAT3融合蛋白。经过SDS-PAGE蛋白质电泳,His-CAT1,His-CAT3融合蛋白有不同的分子量。重组融合蛋白经过透析复性后,通过紫外分光光度法测定其活性,结果表明,复性后的重组蛋白表现出酶的活性,His-CAT1,His-CAT3融合蛋白两者之间的活性差异不明显。以上研究表明,水稻过氧化氢酶基因在植物抵抗外界环境压力中起着重要的作用,过氧化氢酶基因表达与环境应答之间,同工酶之间有一定的差异,变性纯化重组融合蛋白经过透析复性后表现出酶的活性。更多还原

【Abstract】 Plants frequently encounter stresses such as salinity, drought, heavy metal, radiation, low temperature, microbic infection; they adversely affect plant growth, development and productivity. The stability of plant internal redox environment and plant tolerance to stresses are closely related with system of scavenge the reactive oxygen.Catalase （EC 1.11.1.6 CAT） is one of the main antioxidant enzymes in Organisms that catalyzes the dismutation of H₂O₂ to water and dioxygen. CAT, along with hydroperoxidases and superoxide dismutases, serves as an efficient scavenger of reactive oxygen species （ROS）, preventing cellular damage. As the modal plant of molecular biology research, the whole genome information of rice had been known to us, and the CAT gene of rice had been cloned. In rice, three catalase isozymes have been identified and characterized in various tissues. .The developmental expression pattern of each CAT is unique and complex. In this study, we did a particular research for the isoenzymes CAT1; CAT2; CAT 3 genes of riceTo understand the responsive relationship between CAT of rice and environmental stress, the transcription expression profile of CAT was studied by RT-PCR and Northern blot analysis using rice leaf and root in NaCl, NaHCO₃, Na₂CO₃, PEG6000, H₂O₂, and 4℃cool conditions. The results show that the accumulation of CAT mRNA was observed in these stresss, and the intensity of stresses influenced the expression of CAT. The tendency of increases and the time to the greater expression, between three isozymes, in different adverse circumstance demonstrate different. In the leaf, the increase of CAT1 gene expression to NaHCO₃, Na₂CO₃ stress compares CAT2 and CAT3 gene tendency to be obvious, in the root, the increase of CAT2 gene expression to NaCl, NaHCO₃, Na₂CO₃ stress tendency to be obvious, and in leaf and root, the increase of CAT3 gene expression to be more obvious with PEG6000 and cold treatment. The result show that, the identical CAT gene, is more sensitive to several different adverse circumstance stress, but to other adverse circumstance stress was inferior that other CAT is sensitive, in different organization, the CAT can also be different.To understand the genes function in vitro, the CAT1 and CAT3 genes were cloned into the prokaryotic vector. After the inducement with IPTG, the His-CAT1and His-CAT3 fusion protein were obtained.finally purified by Ni⁺-sepharose affinity column from denatured inclusionbody and analyzed by SDS-PAGE. There are some different between His-CAT1and His-CAT3 fusion protein. The recombinant His-CAT1, His-CAT3 protein activity was measured after renaturation. We obtained the protein with biological activities.The above research indicated that, the rice catalase gene is playing the vital role in the plant resistance external environment stress, the responsive relationship between CAT of rice and environmental stress, between the isozymes has certain difference, the fusion protein with biological activities was obtained after denaturation purifies and renaturation.更多还原