海滨锦葵耐盐生理特性及脯氨酸代谢相关基因的研究

发布时间：2018-11-26 10:07

【摘要】：土壤盐渍化已成为一个制约农作物产量和质量的全球性问题。为了充分开发盐土资源和发展盐土农业,耐盐经济植物的筛选和培育越来越受到人们的重视。耐盐植物在长期适应盐渍环境的过程中进化出独特的耐盐机制,也为人类提供了取之不尽的耐盐基因资源库。因此,研究耐盐植物对盐胁迫的生理响应机制,从中挖掘关键耐盐基因,并利用基因工程等技术培育耐盐新品种,对盐土农业和农业的可持续发展都具有重要的理论和现实意义。本论文以耐盐植物海滨锦葵为实验材料,在系统地研究海滨锦葵对盐胁迫的生理响应的基础上,针对盐胁迫导致脯氨酸显著积累的生理特点,我们克隆了脯氨酸代谢相关基因,并对其表达模式和功能进行了初步分析。主要研究结果如下:(1)用不同浓度的Na Cl对海滨锦葵幼苗进行20d的胁迫处理,对其生长参数及相关生理生化指标进行测定。结果表明:100和200 mmol/L Na Cl的盐胁迫对海滨锦葵幼苗的生长影响不大,虽然导致海滨锦葵幼苗叶片和根内的Na+含量显著增加,但更多的Na+被区隔化在根内,而叶片内保持较高的K+/Na+比值和脯氨酸含量,说明海滨锦葵可以利用无机离子和有机溶质的协同渗透调节作用以对抗盐胁迫。同时,抗氧化酶活性的增加能够及时清除活性氧的积累,避免了氧化伤害的发生,表现在叶绿素荧光参数、MDA含量和细胞膜透性与对照没有显著差异。在300 mmol/L和400 mmol/L Na Cl的高盐处理下,海滨锦葵幼苗的生长受到严重抑制,各项生理指标显示海滨锦葵幼苗受到严重的渗透胁迫和离子毒害,光合机构、细胞膜系统受到破坏。由此可见,海滨锦葵幼苗具有一定的耐盐能力,低于200 mmol/L Na Cl胁迫处理基本不影响其生长,具有良好的渗透调节、离子平衡和区隔化及抗氧化能力。(2)针对盐胁条件下海滨锦葵脯氨酸显著积累的生理特征,采用RT-PCR及RACE技术克隆了脯氨酸代谢的相关基因,包括Kv P5CS、Kv OAT、Kv PDH和Kv Pro T基因。生物信息学分析表明,这些基因及其编码的氨基酸序列与其他植物中已知的同源序列均具有较高的同源性。对这些基因在盐胁迫条件下的表达模式进行研究表明,海滨锦葵幼苗叶片中脯氨酸的积累主要是因为盐胁迫促进了脯氨酸的生物合成。其中调控谷氨酸合成途径关键酶基因Kv P5CS相对于调控鸟氨酸合成途径关键酶基因Kv OAT发挥了更重要的作用。Kv Pro T的表达与Kv P5CS类似,被盐胁迫诱导显著上调,推测它可能在脯氨酸从其他部位向叶片中的运输和积累过程中发挥了重要作用,但这需要进一步的试验来证实。(3)成功构建了海滨锦葵脯氨酸合成酶关键基因Kv P5CS的过表达载体p BI121-Kv P5CS,并通过农杆菌介导和叶盘转化法获得了转基因烟草。利用200m M Na Cl对两个株系的T1代转基因烟草和野生型烟草进行14天的盐胁迫处理。结果表明,转Kv P5CS基因烟草比野生型烟草具有较强的耐盐能力,其叶绿素、脯氨酸含量及抗氧化酶活性明显高于野生型,说明过表达Kv P5CS基因而引起脯氨酸的积累可能在其抵抗盐胁迫过程发挥了重要作用。
[Abstract]:The salinization of soil has become a global problem that restricts the yield and quality of crops. In order to fully develop the salt-soil resources and to develop the salt-soil agriculture, the screening and cultivation of salt-tolerant economic plants are becoming more and more important. The salt tolerant plant has evolved a unique salt tolerance mechanism in the process of long-term adaptation to the saline environment, and also provides an inexhaustible resistant gene resource base for human. Therefore, it is of great theoretical and practical significance to study the physiological response mechanism of salt-tolerant plants to salt stress, to explore the key salt-resistant genes from them, and to use the techniques of genetic engineering to develop new varieties of salt-tolerant plants. Based on the study of the physiological response of the sea anemone to salt stress and the physiological characteristics of the significant accumulation of proline in the salt stress, we cloned the relevant genes of proline metabolism. The expression pattern and function were analyzed in this paper. The main results of the study were as follows: (1) The growth parameters and relevant physiological and biochemical indexes were determined by using Na Cl at different concentrations to treat the seedlings of Abelmoschus esculenta. The results showed that the salt stress of 100 and 200 mmol/ L Na Cl had no significant effect on the growth of the seedlings of the sea anemones, although the Na + content in the leaves and roots of the mallow seedlings was significantly increased, but more Na + was separated into the roots, while the higher K +/ Na + ratio and proline content in the leaves were maintained. The co-osmotic effect of inorganic ion and organic solute can be used to counter salt stress. At the same time, the increase of antioxidant enzyme activity can remove the accumulation of active oxygen in time, avoid the occurrence of oxidative damage, and show no significant difference in chlorophyll fluorescence parameter, MDA content and cell membrane permeability and control. Under the high salt treatment of 300 mmol/ L and 400 mmol/ L Na Cl, the growth of the seedlings of the Abelmoschus esculenta was severely inhibited, and various physiological indexes showed that the seedlings of the sea anemone were subjected to severe osmotic stress and ionic toxicity, and the photosynthetic mechanism and the cell membrane system were destroyed. It can be seen that the seedling of the sea anemone has a certain salt tolerance, less than 200 mmol/ L Na Cl stress treatment basically does not affect the growth of the seedling, and has good penetration regulation, ion balance and separation and oxidation resistance. (2) The relative genes of proline metabolism were cloned by RT-PCR and RACE, including Kv P5CS, Kv OAT, Kv PDH and Kv Pro T gene. Bioinformatics analysis shows that these genes and their encoded amino acid sequences have higher homology with the homologous sequences known in other plants. The study of the expression pattern of these genes under the condition of salt stress indicated that the accumulation of proline in the leaves of the moschus manihot was mainly because of salt stress and promoted the biosynthesis of proline. The key enzyme gene Kv P5CS, which regulates the synthesis of glutamic acid, plays a more important role in the control of the key enzyme gene Kv OAT of the synthetic pathway of the ornithine. The expression of Kv Pro T was similar to that of Kv P5CS, and was significantly upregulated by salt stress, suggesting that it could play an important role in the transport and accumulation of proline from other sites to the leaves, but this requires further testing to confirm. (3) The expression vector pBI121-Kv P5CS of Kv P5CS, a key gene of the Kv P5CS, was successfully constructed, and the transgenic tobacco was obtained by Agrobacterium-mediated and leaf-plate transformation. The T1-generation transgenic tobacco and wild-type tobacco of two strains were treated with 200m M Na Cl for 14 days. The results showed that the transgenic tobacco with Kv P5CS had a strong salt tolerance to wild type tobacco, and the content of chlorophyll, proline and antioxidant enzymes was higher than that of wild type, and it was shown that the accumulation of proline caused by the expression of Kv P5CS gene could play an important role in its resistance to salt stress.
【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945.78

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