海滨锦葵耐盐生理特性及脯氨酸代谢相关基因的研究
王红艳
学位类型博士
导师邵宏波
2016-05-16
学位授予单位中国科学院大学
学位授予地点北京
关键词海滨锦葵 盐胁迫 脯氨酸代谢 基因表达调控 盐土农业
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NaCl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 NaCl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 NaCl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 NaCl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KvP5CSKvOATKvPDHKvProT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KvP5CS相对于调控鸟氨酸合成途径关键酶基因KvOAT 发挥了更重要的作用。KvProT的表达与KvP5CS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KvP5CS的过表达载体pBI121-KvP5CS,并通过农杆菌介导和叶盘转化法获得了转基因烟草。利用200 mM NaCl 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KvP5CS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KvP5CS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。
其他摘要
 Soil salinization has become one of the resource and ecological problems all over the world, and it is one of the important abiotic stress factors that affect crop production. In order to fully develop the saline soil resources and saline soil agriculture, the selection and breeding of salt-tolerant plants with economic values has been attracting more and more attention. Salt-tolerant plants have evolved into complicated salt-tolerant mechanisms which enable them to thrive in high-salt environments and they can be either domesticated into new salt-tolerant crops or used as a potent genetic source for improving the salt tolerance of conventional crops through genetic engineering methodology. So it is very important to study the salt-tolerant physiological mechanisms and determine the key genes of salt tolerance for breeding the new salt-tolerant species by use of genetic engineering, which is of extremely important theoretical and practical significance for the sustainable development of saline soil amelioration and saline agriculture. This work firstly studied the physiological response of Kosteletzkya virginica to salt stress and proline accumulation was found in the leaf of seedlings under salt stress. To explore the cause behind proline accumulation , we first cloned proline metabolism-related genes and investigated their expression and function. The main results are as follows:
  (1) After treatment by different concentrations of NaCl for 20 days, the growth parameters of Kosteletzkya virginica seedlings were almostly unaffected under 100 and 200 mmol/L NaCl stress. Although Na+ content in the leaves and roots was significantly increased, more Na+ was compartmented into roots and the leaves had higher K+/Na+ ratio and higher proline content than roots, indicating that both inorganic ions and organic solutes of seedlings were involved in the osmotic adjustment to counter salt stress. Compared with the control, no significant difference was found in chlorophyll fluorescence parameters, MDA content and cell membrane permeability. Maybe the increased activitiy of antioxidant enzymes protected the seedlings from oxidative damage caused by reactive oxygen species. Under 300 and
 

 400 mmol/L NaCl stress, the growth of seedlings was severely inhibited. The physiological indexes showed that the treated seedlings were subjected to severe osmotic stress and ion toxicity which damaged their photosynthetic apparatus and cell membrane system. In summary, Kosteletzkya virginica seedlings had good salt tolerance due to the combination of osmotic adjustment, ion balance and antioxidant capacities, which enable them to grow well under less than 200 mmol/L NaCl stress.
  (2) To explore the causes behind proline accumulation induced by salt stress, we cloned four proline metabolism-related genes including KvP5CS, KvOAT , KvPDH, and KvProT from Kosteletzkya virginica by means of RT-PCR and RACE methods.
Bioinformatics analysis showed that these genes and their encoded amino acid sequences shared high homology with other known homologous sequences from other plants. Their expression profiles under salt stress revealed that proline accumulation in leaves under salt stress was due to the up-regulated expression of KvP5CS and KvOAT which promoted the biosynthesis of proline. In this process, the up-regulated expression of KvP5CS played a more important role than KvOAT. Similar to the KvP5CS expression, the KvProT expression was also significantly up-regulated by salt stress. It was speculated that KvProT might be involved in the proline transport from other parts to the leaves, but this needs to be confirmed.
  (3) The expression vector pBI121-KvP5CS was constructed and transformed into tobacco by Agrobacterium-mediated method to obtain transgenic tobacco. The T1 generation transgenic seedlings and wild tobacco were treated by 200 mmol/L NaCl stress for 14 days. The results showed that transgenic seedlings had better salt tolerance than wild type. The chlorophyll content, proline content and antioxidant enzyme activity of transgenic seedlings were significantly higher than that of wild type, indicating that increased proline content induced by over-expression KvP5CS might play an important role in their responses to salt stress.
Keywords: Kosteletzkya virginica, salt stress, proline accumulation, proline metabolism,gene expression and regulation, salt-soil agriculture
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/13840
专题中国科学院烟台海岸带研究所知识产出_学位论文
作者单位中科院烟台海岸带研究
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王红艳. 海滨锦葵耐盐生理特性及脯氨酸代谢相关基因的研究[D]. 北京. 中国科学院大学,2016.
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