盐胁迫下红树植物—角果木耐盐生理及分子基础研究
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摘要
红树是生长在热带、亚热带海岸潮间带的木本挺水植物,长期生长于高盐生境使红树植物具有一套独特的耐盐机制。本研究以红树科植物角果木(Ceriops tagal (Perr.) C.B.Rob.)为研究对象,从根的显微结构、生理变化、基因表达变化等3个方面对角果木的耐盐机理进行探索。
主要结果如下：
1、角果木幼苗在不同浓度处理一个月后,根尖的中柱鞘变得不明显,且盐浓度越大,中柱鞘越不明显,从而盐胁迫影响了角果木侧根的发生。盐胁迫对角果木根尖皮层也有较大影响：高盐(500 mmol·L-1)胁迫下,角果木根尖外皮层、内皮层明显增厚,以阻止有害离了进入。
2、盐胁迫下,同一处理时间点随着盐浓度的增加角果木根部总蛋白含量下降,但随处理时间的延长各盐处理下的总蛋白含量与对照组的差异逐渐缩小,且在24h后略高于对照组,说明角果木在适应盐环境的过程中有新的蛋白产生。
3、植物受到盐胁迫后会立即引起氧化胁迫,产生氧化胁迫后就导致膜脂过氧化,因此早期(≦9h) MDA含量急剧上升,这种信号的出现诱导植物中抗氧化防御系统的启动,相关酶的表达。因此在24h内各盐处理前期,角果木根部抗氧化酶类及抗氧化物质均有所增加,而处理24 h后,角果木根部MDA含量、ASA含量、POD活性以及SOD活性均恢复至对照水平,表明角果木在受盐胁迫后通过启动抗氧化酶类、抗氧化物质以清除活性氧对根部的损伤,而后开始适应外界盐环境。
4、在高盐(500 mmol·L-1)胁迫下,角果木根部可溶性糖含量和Pro含量在盐胁迫前期(≦72h)均有所增加,而在72h后均处于稳定状态,而多酚含量在盐胁迫初期(≦24h)有所下降,之后也处于稳定状态。表明角果木在高盐胁迫下,在对盐渍的适应过程中需要渗透调节物质进行调节,当角果木适应外界盐环境后,这些渗透调节物质便趋于稳定。
5、利用cDNA-AFLP技术筛选得到69个差异片段,经克隆测序及RT-PCR验证后,筛选获得61个差异片段。对所得到的61个差异片段进行生物信息学分析,经blastx功能分析后,得到38个已知基因,这38个基因按功能分为8大类,由此说明角果木在盐胁迫条件下要涉及大量的基因参与各种代谢途径。对角果木用不同盐浓度处理不同时间,通过半定量RT-PCR技术进行差异表达基因在盐胁迫下的表达谱分析,证实了所获得的基因序列是真正的诱导表达序列。
Mangroves are woody plants which form the dominant vegetation in tidal, saline wetlands along tropical and subtropical coasts. Mangroves show adaptation to the saline environment, either through morphological manifestations or through physiological adjustments that permit metabolic activities at these salt concentrations. In order to understand the salt-tolerant mechanism of the mangrove, Ceriops tagal, the microstrucure, physiological changes and expression of salt-tolerant genes of the root of Ceriops tagal were researched in this study.
The main results as follows:
1. Following the salinity increasing, the pericycle of the root tips decreased, the exodermis and endodermis of the root tips increased, the Ceriops tagal in culture treated with different salinity for one month.
2. Under the salt stress for same time, the contents of total protein in toots reduced, as the salinity increasing. However, under the same salinity, the contents of protein increasing, as the stress time extending. This results show that the new protein were produced in the process of the Ceriops tagal adaptation to the saline environment.
3. The content of MDA in root increased sharply in the early of salt-stress, this signal induced the starting of the antioxidant defense system and the expression of the relevant enzymes in roots. So the activities of antioxidant enzymes and the contents of antioxidants raised firstly, and then decreased in 24 h of salt-stress. After salt-tolerant for 24 h, the contents of MDA, ASA and activities of POD, SOD had the same levels with control. It indicated that, under the salt-tolerant, the mangrove Ceriops tagal eliminate ROS to the root by the increasing of the activities of antioxidant enzymes and the contents of antioxidants, and then decreased after mangrove Ceriops tagal started to adapt the salt environment.
4. In the high salt-stress, the contents of soluble sugar and Pro in roots went up in 72 h, and then kept stable level. However the ployphenol content in roots went down in 24 h, and then kept stable level. This result showed that, in high salt-stress, during the Ceriops tagal adapt to the saline, osmotic substance were needed to regulate the osmolarity, when the Ceriops tagal had adapted to the saline environment, the osmotic substance contents kept stable level.
5. The technique of cDNA-AFLP was occupied to study genes differentially expressed in the roots of Ceriops tagal under salt-stress. Results showed the total of 69 differentially expressed bands was obtained.61 differential bands were picked and successfully cloned, sequenced and verified by RT-PCR analysis. Based on the results of blastx identification in NCBI,38 gene sequences of differentially were known. The 38 gene were into 8 classes by the different function.
主要结果如下：
1、角果木幼苗在不同浓度处理一个月后,根尖的中柱鞘变得不明显,且盐浓度越大,中柱鞘越不明显,从而盐胁迫影响了角果木侧根的发生。盐胁迫对角果木根尖皮层也有较大影响：高盐(500 mmol·L-1)胁迫下,角果木根尖外皮层、内皮层明显增厚,以阻止有害离了进入。
2、盐胁迫下,同一处理时间点随着盐浓度的增加角果木根部总蛋白含量下降,但随处理时间的延长各盐处理下的总蛋白含量与对照组的差异逐渐缩小,且在24h后略高于对照组,说明角果木在适应盐环境的过程中有新的蛋白产生。
3、植物受到盐胁迫后会立即引起氧化胁迫,产生氧化胁迫后就导致膜脂过氧化,因此早期(≦9h) MDA含量急剧上升,这种信号的出现诱导植物中抗氧化防御系统的启动,相关酶的表达。因此在24h内各盐处理前期,角果木根部抗氧化酶类及抗氧化物质均有所增加,而处理24 h后,角果木根部MDA含量、ASA含量、POD活性以及SOD活性均恢复至对照水平,表明角果木在受盐胁迫后通过启动抗氧化酶类、抗氧化物质以清除活性氧对根部的损伤,而后开始适应外界盐环境。
4、在高盐(500 mmol·L-1)胁迫下,角果木根部可溶性糖含量和Pro含量在盐胁迫前期(≦72h)均有所增加,而在72h后均处于稳定状态,而多酚含量在盐胁迫初期(≦24h)有所下降,之后也处于稳定状态。表明角果木在高盐胁迫下,在对盐渍的适应过程中需要渗透调节物质进行调节,当角果木适应外界盐环境后,这些渗透调节物质便趋于稳定。
5、利用cDNA-AFLP技术筛选得到69个差异片段,经克隆测序及RT-PCR验证后,筛选获得61个差异片段。对所得到的61个差异片段进行生物信息学分析,经blastx功能分析后,得到38个已知基因,这38个基因按功能分为8大类,由此说明角果木在盐胁迫条件下要涉及大量的基因参与各种代谢途径。对角果木用不同盐浓度处理不同时间,通过半定量RT-PCR技术进行差异表达基因在盐胁迫下的表达谱分析,证实了所获得的基因序列是真正的诱导表达序列。
Mangroves are woody plants which form the dominant vegetation in tidal, saline wetlands along tropical and subtropical coasts. Mangroves show adaptation to the saline environment, either through morphological manifestations or through physiological adjustments that permit metabolic activities at these salt concentrations. In order to understand the salt-tolerant mechanism of the mangrove, Ceriops tagal, the microstrucure, physiological changes and expression of salt-tolerant genes of the root of Ceriops tagal were researched in this study.
The main results as follows:
1. Following the salinity increasing, the pericycle of the root tips decreased, the exodermis and endodermis of the root tips increased, the Ceriops tagal in culture treated with different salinity for one month.
2. Under the salt stress for same time, the contents of total protein in toots reduced, as the salinity increasing. However, under the same salinity, the contents of protein increasing, as the stress time extending. This results show that the new protein were produced in the process of the Ceriops tagal adaptation to the saline environment.
3. The content of MDA in root increased sharply in the early of salt-stress, this signal induced the starting of the antioxidant defense system and the expression of the relevant enzymes in roots. So the activities of antioxidant enzymes and the contents of antioxidants raised firstly, and then decreased in 24 h of salt-stress. After salt-tolerant for 24 h, the contents of MDA, ASA and activities of POD, SOD had the same levels with control. It indicated that, under the salt-tolerant, the mangrove Ceriops tagal eliminate ROS to the root by the increasing of the activities of antioxidant enzymes and the contents of antioxidants, and then decreased after mangrove Ceriops tagal started to adapt the salt environment.
4. In the high salt-stress, the contents of soluble sugar and Pro in roots went up in 72 h, and then kept stable level. However the ployphenol content in roots went down in 24 h, and then kept stable level. This result showed that, in high salt-stress, during the Ceriops tagal adapt to the saline, osmotic substance were needed to regulate the osmolarity, when the Ceriops tagal had adapted to the saline environment, the osmotic substance contents kept stable level.
5. The technique of cDNA-AFLP was occupied to study genes differentially expressed in the roots of Ceriops tagal under salt-stress. Results showed the total of 69 differentially expressed bands was obtained.61 differential bands were picked and successfully cloned, sequenced and verified by RT-PCR analysis. Based on the results of blastx identification in NCBI,38 gene sequences of differentially were known. The 38 gene were into 8 classes by the different function.
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