脱水蛋白和CBF1转录因子对植物抗旱性和抗冻性作用的研究
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摘要
低温和干旱是影响植物生长、发育和地理分布的重要环境限制因素。低温和脱水诱导大量基因的表达，基因产物可分为两大类：一类是直接保护细胞免受胁迫伤害的功能蛋白；另一类是传递信号和调控基因表达的调节蛋白。本文以植物脱水蛋白和冷诱导转录因子CBF1基因为主要研究对象，探讨它们在低温和干旱胁迫中对植物抗冻性和抗旱性形成的作用。
一、脱水蛋白对植物抗冻性和抗旱性作用研究
以铁皮石斛类原球茎(PLBs)为材料，研究脱水、低温、高渗、高盐和外源ABA处理与诱导热稳定蛋白和脱水蛋白的关系，以及胁迫诱导蛋白的积累与各种抗逆性形成的相关性。主要实验结果如下：
1．用空气干燥法对PLBs进行脱水处理及超低温保存，分别测定不同含水量样品的冻前和冻后存活率，并进行可溶性糖含量测定、热稳定蛋白的SDS-PAGE分析及脱水蛋白的Western-blot检测。结果表明：在本实验体系下，PLBs脱水时间(x)对含水量(y)的回归方程式为y=-0.1208x+9.9636。PLBs含水量为0.1g至0.5H_2O/g DW时，其冻后存活率达到最大值。当含水量达1.0g H_2O/g DW时，电泳图上出现五条明显增强的热稳定蛋白条带，分子量分别为34.3、28.7、19.2、17.0、13.3kDa。经抗脱水蛋白免疫检测发现其中分子量为28.7和34.3kDa的蛋白为脱水蛋白。干燥处理中PLBs的可溶性糖含量增加，其积累主要发生在含水量大于7.2g H_2O/g DW的阶段。可见脱水蛋白的积累要早于糖的积累。外源ABA的预处理可明显提高可溶性糖、热稳定蛋白和脱水蛋白的含量。
2．低温驯化3天开始检测到28.7 kDa脱水蛋白的积累，随后其含量逐渐增加，驯化7天时含量最高。但是驯化过程中，热稳定蛋白水平并无明显变化。经低温驯化PLBs的抗冻性比未经驯化的对照明显提高。在-8.2～-8.0℃温度下冰冻2小时，对照的相对存活率为2.6±0.7％，驯化3天和5天的PLBs具有较高的抗冻性，其冻后相对存活率分别为56.3±22.5％和49.9±19.5％。随着低温驯化时间延长至7天，PLBs的冻后存活率并未进一步上升，为31.4±14.7％。
3．通过对不同浓度PEG和NaCl处理后PLBs存活率的测定，得出PLBs的PEG半致死浓度为40％、NaCl半致死浓度为200mmol/L。PEG处理诱导17.0kDa热稳定蛋白的积累，其含量随PEG浓度的增加而增加。而NaCl处理对热稳定蛋白影响不明显。经抗脱水蛋白免疫检测，发现PEG和NaCl处理均不能诱导PLBs脱水蛋白的积累。
二、转CBF1基因提高植物抗冻性和抗旱性研究
浙江大学博士学位论文
以烟草为材料进行了转CBFI基因研究，并对转基因后代开展了形态变异观
察、抗冻性和抗旱性测定以及遗传稳定性分析。主要结果如下:
1.将C丑尸z基因与Cal城v35s启动子融合构建植物表达载体pBnZI一CBFI，
以农杆菌介导的叶盘法转化烟草，获得转化植株。经C万卢7基因的PCR鉴定和
转基因后代(包括Tl、TZ和T3代)的卡那霉素抗性分析，获得2个单一位点
插入的转基因纯系，证明了外源基因在转基因烟草中的表达及遗传。
2.采用电解质渗漏检测和小苗冻害观察进行转基因后代的抗冻性检测，结果
显示C万卢了基因的表达有利于冰冻条件下细胞膜和叶绿体膜的稳定性，从而使转
基因烟草的抗冻性增强。
3.采用不同PEG处理浓度和处理时间对转基因后代进行了抗旱性测定，发现
可选用15%的PEG浓度处理48小时来测定小苗抗旱性。结果显示转基因烟草
的抗早性比野生型烟草增强。
4.转C万尸了基因烟草出现明显的表型变化，主要有植株矮化、花器官变异、
叶片卷曲或细长和果实数减少等。分析原因可能是与CBFI转录因子所具有的
AP2/ EREBP结构域的功能有关。注尸2基因是拟南芥花器官特征特异性基因，能
在各轮花器官及茎叶中表达，无组织特异性。推测C万尸了基因的组成性表达，干
扰了烟草原有的花器官和茎叶生长发育，导致转基因植株的表型改变。
Low temperature and drought represent major environmental constraints limiting growth, development and distribution of plants. A variety of genes are induced both by low temperature and dehydration. These gene products can be classified into two groups. The first group includes proteins that probably function in protecting cells from stresses. The second group of gene products contains protein factors that are involved in further regulation of gene expression and signal transduction in stress response. In this paper, the roles of dehydrins and CBF1 transcription factor were studied in plants under low temperature and dehydration condition, and their possible mechanisms in the development of freezing and drought tolerance were also discussed. I. Roles of dehydrins in plant tolerance to freezing and drought.
Accumulations of heat-stable proteins and dehydrins induced by dehydration, low temperature, osmotic stress, high salt or ABA treatment were investigated in protocorm-like-bodies (PLBs) ofDendrobium candidum. Moreover, we described the relationship between these accumulations and the development of tolerance to stresses. Our experimental results were shown as followings:
1. PLBs were desiccated and cryopreserved by the air-drying method. Under the controlled system, the dehydration linear equation was expressed at y = -0.1208x + 9.9636. The optimal water content before freezing seemed to be at the range of 0.1 g H2O/g DW to 0.5 g H2O/g DW. Changes in soluble sugars, heat-stable proteins and dehydrins during desiccation of PLBs were analyzed. Extensive accumulation of soluble sugars was observed at water content of about 7.2 g H2O/g DW (after 24 h desiccation), and the sugars contents didn't increased further during the following desiccation. The amount of heat-stable proteins increased significantly when water content decreased to 1.0 g H2O/g DW (after approximately 66 h desiccation). Their molecular masses were 34.3 . 28.7, 19.2,
17.0 and 13.3 kDa. Results from immunological detection showed that two bands of the heat-stable proteins with respective molecular masses of 28.7 and 34.3 kDa were dehydrins, which appeared when water content dropped to 1.0 g H2O/g DW. Therefore, it seemed that accumulation of dehydrins happened later than that of soluble sugars. Interestingly, exogenous ABA treatment of PLBs before desiccation could also induce the accumulation of soluble sugars, heat-stable proteins and dehydrins.
2. Accumulation of 28.7-kDa dehydrin began to be detectable after 3 days of cold acclimation. Then the level of the dehydrin increased and reached to the maximum at the seventh day. However there were little changes in the level of heat-stable proteins. After 2 hr of exposure to -8.2 ~ -8.0C, the relative survival rate of 3 or 5-day-acclimated PLBs increased to 56.3+22.5% 49.9+19.5% respectively, while that of non-acclimated PLBs was 2.6+0.7%. However, freezing tolerance did not seem to be further enhanced when cold acclimation remained to 7 days. It suggested that acclimated PLBs achieved greater freezing tolerance than non-acclimated controls.
3. PEG semilethal concentration was 40% and NaCl semilethal concentration was 200 mmol/L in PLBs by the detection of survival rate of PLBs subjected to various concentrations. Heat-stable protein of 17.0 kDa molecular mass was induced to accumulate by PEG treatment, whose level was increased with the rise of PEG concentration. However, NaCl treatment did not affect the level of heat-stable proteins. Using western blot analysis, it revealed that neither PEG nor NaCl treatment could initiate accumulation of dehydrins in PLBs.
II. Studies on enhancement of drought and freezing tolerance by gene transfer of
CBF1.
1. CBF1 gene was introduced into tobacco (Nicotiana tabacum) by a plant expression vector pBI121-CBFl containing the CaMV35S promoter using Agrobacterium tumefaciens-mediated transformation. By PCR determination of CBF1 and kanamycin selection of transgenic progeny (Tl, T2 and T3), two pure
transgenic lines were obtained
一、脱水蛋白对植物抗冻性和抗旱性作用研究
以铁皮石斛类原球茎(PLBs)为材料，研究脱水、低温、高渗、高盐和外源ABA处理与诱导热稳定蛋白和脱水蛋白的关系，以及胁迫诱导蛋白的积累与各种抗逆性形成的相关性。主要实验结果如下：
1．用空气干燥法对PLBs进行脱水处理及超低温保存，分别测定不同含水量样品的冻前和冻后存活率，并进行可溶性糖含量测定、热稳定蛋白的SDS-PAGE分析及脱水蛋白的Western-blot检测。结果表明：在本实验体系下，PLBs脱水时间(x)对含水量(y)的回归方程式为y=-0.1208x+9.9636。PLBs含水量为0.1g至0.5H_2O/g DW时，其冻后存活率达到最大值。当含水量达1.0g H_2O/g DW时，电泳图上出现五条明显增强的热稳定蛋白条带，分子量分别为34.3、28.7、19.2、17.0、13.3kDa。经抗脱水蛋白免疫检测发现其中分子量为28.7和34.3kDa的蛋白为脱水蛋白。干燥处理中PLBs的可溶性糖含量增加，其积累主要发生在含水量大于7.2g H_2O/g DW的阶段。可见脱水蛋白的积累要早于糖的积累。外源ABA的预处理可明显提高可溶性糖、热稳定蛋白和脱水蛋白的含量。
2．低温驯化3天开始检测到28.7 kDa脱水蛋白的积累，随后其含量逐渐增加，驯化7天时含量最高。但是驯化过程中，热稳定蛋白水平并无明显变化。经低温驯化PLBs的抗冻性比未经驯化的对照明显提高。在-8.2～-8.0℃温度下冰冻2小时，对照的相对存活率为2.6±0.7％，驯化3天和5天的PLBs具有较高的抗冻性，其冻后相对存活率分别为56.3±22.5％和49.9±19.5％。随着低温驯化时间延长至7天，PLBs的冻后存活率并未进一步上升，为31.4±14.7％。
3．通过对不同浓度PEG和NaCl处理后PLBs存活率的测定，得出PLBs的PEG半致死浓度为40％、NaCl半致死浓度为200mmol/L。PEG处理诱导17.0kDa热稳定蛋白的积累，其含量随PEG浓度的增加而增加。而NaCl处理对热稳定蛋白影响不明显。经抗脱水蛋白免疫检测，发现PEG和NaCl处理均不能诱导PLBs脱水蛋白的积累。
二、转CBF1基因提高植物抗冻性和抗旱性研究
浙江大学博士学位论文
以烟草为材料进行了转CBFI基因研究，并对转基因后代开展了形态变异观
察、抗冻性和抗旱性测定以及遗传稳定性分析。主要结果如下:
1.将C丑尸z基因与Cal城v35s启动子融合构建植物表达载体pBnZI一CBFI，
以农杆菌介导的叶盘法转化烟草，获得转化植株。经C万卢7基因的PCR鉴定和
转基因后代(包括Tl、TZ和T3代)的卡那霉素抗性分析，获得2个单一位点
插入的转基因纯系，证明了外源基因在转基因烟草中的表达及遗传。
2.采用电解质渗漏检测和小苗冻害观察进行转基因后代的抗冻性检测，结果
显示C万卢了基因的表达有利于冰冻条件下细胞膜和叶绿体膜的稳定性，从而使转
基因烟草的抗冻性增强。
3.采用不同PEG处理浓度和处理时间对转基因后代进行了抗旱性测定，发现
可选用15%的PEG浓度处理48小时来测定小苗抗旱性。结果显示转基因烟草
的抗早性比野生型烟草增强。
4.转C万尸了基因烟草出现明显的表型变化，主要有植株矮化、花器官变异、
叶片卷曲或细长和果实数减少等。分析原因可能是与CBFI转录因子所具有的
AP2/ EREBP结构域的功能有关。注尸2基因是拟南芥花器官特征特异性基因，能
在各轮花器官及茎叶中表达，无组织特异性。推测C万尸了基因的组成性表达，干
扰了烟草原有的花器官和茎叶生长发育，导致转基因植株的表型改变。
Low temperature and drought represent major environmental constraints limiting growth, development and distribution of plants. A variety of genes are induced both by low temperature and dehydration. These gene products can be classified into two groups. The first group includes proteins that probably function in protecting cells from stresses. The second group of gene products contains protein factors that are involved in further regulation of gene expression and signal transduction in stress response. In this paper, the roles of dehydrins and CBF1 transcription factor were studied in plants under low temperature and dehydration condition, and their possible mechanisms in the development of freezing and drought tolerance were also discussed. I. Roles of dehydrins in plant tolerance to freezing and drought.
Accumulations of heat-stable proteins and dehydrins induced by dehydration, low temperature, osmotic stress, high salt or ABA treatment were investigated in protocorm-like-bodies (PLBs) ofDendrobium candidum. Moreover, we described the relationship between these accumulations and the development of tolerance to stresses. Our experimental results were shown as followings:
1. PLBs were desiccated and cryopreserved by the air-drying method. Under the controlled system, the dehydration linear equation was expressed at y = -0.1208x + 9.9636. The optimal water content before freezing seemed to be at the range of 0.1 g H2O/g DW to 0.5 g H2O/g DW. Changes in soluble sugars, heat-stable proteins and dehydrins during desiccation of PLBs were analyzed. Extensive accumulation of soluble sugars was observed at water content of about 7.2 g H2O/g DW (after 24 h desiccation), and the sugars contents didn't increased further during the following desiccation. The amount of heat-stable proteins increased significantly when water content decreased to 1.0 g H2O/g DW (after approximately 66 h desiccation). Their molecular masses were 34.3 . 28.7, 19.2,
17.0 and 13.3 kDa. Results from immunological detection showed that two bands of the heat-stable proteins with respective molecular masses of 28.7 and 34.3 kDa were dehydrins, which appeared when water content dropped to 1.0 g H2O/g DW. Therefore, it seemed that accumulation of dehydrins happened later than that of soluble sugars. Interestingly, exogenous ABA treatment of PLBs before desiccation could also induce the accumulation of soluble sugars, heat-stable proteins and dehydrins.
2. Accumulation of 28.7-kDa dehydrin began to be detectable after 3 days of cold acclimation. Then the level of the dehydrin increased and reached to the maximum at the seventh day. However there were little changes in the level of heat-stable proteins. After 2 hr of exposure to -8.2 ~ -8.0C, the relative survival rate of 3 or 5-day-acclimated PLBs increased to 56.3+22.5% 49.9+19.5% respectively, while that of non-acclimated PLBs was 2.6+0.7%. However, freezing tolerance did not seem to be further enhanced when cold acclimation remained to 7 days. It suggested that acclimated PLBs achieved greater freezing tolerance than non-acclimated controls.
3. PEG semilethal concentration was 40% and NaCl semilethal concentration was 200 mmol/L in PLBs by the detection of survival rate of PLBs subjected to various concentrations. Heat-stable protein of 17.0 kDa molecular mass was induced to accumulate by PEG treatment, whose level was increased with the rise of PEG concentration. However, NaCl treatment did not affect the level of heat-stable proteins. Using western blot analysis, it revealed that neither PEG nor NaCl treatment could initiate accumulation of dehydrins in PLBs.
II. Studies on enhancement of drought and freezing tolerance by gene transfer of
CBF1.
1. CBF1 gene was introduced into tobacco (Nicotiana tabacum) by a plant expression vector pBI121-CBFl containing the CaMV35S promoter using Agrobacterium tumefaciens-mediated transformation. By PCR determination of CBF1 and kanamycin selection of transgenic progeny (Tl, T2 and T3), two pure
transgenic lines were obtained
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