低温胁迫过程中入侵植物紫茎泽兰热激蛋白基因的作用
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摘要
紫茎泽兰(Ageratina adenophora)是一种外来入侵杂草,分布于我国西南部,危害严重。紫茎泽兰喜温暖湿润的环境,但其适应能力极强,可对生存地的纬度或海拔等环境梯度作出适应成为紫茎泽兰成功入侵的主要原因。但目前对紫茎泽兰低温适应性以及入侵扩张的分子机理方面的相关研究较少,因此入侵物种的分子机理研究已经成为生物学界关注的热点。热激蛋白(Heat Shock Proteins, HSPs)是原核和真核生物在应激状态下被诱导表达的一类具有重要生理功能的高度保守多肽类蛋白质,是植物对逆境胁迫短期适应的必需组成,它们以分子伴侣的形式减轻逆境胁迫引起的伤害。
本研究针对紫茎泽兰的低温反应采集高海拔的紫茎泽兰种群,通过模拟自然条件的实验方法鉴定紫茎泽兰高海拔种群的耐寒性,通过测定生理生化指标分析明确高海拔紫茎泽兰种群在低温胁迫过程中的耐性强弱。重点采用了分子生物学手段研究紫茎泽兰在高温与低温逆境下四种分子量(hsp17.6、hsp60、hsp70和hsp90)热激蛋白基因表达特性及其在紫茎泽兰低温胁迫过程中的作用。
采用人工模拟气候鉴定了采自四川和云南的高海拔紫茎泽兰种群对低温的不同适应性,从生理生化角度表明紫茎泽兰种群在入侵我国过程中对温度表现出的适应性差异。结果表明丙二醛(MDA)、可溶性糖、抗氧化酶指标(SOD、CAT、APX)在低温下均出现不同程度的升高,不同种群指标变化幅度说明紫茎泽兰高海拔种群对低温适应性存在差异,其中采自四川西昌的磨盘山和泸山的种群耐寒性最强,西昌的大青山种群和云南的龙陵种群则为低温敏感种群,从而为干扰或是抑制其保护机制来降低其适应能力提供思路,控制其进一步向北部地区扩散。
根据植物热激蛋白高度的保守性对紫茎泽兰的hsp17.6、hsp60、hsp70和hsp90的全长进行同源克隆,命名为Aahsp17.6、Aahsp60、Aahs70和Aahsp90。序列全长分别为691、1581、2074和2094bp,登陆Genebank注册号为EU209067、EU209068、EU209069和EU209070。运用生物信息学软件对这四个热激蛋白的氨基酸序列同源性以及结构的分析充分表明了所克隆的hsps编码的氨基酸是属于热激蛋白家族的。进化树分析结果表明在进化上这四类HSPs与拟南芥、烟草、番茄的HSPs表现较近的遗传距离。
半定量PCR结果显示紫茎泽兰的四个热激蛋白基因中Aahsp17.6、Aahsp60、Aahs70和Aahsp90均在幼苗茎、叶中的表达受热激的诱导,该基因的表达在植物器官中可能具有普遍性。Northern杂交检测到在高、低温逆境下紫茎泽兰的这四类热激蛋白在mRNA水平上发生明显的变化。一般需要适当时间达到转录的最高水平和最大表达量的恢复才达到最大表达量,而且高温胁迫下并不是在胁迫过程中或胁迫刚结束时即消失,合成还会持续在恢复后的一段时间内,并且紫茎泽兰Aahsp70和Aahsp90对高温和低温的表达模式相似,二者协同表达。Southern结果显示Aahsp17.6和Aahsp60为单拷贝,Aahsp70和Aahsp90为3-4个拷贝数。
通过分析异源表达的紫茎泽兰的HSPs在高温和低温下对大肠杆菌生存能力的影响,初步研究在高温和低温条件下紫茎泽兰Aahsp17.6、Aahsp60、Aahsp70和Aahsp90基因在活体中的功能。用pET-30a表达载体与Aahsp17.6、Aahsp60、Aahsp70和Aahsp90基因进行重组并在大肠杆菌BL21(DE3)中进行诱导表达。高温处理后,重组细胞的存活力较对照相比有明显的提高;并且对热激后的重组菌的可溶性蛋白SDS-PAGE检测表明热激蛋白的表达延缓了可溶性蛋白的降解。在4°C处理8d后,重组细胞的存活力较对照相比有明显的提高。据此推测这四个热激蛋白对低温条件下保护大肠杆菌的功能。
采用染色体步移方法的两个技术获得紫茎泽兰hsp90长864bp的启动子序列(GenBank No. FJ434253)和hsp17.6长1485bp的启动子序列(GenBank No. FJ434252),序列分析得出两个启动子的序列均含有HSPs特异应答元件HSE(nGAAn)以及其它保守的启动子元件。在紫茎泽兰hsp90和hsp17.6启动子中分别存在2个重复以上的,尤其是hsp17.6启动子序列中存在10个此类的重复,因而在响应温度等胁迫时可以快速的与HSF结合,激活hsp基因的表达。
通过体外实验检测了紫茎泽兰HSP17.6和HSP17.7的重组蛋白作为分子伴侣对柠檬酸合酶(CS)的热聚集和热降解的抑制作用。对异源表达的重组蛋白进行Ni-NTA柱纯化后,从1L的菌液中得到浓度为33.6mg的HSP17.6和20.2mg的HSP17.7重组蛋白。体外实验表明HSP17.6与HSP17.7均可减慢45°C处理下CS的热变性,抑制热聚集的最佳比例为HSP17.6/HSP17.7-to-CS为5:1,说明紫茎泽兰的sHSPs具有分子伴侣的活性。此外HSP17.6与HSP17.7均加速热变性CS的复性,38°C处理后的CS活性仅为12%,处理60min后恢复到25°C,加入纯化的重组蛋白后CS活性可以恢复到60%以上。两种sHSPs进行等比例混合后检测其保护功能也被观察到。根据紫茎泽兰的HSP17.6和HSP17.7体外分子伴侣功能推测其在低温胁迫下保护了功能蛋白的活性进而增强了低温适应能力。
将Aahsp17.6定向克隆于带有组成性表达启动子CaMV35S和NPTⅡ基因(带有卡那霉素抗性基因)的植物表达载体pBI121中,冻融法转化农杆菌LBA4404,利用叶圆盘法对番茄进行Ti质粒介导的遗传转化,PCR与Sourthern杂交鉴定出阳性转基因植株。在低温处理下,转基因植株小苗表现出明显优于野生型植株的耐寒性状。转基因植株MDA含量明显低于野生型植株,可溶性糖的含量比野生型植株高出0.5倍,但测定的SOD的活性没有显著差异。结果说明sHSPs提高了植物的耐寒性。
在本研究中紫茎泽兰高海拔种群低温适应性的差异为紫茎泽兰在我国的适应性进化提供了参考价值。对于紫茎泽兰而言,其体内HSPs的多样性、基因结构的特点可能是其对温度逆境尤其是低温的适应能力的重要内在因素,因而对紫茎泽兰sHSP或是其它分子量HSPs的研究提供了紫茎泽兰面临温度胁迫时内在基因表达变化的一个线索,有利于从分子水平解释紫茎泽兰对温度逆境的适应性。
An invasive aline weed, Ageratina adenophora,has become widespread in Southwest of China due to its high environmental adaptation. The invasion of A. adenophora in China, is due to the gradual acclimation of active factor induced by temperature conditions. The alien plants evolutionarily adapt to environmental changes related to different latitudes and altitudesin introduced ranges. However, little is known about the low temperature response of A. adenophora and molecular and genetic aspects that influence resistance to environmental variability. Heat shock proteins genes (hsps) have an essential role in stress resistance and adaptation to the environment. The gene provides an accessible and useful tool for generating insights about molecular mechanisms by how the weeds become successfully invaders.
In this study, with respect to adaptation to abiotic environments, we collected and compared response of A. adenophora populations to low temperature from different high altitude in Sichuan Yunnan of China where the infestation of A. adenophora is quite severe. Meanwhile, we have studied hsps genes that are important in relation to stress resistance and adaptation to the environment to reveal their contribution to invasion of A. adenophora in China.
The physiological responses to low temperature were determined to elucidate mechanisms chilling tolerance. The study made use of artificial climate chamber to compare the A. adenophora chilling tolerance from different high altitude populations from Sichuan and Yunnan province of China. With the decrease of temperature, the physiological changes including increases in malondialdehyde (MDA) and total soluble protein contents, reductions in total soluble sugar, fluctuation of superoxide dismutase (SOD) activity, and catalase activity (CAT) were observed among all the eight populations. However, different extents of low temperature sensitivities were found among the high altitude populations. The results revealed that after the trestment with gradually decreasing temperature, the A. adenophora from the population of Mopanshan and Lushan of Sichuan showed the lowest chilling damage and high tolerance. The populations from Daqingshan of Sichuan and Longling of Yunnan both presented a higher chilling damage and lower tolerance. These indicated that A. adenophora populations from high altitudes were insensitive to chilling tolerance. These data indicate that the invasive plant from high altitude populations with different chilling tolerance reflected a higher capacity to its infested environments. These results may be a manifest A. adenophora expanded to Northern China much faster.
In order to know molecular and genetic aspects of adaption, we cloned full length of coding sequences of invasive alien weed A. adenophora heat shock proteins by the combination of homology cloning in the present study, renamed as Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90. Full-length of Aahsp17, Aahsp60, Aahsp70 and Aahsp90 were 691, 1,581, 2,074 and 2,094 bases of nucleotide sequence, which have been deposited in Genebank database (Accession no. EU209067, EU209068, EU209069, EU209070). The four genes were found highly to be conserved when compared at the amino acid level with the corresponding HSPs from other plants. Sequence analysis, identification, alignments and phylogenetic analyses of the hsps showed that these HSPs shared high levels of identity with corresponding proteins from other species. The phylogenetic analysis suggested that the four hsps were present closed to some other HSPs from Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum et al.
We investigated the characterization and expression of Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 of A. adenophora. After heat treatment, Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 transcripts were not detected in roots, but high levels were found in the stems and leaves. Tissue specificity showed a similar pattern of response to heat shock. The kinetics of regulation of Aahsps was analysed by Northern blot hybridisation, which revealed the significantly changes at mRNA level for Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90. Transcripts of these genes were considerably induced underheat and cold stress. Although the gene expression responses induced by high and low temperture stress shared several common features, there we still observed some differences among Aahsps. The four hsps expression response to heat exposure was robust, and differed from the response to low temperture stress. These results indicate that all of the genes are stress-inducible forms or cognate forms that are constitutively expressed. The sourthern results show that Aahsp17.6 and Aahsp60 appear to be present as a single copy and Aahsp70 and Aahsp90 are encoded by a multi-gene family.
We made investigations on the possible function of Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 in vivo under heat and cold sress, and SDS-PAGE analysis of cell lysates revealed their protective effect is associated with an increase in the thermo stability of soluble proteins. The PCR products of ORF sequences encoding Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 were inserted into the same digested pET-30a vector to generate pET-30a-Aahsps. These plasmids were transformed into Escherichia coli BL21 (DE3) plyss competent cells. Aahsps were expressed in E. coli cells. The recombinant Aahsps proteins produced in bacterial expression were separated by 12% SDS-PAGE.The recombinant Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 over-expression improved viability in comparison with control cultures in E. coli under heat stress conditions. SDS-PAGE analysis of cell lysates suggested that the protective effect in vivo was due to increased thermol stability of soluble cytosolic proteins. The recombinant Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 over-expression improved viability in comparison with control cultures in E. coli under cold stress conditions.
Cloning and sequence analysis of promoters in hsp90 and hsp17.6 genes from A. adenophora by two skills of chromosome walking from a known sequence to an unknown region based on PCR methods. The promoter region of hsp90 with 864bp (GenBank No. FJ434253), from invasive weed A.adenophora was amplified based on PCR methods. Another promoter region of hsp17.6 with 1,485 bp (GenBank No. FJ434252) was cloned by TAIL-PCR. Both of the 5′-flanking regions of hsp90 and hsp17.6 contained specific heat shock element (HSE) (nGAAn) and several cis-acting elements, such as TATA-box, CAAT-box. Especially, the promoter region of hsp17.6 has ten of HSE, which could be bound to heat shock transcription factors (HSFs). Then hsp17.6 of A. adenophora wae robustly induced and accumilated in response to low temperture stress.
The recombinant HSP17.6 and HSP17.7 from A.adenophora were applied to determine their chaperone function. The recombinant HSP17.6 and HSP17.7 were applied to determine their chaperone function. 33.6 mg of HSP17.6 and 20.2 mg of HSP17.7 was purified from 1l of each respective cell culture using a Ni-NTA column. In vitro, HSP17.6 and HSP17.7 actively participated in the refolding of the model substrate citrate synthase (CS) and effectively prevented the thermal aggregation of CS at 45°C HSP17.6 and HSP17.7 prevented this process at different ratios. The maximal suppression by the HSP17.6/HSP17.7-to-CS monomer was also observed at a ratio of 5:1. The effect of the recombinant HSP17.6 and HSP17.7 on the thermal inactivation of CS was investigated. When the CS was incubated at 38°C alone or in presence of 50μg/ml lysozyme, less than 12% of CS activity remained after 60min. After shifting the temperature of the samples to 25°C, the CS activity remained low after 60min in the presence or absence of 50μg/ml lysozyme. They both prevented the irreversible inactivation of CS at 38°C at stoichiometric levels. Therefore, this report confirms the chaperone activity of HSP17.6 and HSP17.7 and their potential as a protectant for active proteins.
To confirm the role of Aahsp17.6 in low temperture condition, an integratedvector pBI121of the Aahsp17.6 and NPTⅡgene(a kanamycin resistant gene)with CaMV35S promoter was constructed and introduced into tobacco mediated by Agrobacterium tumefaciens LBA4404. At low-temperature, the soluble sugar content of transgenic seedlings was higher than that of wild-type plants, whereas the MDA content and the SOD activity were lower than that of wild-type plants. Based on these results, the Aahsp17.6 transgenic tomato plants were more tolerable against low temperture stress than the wild-type plants. Thus, we suggest that Aahsp17.6 may play a pivotal role in chilling tolerance and has potential consequences in this invasive plant for expansion to non-freezing low temperatures zones.
In conculsion, these results may be important for analysis that A. adenophora from high altitude in southwest regions of China has evolved into different ecotypes through physiological adaptation. And the inducible hsps provide a unique system to measure the molecular mechanism by which the invasive alien plants respond to changing environmental and developmental cues. Moreover we could evaluate the molecular capacity of A. adenophora to acclimatise to high and low temperatures in the future according to the results.
本研究针对紫茎泽兰的低温反应采集高海拔的紫茎泽兰种群,通过模拟自然条件的实验方法鉴定紫茎泽兰高海拔种群的耐寒性,通过测定生理生化指标分析明确高海拔紫茎泽兰种群在低温胁迫过程中的耐性强弱。重点采用了分子生物学手段研究紫茎泽兰在高温与低温逆境下四种分子量(hsp17.6、hsp60、hsp70和hsp90)热激蛋白基因表达特性及其在紫茎泽兰低温胁迫过程中的作用。
采用人工模拟气候鉴定了采自四川和云南的高海拔紫茎泽兰种群对低温的不同适应性,从生理生化角度表明紫茎泽兰种群在入侵我国过程中对温度表现出的适应性差异。结果表明丙二醛(MDA)、可溶性糖、抗氧化酶指标(SOD、CAT、APX)在低温下均出现不同程度的升高,不同种群指标变化幅度说明紫茎泽兰高海拔种群对低温适应性存在差异,其中采自四川西昌的磨盘山和泸山的种群耐寒性最强,西昌的大青山种群和云南的龙陵种群则为低温敏感种群,从而为干扰或是抑制其保护机制来降低其适应能力提供思路,控制其进一步向北部地区扩散。
根据植物热激蛋白高度的保守性对紫茎泽兰的hsp17.6、hsp60、hsp70和hsp90的全长进行同源克隆,命名为Aahsp17.6、Aahsp60、Aahs70和Aahsp90。序列全长分别为691、1581、2074和2094bp,登陆Genebank注册号为EU209067、EU209068、EU209069和EU209070。运用生物信息学软件对这四个热激蛋白的氨基酸序列同源性以及结构的分析充分表明了所克隆的hsps编码的氨基酸是属于热激蛋白家族的。进化树分析结果表明在进化上这四类HSPs与拟南芥、烟草、番茄的HSPs表现较近的遗传距离。
半定量PCR结果显示紫茎泽兰的四个热激蛋白基因中Aahsp17.6、Aahsp60、Aahs70和Aahsp90均在幼苗茎、叶中的表达受热激的诱导,该基因的表达在植物器官中可能具有普遍性。Northern杂交检测到在高、低温逆境下紫茎泽兰的这四类热激蛋白在mRNA水平上发生明显的变化。一般需要适当时间达到转录的最高水平和最大表达量的恢复才达到最大表达量,而且高温胁迫下并不是在胁迫过程中或胁迫刚结束时即消失,合成还会持续在恢复后的一段时间内,并且紫茎泽兰Aahsp70和Aahsp90对高温和低温的表达模式相似,二者协同表达。Southern结果显示Aahsp17.6和Aahsp60为单拷贝,Aahsp70和Aahsp90为3-4个拷贝数。
通过分析异源表达的紫茎泽兰的HSPs在高温和低温下对大肠杆菌生存能力的影响,初步研究在高温和低温条件下紫茎泽兰Aahsp17.6、Aahsp60、Aahsp70和Aahsp90基因在活体中的功能。用pET-30a表达载体与Aahsp17.6、Aahsp60、Aahsp70和Aahsp90基因进行重组并在大肠杆菌BL21(DE3)中进行诱导表达。高温处理后,重组细胞的存活力较对照相比有明显的提高;并且对热激后的重组菌的可溶性蛋白SDS-PAGE检测表明热激蛋白的表达延缓了可溶性蛋白的降解。在4°C处理8d后,重组细胞的存活力较对照相比有明显的提高。据此推测这四个热激蛋白对低温条件下保护大肠杆菌的功能。
采用染色体步移方法的两个技术获得紫茎泽兰hsp90长864bp的启动子序列(GenBank No. FJ434253)和hsp17.6长1485bp的启动子序列(GenBank No. FJ434252),序列分析得出两个启动子的序列均含有HSPs特异应答元件HSE(nGAAn)以及其它保守的启动子元件。在紫茎泽兰hsp90和hsp17.6启动子中分别存在2个重复以上的,尤其是hsp17.6启动子序列中存在10个此类的重复,因而在响应温度等胁迫时可以快速的与HSF结合,激活hsp基因的表达。
通过体外实验检测了紫茎泽兰HSP17.6和HSP17.7的重组蛋白作为分子伴侣对柠檬酸合酶(CS)的热聚集和热降解的抑制作用。对异源表达的重组蛋白进行Ni-NTA柱纯化后,从1L的菌液中得到浓度为33.6mg的HSP17.6和20.2mg的HSP17.7重组蛋白。体外实验表明HSP17.6与HSP17.7均可减慢45°C处理下CS的热变性,抑制热聚集的最佳比例为HSP17.6/HSP17.7-to-CS为5:1,说明紫茎泽兰的sHSPs具有分子伴侣的活性。此外HSP17.6与HSP17.7均加速热变性CS的复性,38°C处理后的CS活性仅为12%,处理60min后恢复到25°C,加入纯化的重组蛋白后CS活性可以恢复到60%以上。两种sHSPs进行等比例混合后检测其保护功能也被观察到。根据紫茎泽兰的HSP17.6和HSP17.7体外分子伴侣功能推测其在低温胁迫下保护了功能蛋白的活性进而增强了低温适应能力。
将Aahsp17.6定向克隆于带有组成性表达启动子CaMV35S和NPTⅡ基因(带有卡那霉素抗性基因)的植物表达载体pBI121中,冻融法转化农杆菌LBA4404,利用叶圆盘法对番茄进行Ti质粒介导的遗传转化,PCR与Sourthern杂交鉴定出阳性转基因植株。在低温处理下,转基因植株小苗表现出明显优于野生型植株的耐寒性状。转基因植株MDA含量明显低于野生型植株,可溶性糖的含量比野生型植株高出0.5倍,但测定的SOD的活性没有显著差异。结果说明sHSPs提高了植物的耐寒性。
在本研究中紫茎泽兰高海拔种群低温适应性的差异为紫茎泽兰在我国的适应性进化提供了参考价值。对于紫茎泽兰而言,其体内HSPs的多样性、基因结构的特点可能是其对温度逆境尤其是低温的适应能力的重要内在因素,因而对紫茎泽兰sHSP或是其它分子量HSPs的研究提供了紫茎泽兰面临温度胁迫时内在基因表达变化的一个线索,有利于从分子水平解释紫茎泽兰对温度逆境的适应性。
An invasive aline weed, Ageratina adenophora,has become widespread in Southwest of China due to its high environmental adaptation. The invasion of A. adenophora in China, is due to the gradual acclimation of active factor induced by temperature conditions. The alien plants evolutionarily adapt to environmental changes related to different latitudes and altitudesin introduced ranges. However, little is known about the low temperature response of A. adenophora and molecular and genetic aspects that influence resistance to environmental variability. Heat shock proteins genes (hsps) have an essential role in stress resistance and adaptation to the environment. The gene provides an accessible and useful tool for generating insights about molecular mechanisms by how the weeds become successfully invaders.
In this study, with respect to adaptation to abiotic environments, we collected and compared response of A. adenophora populations to low temperature from different high altitude in Sichuan Yunnan of China where the infestation of A. adenophora is quite severe. Meanwhile, we have studied hsps genes that are important in relation to stress resistance and adaptation to the environment to reveal their contribution to invasion of A. adenophora in China.
The physiological responses to low temperature were determined to elucidate mechanisms chilling tolerance. The study made use of artificial climate chamber to compare the A. adenophora chilling tolerance from different high altitude populations from Sichuan and Yunnan province of China. With the decrease of temperature, the physiological changes including increases in malondialdehyde (MDA) and total soluble protein contents, reductions in total soluble sugar, fluctuation of superoxide dismutase (SOD) activity, and catalase activity (CAT) were observed among all the eight populations. However, different extents of low temperature sensitivities were found among the high altitude populations. The results revealed that after the trestment with gradually decreasing temperature, the A. adenophora from the population of Mopanshan and Lushan of Sichuan showed the lowest chilling damage and high tolerance. The populations from Daqingshan of Sichuan and Longling of Yunnan both presented a higher chilling damage and lower tolerance. These indicated that A. adenophora populations from high altitudes were insensitive to chilling tolerance. These data indicate that the invasive plant from high altitude populations with different chilling tolerance reflected a higher capacity to its infested environments. These results may be a manifest A. adenophora expanded to Northern China much faster.
In order to know molecular and genetic aspects of adaption, we cloned full length of coding sequences of invasive alien weed A. adenophora heat shock proteins by the combination of homology cloning in the present study, renamed as Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90. Full-length of Aahsp17, Aahsp60, Aahsp70 and Aahsp90 were 691, 1,581, 2,074 and 2,094 bases of nucleotide sequence, which have been deposited in Genebank database (Accession no. EU209067, EU209068, EU209069, EU209070). The four genes were found highly to be conserved when compared at the amino acid level with the corresponding HSPs from other plants. Sequence analysis, identification, alignments and phylogenetic analyses of the hsps showed that these HSPs shared high levels of identity with corresponding proteins from other species. The phylogenetic analysis suggested that the four hsps were present closed to some other HSPs from Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum et al.
We investigated the characterization and expression of Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 of A. adenophora. After heat treatment, Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 transcripts were not detected in roots, but high levels were found in the stems and leaves. Tissue specificity showed a similar pattern of response to heat shock. The kinetics of regulation of Aahsps was analysed by Northern blot hybridisation, which revealed the significantly changes at mRNA level for Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90. Transcripts of these genes were considerably induced underheat and cold stress. Although the gene expression responses induced by high and low temperture stress shared several common features, there we still observed some differences among Aahsps. The four hsps expression response to heat exposure was robust, and differed from the response to low temperture stress. These results indicate that all of the genes are stress-inducible forms or cognate forms that are constitutively expressed. The sourthern results show that Aahsp17.6 and Aahsp60 appear to be present as a single copy and Aahsp70 and Aahsp90 are encoded by a multi-gene family.
We made investigations on the possible function of Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 in vivo under heat and cold sress, and SDS-PAGE analysis of cell lysates revealed their protective effect is associated with an increase in the thermo stability of soluble proteins. The PCR products of ORF sequences encoding Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 were inserted into the same digested pET-30a vector to generate pET-30a-Aahsps. These plasmids were transformed into Escherichia coli BL21 (DE3) plyss competent cells. Aahsps were expressed in E. coli cells. The recombinant Aahsps proteins produced in bacterial expression were separated by 12% SDS-PAGE.The recombinant Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 over-expression improved viability in comparison with control cultures in E. coli under heat stress conditions. SDS-PAGE analysis of cell lysates suggested that the protective effect in vivo was due to increased thermol stability of soluble cytosolic proteins. The recombinant Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 over-expression improved viability in comparison with control cultures in E. coli under cold stress conditions.
Cloning and sequence analysis of promoters in hsp90 and hsp17.6 genes from A. adenophora by two skills of chromosome walking from a known sequence to an unknown region based on PCR methods. The promoter region of hsp90 with 864bp (GenBank No. FJ434253), from invasive weed A.adenophora was amplified based on PCR methods. Another promoter region of hsp17.6 with 1,485 bp (GenBank No. FJ434252) was cloned by TAIL-PCR. Both of the 5′-flanking regions of hsp90 and hsp17.6 contained specific heat shock element (HSE) (nGAAn) and several cis-acting elements, such as TATA-box, CAAT-box. Especially, the promoter region of hsp17.6 has ten of HSE, which could be bound to heat shock transcription factors (HSFs). Then hsp17.6 of A. adenophora wae robustly induced and accumilated in response to low temperture stress.
The recombinant HSP17.6 and HSP17.7 from A.adenophora were applied to determine their chaperone function. The recombinant HSP17.6 and HSP17.7 were applied to determine their chaperone function. 33.6 mg of HSP17.6 and 20.2 mg of HSP17.7 was purified from 1l of each respective cell culture using a Ni-NTA column. In vitro, HSP17.6 and HSP17.7 actively participated in the refolding of the model substrate citrate synthase (CS) and effectively prevented the thermal aggregation of CS at 45°C HSP17.6 and HSP17.7 prevented this process at different ratios. The maximal suppression by the HSP17.6/HSP17.7-to-CS monomer was also observed at a ratio of 5:1. The effect of the recombinant HSP17.6 and HSP17.7 on the thermal inactivation of CS was investigated. When the CS was incubated at 38°C alone or in presence of 50μg/ml lysozyme, less than 12% of CS activity remained after 60min. After shifting the temperature of the samples to 25°C, the CS activity remained low after 60min in the presence or absence of 50μg/ml lysozyme. They both prevented the irreversible inactivation of CS at 38°C at stoichiometric levels. Therefore, this report confirms the chaperone activity of HSP17.6 and HSP17.7 and their potential as a protectant for active proteins.
To confirm the role of Aahsp17.6 in low temperture condition, an integratedvector pBI121of the Aahsp17.6 and NPTⅡgene(a kanamycin resistant gene)with CaMV35S promoter was constructed and introduced into tobacco mediated by Agrobacterium tumefaciens LBA4404. At low-temperature, the soluble sugar content of transgenic seedlings was higher than that of wild-type plants, whereas the MDA content and the SOD activity were lower than that of wild-type plants. Based on these results, the Aahsp17.6 transgenic tomato plants were more tolerable against low temperture stress than the wild-type plants. Thus, we suggest that Aahsp17.6 may play a pivotal role in chilling tolerance and has potential consequences in this invasive plant for expansion to non-freezing low temperatures zones.
In conculsion, these results may be important for analysis that A. adenophora from high altitude in southwest regions of China has evolved into different ecotypes through physiological adaptation. And the inducible hsps provide a unique system to measure the molecular mechanism by which the invasive alien plants respond to changing environmental and developmental cues. Moreover we could evaluate the molecular capacity of A. adenophora to acclimatise to high and low temperatures in the future according to the results.
引文
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