棉花蔗糖非酵解型蛋白激酶GhSnRK2的克隆及功能分析
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摘要
非生物胁迫会导致全球范围内大多作物减产。植物所处的环境在不断变化中,当植物遭受到压力时,会对它们的生长发育不利。非生物胁迫在植物代谢中出现的频率、效率和持续性已经是当代社会关注的焦点。植物中非生物胁迫起始时会激活一些起始原件,然后激活信号转导通路,导致压力响应相关基因的表达和生理变化。植物持续遭受非生物胁迫会导致代谢改变和生物分子的损伤。植物通过上调抗氧化酶,可溶性糖和渗透分子来应对这些胁迫,从而做出防御机制。植物学家已经充分利用现代分子遗传手段来解决非生物胁迫带来的难题,这已经是大家关注的焦点和热点。在我们的研究中,GhSnRK2基因克隆到载体pCAMBIA2301后通过农杆菌GV3101介导的蘸花法转化拟南芥。我们获得了40个阳性植株。RT-PCR分析表明GhSnRK2已经转化到拟南芥中,在不同的转基因株系的后代中都有检测到表达。GhSnRK2-GFP的亚细胞定位表明,GFP信号显示该蛋白定位在细胞核中。过表达GhSnRK2基因的拟南芥植株在抗旱,抗冷,耐ABA和氯化钠能力方面都有提高。相对于非转基因植株,转基因植株的种子对ABA和氯化钠有更好的耐受能力。用10%PEG处理后,GhSnRK2被诱导表达。通过比较过表达GhSnRK2植株和野生型植株,发现转基因植株的相对含水量和脯氨酸含量都比野生型高。组织特异性分析,发现GhSnRK2主要在根中表达。过表达GhSnRK2的转基因植株在正常条件和压力诱导下,压力响应基因RD29A,RD29B, P5CS1, ABI3和ABI5的表达量都有上调。病毒介导的基因沉默进一步解释了GhSnRK2的功能。在VIGS干扰的植株中,GhSnRK2的表达量下调,说明VIGS技术通过干扰基因表达能有效的研究基因的功能。我们的研究表明,通过基因操作,GhSnRK2基因可以提高植物对非生物胁迫的耐受能力。
Abiotic stress has been responsible for the reduction in yield of most major crop plant worldwide; Plants are regularly exposed to changes in environmental conditions which they perceive as stresses when it becomes detrimental. However, the occurrence of abiotic stress response in plants metabolism, efficiency and sustainability is attaining substantial significance in the contemporary world. Initiation of abiotic stress in plant activate some initial sensors, which then activate protein signaling pathways, resulting in stress-responsive gene expression and physiological changes. Continuous exposure to these abiotic stresses results in altered metabolism and damage to biomolecules. Plants develop defense mechanisms to tolerate these stresses by upregulation of enzymatic and non-enzymatic antioxidants, compatible solutes and osmolytes molecules. However, the use of modern genetics approaches in effectively dealing with abiotic stress complications has been the major focus of plant molecular biologists.In the present study, expression vector pCAMBIA2301, that carries GhSnRK2gene, was transformed into Arabidopsis thaliana by Agrobacterium tumifascen GV3101mediated transformation. About40resistant transgenic plants were obtained. RT-PCR analyses showed that GhSnRK2gene had been integrated into the Arabidopsis genome of the transgenic plants and was well expressed in offspring seed of different transgenic lines. Subcellular localization of GhSnRK2-GFP protein revealed that, green fluorescent protein (GFP) signals were localized in the nuclei. In contrast to wild-type plants, the transgenic plants overexpressing GhSnRK2showed increased tolerance to drought, cold, abscisic acid and salt stress, suggesting that GhSnRK2acts as a positive regulator in response to cold and drought stress. GhSnRK2gene was induced by10%PEG treatment. Plants overexpressing GhSnRK2revealed evidence of reduced water loss, elevated relative water content and proline accumulation. Tissue specificity analysis revealed that GhSnRK2gene is abundantly expressed in the root. Under normal and stressed conditions, stress inducible genes; RD29A, RD29B, P5CS1, ABI3, CBF1and ABI5showed increased expression level in the GhSnRK2overexpressed plants than Wild-type. Virus induced gene silencing was further used to elucidate the function of GhSnRK2gene in cotton plants. It was observed from the result that transcript level of GhSnRK2gene was downregulated in VIGS plant. Drought stress assay demonstrated that silencing GhSnRK2gene alleviated drought tolerance in cotton plant. Hence, VIGS technique can inevitably be used as an effective means to study gene function by knocking down expression of distinctly expressed genes. This research suggested that through gene manipulation approaches, inception of GhSnRK2gene functions will permit enhancement of plant tolerance to abiotic stress
Abiotic stress has been responsible for the reduction in yield of most major crop plant worldwide; Plants are regularly exposed to changes in environmental conditions which they perceive as stresses when it becomes detrimental. However, the occurrence of abiotic stress response in plants metabolism, efficiency and sustainability is attaining substantial significance in the contemporary world. Initiation of abiotic stress in plant activate some initial sensors, which then activate protein signaling pathways, resulting in stress-responsive gene expression and physiological changes. Continuous exposure to these abiotic stresses results in altered metabolism and damage to biomolecules. Plants develop defense mechanisms to tolerate these stresses by upregulation of enzymatic and non-enzymatic antioxidants, compatible solutes and osmolytes molecules. However, the use of modern genetics approaches in effectively dealing with abiotic stress complications has been the major focus of plant molecular biologists.In the present study, expression vector pCAMBIA2301, that carries GhSnRK2gene, was transformed into Arabidopsis thaliana by Agrobacterium tumifascen GV3101mediated transformation. About40resistant transgenic plants were obtained. RT-PCR analyses showed that GhSnRK2gene had been integrated into the Arabidopsis genome of the transgenic plants and was well expressed in offspring seed of different transgenic lines. Subcellular localization of GhSnRK2-GFP protein revealed that, green fluorescent protein (GFP) signals were localized in the nuclei. In contrast to wild-type plants, the transgenic plants overexpressing GhSnRK2showed increased tolerance to drought, cold, abscisic acid and salt stress, suggesting that GhSnRK2acts as a positive regulator in response to cold and drought stress. GhSnRK2gene was induced by10%PEG treatment. Plants overexpressing GhSnRK2revealed evidence of reduced water loss, elevated relative water content and proline accumulation. Tissue specificity analysis revealed that GhSnRK2gene is abundantly expressed in the root. Under normal and stressed conditions, stress inducible genes; RD29A, RD29B, P5CS1, ABI3, CBF1and ABI5showed increased expression level in the GhSnRK2overexpressed plants than Wild-type. Virus induced gene silencing was further used to elucidate the function of GhSnRK2gene in cotton plants. It was observed from the result that transcript level of GhSnRK2gene was downregulated in VIGS plant. Drought stress assay demonstrated that silencing GhSnRK2gene alleviated drought tolerance in cotton plant. Hence, VIGS technique can inevitably be used as an effective means to study gene function by knocking down expression of distinctly expressed genes. This research suggested that through gene manipulation approaches, inception of GhSnRK2gene functions will permit enhancement of plant tolerance to abiotic stress
引文
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