大豆DREB基因GmDREB1改良紫花苜蓿耐盐性的研究
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摘要
盐胁迫严重影响作物和牧草产量。在我国由于水资源日益短缺,干旱半干旱地区土壤干旱和盐渍化问题越发严峻。紫花苜蓿(Medicago sativa. L)作为一种营养丰富的豆科牧草在我国西北干旱半干旱及吉林省西部地区农牧业生产和生态建设中发挥重要作用。可是,大多数苜蓿品种对盐胁迫的耐受能力不强,产量受到很大限制。在改良作物抗逆能力方面,传统育种方法的成功案例并不多。相对而言,应用基因工程技术提高植物抗逆性更快捷更有效。在逆境胁迫下,DREB转录因子以与DRE顺式作用元件特异结合的方式,可以调控一系列逆境相关下游基因表达。此类基因已被证明在改良植物抗逆性方面具有相当重要的应用价值。
本论文实验以基因工程技术为手段扩增了大豆GmDREB1基因,构建了重组植物表达载体Rd29A:GmDREB1:GFP。将这个表达载体转入农杆菌LBA4404并以农杆菌介导的方法转化到紫花苜蓿栽培种公农1号中,获得了四个株系200株再生苗。PCR、Southern杂交结果表明外源基因已经整合到苜蓿核基因组中,且29A-2,29A-4植株的外源基因整合方式为单拷贝,外源基因在其后代中遗传等为稳定。Northern杂交表明在盐胁迫条件下,外源基因杂交信号明显,而在非盐胁迫条件下,杂交信号很弱,无法检测到。且转基因植株在正常条件下,并没有出现矮化、产量降低的现象,由此证明Rd29A启动子的作用如预期一致。对再生植株的生理检测表明,GmDREB1转基因苜蓿对中度盐胁迫(200mM NaCl)具有很强的耐受性。生理指标分析发现:在盐胁迫处理后,转基因植株的膜透性和光合系统II活性明显好于对照,且累积了大量的游离脯氨酸和可溶糖,使得转基因植株能较好的应对盐胁迫逆境。除此之外,我们确定GmDREB1通过提升转基因植株体内P5CS基因的转录水平而导致大量游离脯氨酸的累积。
总之,本研究首次将DREB类基因转入苜蓿中,证明转基因植株获得较好的耐盐能力。同时确定以诱导型启动子Rd29A启动子驱动DREB基因表达是提高植物抗逆能力的一个有效手段。这些研究结果为深入研究植物耐盐机制,培育耐盐转基因植株等方面的应用提供了一定的基础和依据。进一步的筛选GmDREB1下游目的基因以及GmDREB1调控下游基因的具体方式等工作正在进行中。
Salt stress adversely affects crop productivity and forage yield. With ingravescent fresh water scarcity, the situation of drought and soil salinity get even worse in the arid and semi-arid areas of China. Alfalfa (Medicago sativa.L) is a protein-riched legume forage, acting an important role in agriculture and animal husbandry and ecological construction in arid and semi-arid areas of western JiLin Province and Northeast of China. However, salt stress at 50-200 mM NaCl significantly limits the productivity of alfalfa. Traditional breeding strategies have generated very few crop varieties with improved stress tolerance. Contrary to the classical breeding approaches, direct introduction of genes by genetic engineering seems a more attractive and quick solution for improving stress tolerance. Dehydration-responsive element binding proteins (DREB), specifically interacting with DRE element, could up-regulation of a whole array of downstream genes in response to stress and are regarded as important genetic resources for improving plant stress tolerance.
In the current study, a soybean DREB orthologue, GmDREB1, was amplified by using genetic method. Subsequently, we constructed the recombinant expression vectors Rd29A: GmDREB 1:GFP and transformed it into Agrobacterium tumefaciens LBA4404. With Agrobacterium tumefaciens mediated transformation method, GmDREB1 was introduced into alfalfa cultiva, Gongnong1, and thereafter 200 regenerated plants from 4 lines were gained. PCR and Southren blot analysis demonstrated that the exogenous gene had been integrated into the chromosome of all the four transgenic lines. Furthermore, plants from line 29A-2 and 29A-4 carried a single active T-DNA locus and were applied for further analysis. Northern blot analysis shown thant GmDREB1 transcripts were stably detected in salt-treated line 29A-2 and 29A-4 plants. Together with the facts that under normal conditions no morphological differences were found between transgenic plants and wild-type plants, Rd29A promoter was comfirmed to play its role as expected. Moreover, GmDREB1 transgenic plants displayed strong tolerance to moderated salt stress(200mM NaCl). The four physiological indices of transgenic plants were all better than those of wild control under moderated salt stress treatments (200mM NaCl). Northern bolt analysis furthern confirmed thant GmDREB1 increased P5CS transcript levels thus led to more accumulation of free proline in transgenic plants.
Overall, we the first to intruduce a DREB gene into alfalfa and improve its salt tolerance. Also we make sure that incorporating a key regulator gene such as DREB gene under the control of Rd29A promoter is an efficient approach to minimize stress damage to crops. Futher study on screening the downstream genes of GmDREB1 and the concrete way of GmDREB1 to affect its downstream genes will be carried out soon in the future.
本论文实验以基因工程技术为手段扩增了大豆GmDREB1基因,构建了重组植物表达载体Rd29A:GmDREB1:GFP。将这个表达载体转入农杆菌LBA4404并以农杆菌介导的方法转化到紫花苜蓿栽培种公农1号中,获得了四个株系200株再生苗。PCR、Southern杂交结果表明外源基因已经整合到苜蓿核基因组中,且29A-2,29A-4植株的外源基因整合方式为单拷贝,外源基因在其后代中遗传等为稳定。Northern杂交表明在盐胁迫条件下,外源基因杂交信号明显,而在非盐胁迫条件下,杂交信号很弱,无法检测到。且转基因植株在正常条件下,并没有出现矮化、产量降低的现象,由此证明Rd29A启动子的作用如预期一致。对再生植株的生理检测表明,GmDREB1转基因苜蓿对中度盐胁迫(200mM NaCl)具有很强的耐受性。生理指标分析发现:在盐胁迫处理后,转基因植株的膜透性和光合系统II活性明显好于对照,且累积了大量的游离脯氨酸和可溶糖,使得转基因植株能较好的应对盐胁迫逆境。除此之外,我们确定GmDREB1通过提升转基因植株体内P5CS基因的转录水平而导致大量游离脯氨酸的累积。
总之,本研究首次将DREB类基因转入苜蓿中,证明转基因植株获得较好的耐盐能力。同时确定以诱导型启动子Rd29A启动子驱动DREB基因表达是提高植物抗逆能力的一个有效手段。这些研究结果为深入研究植物耐盐机制,培育耐盐转基因植株等方面的应用提供了一定的基础和依据。进一步的筛选GmDREB1下游目的基因以及GmDREB1调控下游基因的具体方式等工作正在进行中。
Salt stress adversely affects crop productivity and forage yield. With ingravescent fresh water scarcity, the situation of drought and soil salinity get even worse in the arid and semi-arid areas of China. Alfalfa (Medicago sativa.L) is a protein-riched legume forage, acting an important role in agriculture and animal husbandry and ecological construction in arid and semi-arid areas of western JiLin Province and Northeast of China. However, salt stress at 50-200 mM NaCl significantly limits the productivity of alfalfa. Traditional breeding strategies have generated very few crop varieties with improved stress tolerance. Contrary to the classical breeding approaches, direct introduction of genes by genetic engineering seems a more attractive and quick solution for improving stress tolerance. Dehydration-responsive element binding proteins (DREB), specifically interacting with DRE element, could up-regulation of a whole array of downstream genes in response to stress and are regarded as important genetic resources for improving plant stress tolerance.
In the current study, a soybean DREB orthologue, GmDREB1, was amplified by using genetic method. Subsequently, we constructed the recombinant expression vectors Rd29A: GmDREB 1:GFP and transformed it into Agrobacterium tumefaciens LBA4404. With Agrobacterium tumefaciens mediated transformation method, GmDREB1 was introduced into alfalfa cultiva, Gongnong1, and thereafter 200 regenerated plants from 4 lines were gained. PCR and Southren blot analysis demonstrated that the exogenous gene had been integrated into the chromosome of all the four transgenic lines. Furthermore, plants from line 29A-2 and 29A-4 carried a single active T-DNA locus and were applied for further analysis. Northern blot analysis shown thant GmDREB1 transcripts were stably detected in salt-treated line 29A-2 and 29A-4 plants. Together with the facts that under normal conditions no morphological differences were found between transgenic plants and wild-type plants, Rd29A promoter was comfirmed to play its role as expected. Moreover, GmDREB1 transgenic plants displayed strong tolerance to moderated salt stress(200mM NaCl). The four physiological indices of transgenic plants were all better than those of wild control under moderated salt stress treatments (200mM NaCl). Northern bolt analysis furthern confirmed thant GmDREB1 increased P5CS transcript levels thus led to more accumulation of free proline in transgenic plants.
Overall, we the first to intruduce a DREB gene into alfalfa and improve its salt tolerance. Also we make sure that incorporating a key regulator gene such as DREB gene under the control of Rd29A promoter is an efficient approach to minimize stress damage to crops. Futher study on screening the downstream genes of GmDREB1 and the concrete way of GmDREB1 to affect its downstream genes will be carried out soon in the future.
引文
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