【作者】 段晓光；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 博士

【摘要】 盐胁迫是自然界中主要的非生物胁迫之一,目前已经严重影响到世界范围内许多重要作物的产量。利用基因工程技术提高作物耐盐性是解决这一问题的最有效途径之一。目前,通过向植物中转入耐盐相关基因可在一定程度上改善植物的耐盐性。然而,由于植物的耐盐性是受多基因控制的复杂遗传性状,依赖于多个基因之间的相互作用,是多种耐盐生理性状的综合体现,因此,转入单个耐盐相关基因只能部分提高植物的耐盐性。一般认为,将多个耐盐相关基因聚合到同一植物中有可能会大大提高转基因植物抵抗盐胁迫的能力。来源于大肠杆菌的betA基因,编码胆碱脱氢酶,转入植物后可提高细胞甘氨酸甜菜碱含量;来自于盐芥的TsVP基因编码V-H~+-PPase,该基因过表达可提高液泡膜逆浓度梯度转运离子的能力;AtNHX1基因编码拟南芥液泡膜Na~+/H~+反向转运蛋白,该蛋白可将胞质中过高的Na~+转运到液泡内积累。已有的基因工程研究表明,分别过量表达这三个单基因都可以明显改善植物的耐盐性。本工作以分别转betA基因烟草(BL)、转TsVP基因烟草(TL)和转AtNHX1基因烟草(AL)为材料,通过有性杂交方法获得聚合有2个不同转基因的烟草植株,即betA×TsVP(BT)、betA×AtNHX1(BA)。PCR、Southern blot、RT-PCR和检测甘氨酸甜菜碱含量、V-H~+-PPase水解活性及Na~+含量的测定结果都表明,两个转基因稳定存在于转基因聚合的烟草植株中,并进行了活跃表达。通过对转单基因植株和转基因聚合植株在植株和细胞水平上的一系列耐盐性分析,确定了细胞水平与植株水平的耐盐性存在一致性,转基因聚合植株与转单基因植株相比具有较高的耐盐性,但未表现出耐盐程度大幅度提高。主要结果和结论如下:转基因聚合烟草BT(betA×TsVP)的耐盐性分析在加有相同浓度NaCl的培养基上,转基因聚合株系(BT)表现出相对较高的种子萌发率和较好的小苗长势,植株单株鲜重最大;而转单基因株系(分别转betA基因和TsVP基因)次之,未转基因对照最差。表明转基因聚合相比转单基因提高了烟草在种子萌发阶段及小苗生长阶段的耐盐性。在正常的生长条件下,转betA基因植株与未转基因植株的生物量相似;250mM NaCl胁迫处理14天后,转betA基因植株的生物量高于未转基因植株。而无论在正常生长条件下还是盐胁迫处理后,转TsVP基因植株和转基因聚合植株都比未转基因植株生长快,具有旺盛的地上部分和发达的根系,其中转基因聚合植株的生物量最高。在正常生长条件下,转TsVP基因植株和转基因聚合植株的净光合速率显著高于未转基因对照和转betA基因植株的,而TsVP转基因植株和转基因聚合植株之间以及未转基因植株和转betA基因植株之间在光合能力上没有显著差异。250 mM NaCl的胁迫处理导致所有株系植株的光合活性下降,但转基因植株的净光合速率和Fv/Fm都比未转基因植株的高,表明转基因植株的光合效率较高。其它光合作用参数的测定结果也表明转基因植株在盐胁迫条件下比未转基因植株具有更好的光合能力,但转基因聚合植株与转单基因植株相比差异不大。在盐胁迫条件下,转基因植株和未转基因植株叶片内Na~+和Cl~-含量都有所增加,但含有TsVP基因的烟草植株中积累了更多的Na~+和Cl~-,Na~+和Cl~-含量显著高于未转基因植株和转betA基因植株的。转基因聚合植株与转TsVP基因植株相比Na~+和Cl~-含量差异不显著。利用激光扫描共聚焦显微镜结合Sodium Green荧光指示剂对液泡中Na~+的积累进行检测,发现盐胁迫导致所有株系的细胞中荧光强度明显升高,意味着Na~+的积累量增加。转TsVP基因细胞和转基因聚合细胞液泡中的Na~+积累量显著高于未转基因对照的和转betA基因细胞的,而转基因聚合细胞与转TsVP基因细胞相比,荧光强度无显著差异,说明二者的Na~+含量相似。这与盐胁迫后植株叶片中Na~+含量的测定结果是相一致的,表明TsVP的表达增加了转基因细胞中Na~+在液泡内的积累量。盐分胁迫使膜系统受到伤害,导致不同株系植株的叶片MDA含量和细胞电解质外渗率大幅度升高。未转基因植株的MDA含量和细胞电解质外渗值最大,而转基因聚合植株的最小,并且与转单基因植株间存在不同程度的差异,表明未转基因植株细胞受到的伤害最重,而转基因聚合细胞受害程度最小。在细胞学水平上对盐胁迫下细胞活力和线粒体膜完整性的测定结果得出同样的结论,即在相同的胁迫条件下,转基因株系受害程度低,原生质体活力高和线粒体膜完整性好。利用pH敏感的荧光探针BCECF-AM结合激光扫描共聚焦显微镜技术检测了盐胁迫对叶肉细胞质和液泡pH值的影响。在相同浓度的NaCl胁迫下,转基因细胞,尤其是TsVP基因过表达的细胞,pH变化幅度较小,说明其能够更好的维持细胞内环境的相对稳定。根据能斯特方程计算跨液泡膜的质子电势(△Ψ(H~+))。在胁迫前和胁迫后,转TsVP基因细胞和转基因聚合细胞中的△Ψ(H~+)值都要显著高于未转基因的和转betA基因细胞的。表明在含有外源TsVP基因的细胞中,V-H~+-PPase通过水解PPi来酸化液泡,从而维持了细胞中高的跨液泡膜质子电势。高的跨液泡膜电势能够促进Na~+在液泡中的区隔化,这与Na~+在液泡中积累增加的实验结果相一致。转基因聚合烟草BA(betA×AtNHX1)的耐盐性分析在含有相同盐浓度的培养基上,转基因株系的种子萌发率和小苗生长状态优于未转基因植株的。在相同的盐浓度下,转基因聚合烟草种子(BA)的萌发率最高,其单株鲜重略高于转单基因植株的(分别转betA基因和AtNHX1基因),但差异达不到显著水平。表明在种子萌发和幼苗生长阶段转基因聚合株系的耐盐性略高于转单基因株系的。盐胁迫增加了所有植株中的Na~+含量,含有AtNHX1基因的植株叶片中的Na~+含量显著高于未转基因植株和转betA基因植株的。Sodium Green标记显示,在盐胁迫条件下液泡内Na~+积累量在转AtNHX1基因的烟草细胞中显著高于未转基因细胞和转betA基因细胞的,而转基因聚合细胞与转AtNHX1基因细胞在液泡内Na~+积累量上差异不显著。这与叶片Na~+含量的测定结果是相一致的,表明在含有AtNHX1基因的细胞中,AtNHX1基因的表达加速了Na~+在液泡中的积累。对胞质和液泡pH值及跨液泡膜电势的计算结果也表明转基因细胞维持内环境稳定的能力要高于未转基因细胞的。转基因细胞在盐胁迫条件下表现出较高活力和较好的线粒体膜的完整性,但转基因聚合细胞和转单基因细胞间差异不显著。盐处理使植株叶片的丙二醛含量和离子渗漏率都有所上升,但转基因植株的测定值显著低于未转基因植株。这些结果表明在盐胁迫下转基因细胞与未转基因对照相比具有明显提高的抗逆性,但betA×AtNHX1植株与转单基因植株相比耐盐性提高幅度不大。尽管在转基因聚合植株中,各外源基因分别通过各自的方式对提高植株的耐盐性做出了贡献,但转基因聚合植株的耐盐性与转单基因植株相比提高幅度有限。虽然转基因聚合在一定程度上改善了转单基因植株的耐盐性,但与单基因分别提高植株耐盐程度之和相差很大,有可能植物的耐盐性是多个代谢活动的综合反应,而某些制约环节决定植株对盐胁迫的敏感性。因此,尽管将控制不同代谢途径的耐盐相关基因聚合是提高植物耐盐性的重要途径,但深入了解与不同植株耐盐性相关的代谢活动及其调控机制十分必要,从而拟订出针对性强易于操作的植物抗逆基因工程策略。另外,本工作结果提示,利用突变体来确定某个基因的生物学功能时有可能因调控网络和代谢网络的破坏导致基因功能的放大,因此,在基因功能分析中应注意不同遗传背景下目标基因的功能差异。更多还原

【Abstract】 Salt stress is one of the major abiotic stresses in nature,and can severely affect the productivity of crops all over the world.Improving the salt tolerance of plants through genetic engineering is one of the most effective strategies to solve this problem.Until now,improved salt tolerance of plants has been achieved by genetic transformation of salt tolerance-related genes.The tolerance to salt stress of plants is a complex trait that is modulated by multiple genes and requires the coordination of many genes.Therefore,the transformation of single gene into a plant can only enhance salt tolerance partially.In general introducing genes from different salt responses into a single plant by gene pyramiding is considered as an efficient approach for engineering extreme salt tolerance,which could be achieved either by cross-breeding plants containing different salt-tolerant genes or by transformation with multiple genes.The betA gene from Escherichia coli,which encodes chroline dehydrogenase (CDH),could increase the betaine content in transgenic plants;the TsVP gene from salt cress(Thellungiella halophila),which encodes vacuolar H~+-translocating inorganic pyrophosphatase,would enhance the transport across the vacuolar membrane;the AtNHX1 gene from Arabidopsis thaliana,which encodes vacuolar Na~+/H~+ antiporter,could sequestrate excessive Na~+ into vacuoles.It has been reported that overexpressing each gene alone can improve the salt tolerance of plants.In this study,the beta gene,TsVP gene and AtNHX1 gene transformed tobaccos were named BL,TL and AL,respectively.The transgene pyramiding tobacco plants were obtained by sexual crossing,including two genes that undergo different mechanisms of salt tolerance(betA×TsVP(named BT),betA×AtNHX1(named BA)). PCR,Southern blot,RT-PCR,and the determination of betaine content,V-H~+-PPase hydrolytic activity and Na~+ concentration indicated that the two genes were integrated into the genome and expressed functionally in transgene pyramiding tobacco plants. The analysis of salt tolerance at the whole plant level and cellular level were performed,which confirmed the consistency between the cellular level and the whole plant level.The transgene pyramiding plants showed higher salt tolerance than single gene transgenics,though the extent was not great.The main results of this study were summarized as follows:Studies on salt tolerance of betA×TsVP(BT)tobaccoOn the medium containing the same concentration of NaCl,the transgene pyramiding tobacco line(BT)showed the highest percentage of seed germination,the best growth and the highest seedling fresh weight.In contrast those of the single gene transgenic lines(BL and TL)were moderate and those of the non-transgenic control line were the lowest.These results indicated that transgene pyramiding could further enhance tobacco salt tolerance than the transformation of single gene at the seed germination and seedlings growth stage.Under normal conditions,the biomass of betA-transgenics(BL)and the wild-type(WT)plants were similar.The fresh weight and dry weight of BL were both higher than those of WT after the treatment of 250 mM NaCl for 14 days.The TsVP-transgenics and the transgene pyramiding plants showed more developed shoot and root systems compared with WT under both normal and salt stress conditions,and the biomass of betA×TsVP plants was the highest.The TsVP-transgenic tobacco plants (TL and BT)showed significantly higher net photosynthetic rate than wild-type and betA-transformed plants under normal conditions.There was no significant difference on the photosynthesis between TL and BT,and the photosynthesis of WT and BL plants were also comparable in the absence of NaCl.Salt stress decreased the photosynthetic activities of both transgenic and wild-type plants,while the transgenic plants exhibited higher photosynthetic rate and Fv/Fm ratio than wild-type,implying the higher photosynthetic efficiency in transgenies.The results of the measurement on other photosynthesis features also indicated the transgenic plants had higher photosynthetic capacity than the wild-type ones under salt stress conditions,but the difference in photosynthesis between transgene pyramiding plants and single gene transgenics was not significant.Salinity stress increased the Na~+ and Cl~- concentrations in all plants,but the TsVP-transgenics(TL)and the transgene pyramiding plants(BT)accumulated significantly more Na~+ and Cl~- than wild-type and betA-transgenic plants.There was no significant difference on Na~+ and Cl~- contents between BT and TL plants.The accumulation of Na~+ in vacuoles was measured using Sodium Green fluorescent indicator and laser scanning confocal microscopy.Higher fluorescent signals(i.e. higher Na~+ concentration)were observed in both transgenic and wild-type cells under salt stress than in the absence of NaCl.It was found that the Ts VP-transgenic and the transgene pyramiding cells accumulated significantly higher Na~+ than wild-type and betA-transgenic cells under salinity stress,but there was no obvious difference on Na~+ accumulation between the transgene pyramiding cells and TsVP-transgenic cells, which was consistent with the measurement result of Na~+ in the leaves.These results suggested that the functional expression of TsVP enhanced the sequestration of Na~+ in vacuoles.Salt stress resulted in membrane injury and caused MDA content and electrolyte leakage rising in both transgenic and wild-type plants.The wild-type plants exhibited higher MDA content and relative conductance than the transgenics under saline conditions,suggesting the higher salt tolerance in transgenic tobacco plants.The transgene pyramiding plants showed the highest salt tolerance for the lowest MDA content and ion leakage of leaves under salinity stress.Such results were in line with the observations of cell viability and the integrity of mitoehondrial membrane at the cellular level.The transgenic tobacco cells showed higher cell viability and better integrity of mitochondrial membrane than wild-type cells under the same NaCl treatment,which also suggested the improved salt tolerance in transgenic cells.The cytoplasmic and vacuolar pH values were recorded by means of the pH-sensitive,cell-permeant fluorescent indicator BCECF-AM.The transgenic cells, especially TsVP transgenic cells,showed the less change of pH,which suggested that they could maintain the intracellular environment homeostasis better than wild-type under salt stress.The vacuolar pH values in TL and BT cells were lower than those in wild-type ones.The membrane potential produced by pH difference across the vacuolar membrane(△Ψ(H~+))was calculated using Nernst equation.The△Ψ(H~+) values of TsVP-transgenic and transgene pyramiding cells were significantly higher than those of wild-type and betA-transgenic cells,both before and after salt stress. These results suggested that the V-H~+-PPase could acidify vacuoles and maintain higher proton electrochemical gradient across the vacuolar membrane in TsVP transgenic cells.The higher△Ψ(H~+)would provide more motive force for the V-Na~+/H~+ antiporter to sequestrate Na~+ within vacuoles,which was proved by the determination of sodium ion in vacuoles.Studies on salt tolerance of BA(betA×AtNHX1)tobaccoThe transgenic tobacco lines exhibited higher percentage of seed germination.In addition,they grew better than the wild-type when placed on medium supplemented with the same concentration of NaCl.The seed germination percentage of the transgene pyramiding line(BA)was the highest,and the BA fresh weight per seedling was slightly higher than single gene transgenics(BL and AL),suggesting improved salt tolerance in the transgene pyramiding line.The AtNHX1-transgenic plants(AL)and the betA×AtNHX1 transgene pyramiding plants(BA)accumulated more Na~+ content than wild-type and betA-transgenic plants under salinity stress.Salt stress resulted in increasing Na~+ accumulation in the AtNHX1 transgenie cells(AL and BA)compared with that in WT or BL cells measured with Sodium Green,and the difference was significant. Alternatively,there was no distinct difference between AL and BA cells in the Na~+ content.The results were in agreement with the findings of Na~+ in leaves,indicating that the heterologous expression of AtNHX1 accelerated the sequestration of Na~+ within vacuoles.The calculation of cytoplasmic pH,vacuolar pH and the△Ψ(H~+) across the vacuolar membrane also indicated that the transgenic cells could maintain the homeostasis of intracellular environment better than WT ones.The transgenic cells showed higher protoplast viability and better mitochondrial integrity than wild-type cells under saline conditions.But no significant difference was found between the transgene pyramiding and single gene transgenic cells.Salt stress increased MDA content and relative conductance in all plants.However,the values of transgenic plants measured were much lower than those of wild-type.These results indicated that the transgenic cells showed enhanced salt tolerance compared with wild-type under salt stress conditions,but the improved salt tolerance of betA×AtNHX1 transgene pyramiding plants was not greater than that of single gene transgenics(BL and AL).In transgene pyramiding plants,the transgenes increased the salt tolerance of tobacco by different tolerance mechanisms,respectively.But the improved salt tolerance of transgene pyramiding plants compared with single gene transformed plants was much less than the additive effects of the two genes alone.It is presumably because plant salt tolerance is a polygenie trait which requires the cooperation of many genes,and some elements restricted the sensitivity of plants to salinity stress. Although the pyramiding of genes involved in different salt tolerance mechanisms is an important approach for enhanced plant salinity tolerance,it is necessary to deeply understand the metabolic and regulatory mechanisms for plant tolerance to salt stress. As a consequence,strategies for plant stress-tolerance genetic engineering could be made with better pertinency and maneuverability.Furthermore,our work implied that the roles of genes might be enlarged due to the damage of regulatory networks when their biological functions were studied by mutants.Therefore,functional differences of target genes should be considered under different genetic backgrounds.更多还原