【作者】 魏爱英；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 硕士

【摘要】 随着世界人口的增加和水资源亏缺以及耕地面积的日益减少,开发利用干旱地和盐碱地越来越重要。已报道的提高植物对非生物逆境抗性的基因工程大多数是将单个目的基因转入植物获得工程植株,但转基因植物抗逆性增加水平仍有待提高。一般认为植物的抗逆性是由多基因控制的,所以多基因组装与转化技术便应运而生。将多个目的基因导入到植物基因组中并在植物中进行表达的策略之一为转基因聚合法,即先用不同基因转化两亲本,获得转基因稳定纯合系后进行杂交,聚合双亲的转基因。该途径虽比较费时,但与同时转入多基因相比,转基因表达相对稳定,易获得多个转基因高效表达的遗传修饰体。本实验以将分别含有betA（大肠杆菌的编码胆碱脱氢酶的基因）和TsVP（盐芥的编码焦磷酸酶的基因）基因的转基因玉米植株（来自自交系DH4866）杂交,得到含有betA和TsVP基因的玉米植株为材料;通过PCR检测和Southern杂交确定了目标基因在杂交植株中存在,并利用RT-PCR检测肯定了两个目标基因在转基因聚合植株中稳定表达。从转基因聚合的F1代株系中选取基因聚合株系,以非转基因自交系DH4866为WT（野生型）对照,以转beta株系和转TsVP的株系分别为转单基因对照,在严格控制条件下进行抗旱耐盐性检测试验。转基因聚合植株的抗旱性分析种子分别播种在大小一致的花盆中,在植株10叶期时开始进行干旱胁迫处理,每天控制浇水量,使其土壤相对含水量保持在15-16%左右,持续处理16天,然后恢复正常浇水（干旱期间避免淋雨）。在干旱处理期间,转基因聚合植株两个目的基因的转录本丰度都明显高于转单基因株系的。干旱胁迫处理后,转基因聚合植株和转单基因植株以及野生型对照植株在株高、雄穗分支数、开花散粉时间、散粉-吐丝间隔时间、花粉活力、果穗长度、百粒重等性状均出现差异。野生型植株雄穗分支数少、雄蕊发育不良、花粉败育、散粉-吐丝间隔时间长、单株籽粒产量低,而转基因聚合植株表现出显著提高的耐旱性,花粉发育基本正常,单株籽粒产量明显高于转单基因对照株系,大幅度高于二野生型的。即转基因聚合植株在干旱处理过程中表现明显增强的抗旱性,生长发育正常。生理测定结果显示:转基因聚合植株叶片相对含水量在干旱胁迫处理中降低速率低于同条件下的转单基因植株的,即保水能力较强,可溶性糖及脯氨酸含量也明显高于转单基因植株,即转基因聚合植株细胞的溶质势更低;离子渗漏率和丙二醛含量表明转基因聚合植株在干旱胁迫处理中叶片细胞膜损伤较小。这些结果表明基因聚合植株在干旱胁迫下可以通过积累更多的渗透保护性物质来维持正常代谢,使植株在干旱胁迫下受损程度轻,与转单基因植株相比具有更强的抗旱性。转基因聚合植株的耐盐性分析将不同株系的种子播于沙盆后分别浇灌不同浓度的盐水,连续浇灌30天,统计出苗率和小苗成活率。转基因聚合植株的种子在高NaCl浓度下出苗率高,小苗在盐胁迫处理中存活率和植株长势要好于转单基因植株的,生物量也明显高于转单基因植株的,细胞Na⁺和Cl^-含量明显提高,但是K⁺和Ca²⁺变化不大,说明转基因聚合植株可以更有效的促使Na⁺在细胞中的区隔化,从而减少过量的Na⁺对植物细胞的伤害。对溶液培养的小苗进行了耐盐性分析,发现在盐胁迫处理条件下,转基因聚合植株长势优于转单基因植株的,叶片离子含量和丙二醛含量较低,叶绿素含量降低速率小于转单基因株系的。转基因表达产物检测表明,在盐胁迫条件下,转基因聚合植株的目的基因的表达产物的丰度分别高于转单基因株系的,推测两个目的基因的共存产生协同效应,使转基因处于一个相对适宜的环境中实现稳定表达,从而提高植株的耐盐性。综上所述,betA和TsVP双基因在玉米中共表达,有效提高了转基因玉米植株的抗旱耐盐性。在干旱胁迫下聚合植株能够通过无机离子和有机溶质的大量积累维持较低的溶质势,从而有利于植物细胞维持膨压,保持水分,免受干旱胁迫的伤害。在盐胁迫条件下,TsVP基因可增强在液泡中储存Na⁺的能力,从而避免了细胞质中过量的Na⁺对细胞的伤害,而betA基因增加了细胞甘氨酸甜菜碱含量,提高了细胞抗胁迫能力,双基因的共同作用明显增强了植株的耐盐性。此外,与野生型植株相比较,转单基因植株抗逆性提高幅度大,而转基因聚合植株与转单基因植株相比抗逆性提高幅度较小,这表明植株抗逆性提高还受到其它代谢途径的制约。本工作一方面为培育抗旱、耐盐玉米新品种创造了优异材料,为我国玉米生产和大面积的盐碱地开发利用做出了力所能及的贡献;另一方面为深入了解玉米抗旱耐盐的分子机制提供了重要资料。更多还原

【Abstract】 In the face of a global increase of population and scarcity of water resources and the decreasing area under cultivation,it is more and more important to exploit and utilize arid and saline-alkali soils.Lots of the genic engineering of improving the stress tolerance of plants are the introduction of single gene into plants to gain engineering plants,and the enhanced level of the stress tolerance is still awaiting to incresae.The plant resitance to abiotic stress is controlled by polygene,so multi-gene assembling and transformation technology emerged as a new strategy.One of the most effective pathway to express multi-transgene in plants appears to be gene stacking.First,individual gene is transformed into parent plants,respectively,and then,the plants with different transgene from one genetype are hybridized each other to generate multi-transgene plants when they are homozygote transgenes.This approach is relatively taking time compered with the introduction of several genes into a plants at one time,but the gene expression is comparatively stabilization and it is more easy to obtain the transgenic plants with high-expression multi-trangene.In our experiment,the betA（coding glycine betaine of E.coil）and TsVP（coding ppase of Thellungiella salsugniea）genes were pyramidsed into maize using cross-pollinated method.The transgene pyramiding plants of the F1 generation were identified by PCR and Southern blotting analysis.And the stable expression of two target transgenes in the pyramiding plants were confirmed using real-time RT-PCR analysis.We chosed the transgene pyramiding lines,and their perent lines that each had a target transgene,and non-transformed DH4866 as wild type control to analysis the tolerance to drought and salt in experimental conditions.Analysis of drought tolerance of the transgene pyramiding plantsThe seeds were planted in unified flowerpots,the drought stress was carried out at the ten leaves stage.Soil water content was maintained at 15-16%.The drought stress was lasted for 16 days before restoring watering the plants.In the period of drought stress,the transcriptional quantity of the two targart genes in the transgene pyramiding plants were distinctive higher than the single transgene.After the drought stress,the transgene pyramiding lines appeared difference with their parent lines and wild type control line in the traits of plant height,branches of tassels,anthesis, anthesis-silking interval,pollen viability,ear length,100 grain weight and so on.The wild type plants had less branches of tassels,abnormal stamen,the sterility of pollen, long anthesis-silking interval,and less grain weight per plant,while the transgene pyramiding plants showed obviously improved drought tolerance,the normal pollen development on the whole,the grain weight per plant was substantially higher than that of their parents,and much higher than that of wild type plants.So the transgene pyramiding plants exhibited significantly improved drought tolerance and grew better under drought sress.The examination of physiology displayed that the decrease rate of relative water content in the leaves of transgene pyramiding plants were lower than that of their parents in the same environment during drought stress,so they had better water capacity.And the soluble carbohydrate and proline content were accumulated more in transgene pyramiding maize than that in their parents and wild type control,and this indicated the transgene pyramiding plants had lower solute potential during drought stress,and had lower ion leakage and MDA content,indicating less damage of cell membrane under drought stress.These results suggested the transgene pyramiding plants could maintain normal metabolism by accumulated osmotic substance under drought stress condition,and undergo moderate damage,so the enhanced drought tolerance is displayed.Analysis of salt tolerance of the transgene pyramiding maizeThe seeds from different lines were planted in unified sand plots and then watered with different concentration NaCl solution.After 30 days,we counted the ratio of seeds germinated and the livability of seedling.The transgene pyramiding seeds had germinated in a relatively higher frequencies under high NaCl condition, and the livabilities and growth of transgene pyramiding seedlings were better than that of their parents,and the biomass of the transgene pyramiding ones were higher too.In the transgene pyramiding seedlings Na⁺ and Cl^- contents had increased more comparing with their parents,but the K⁺ and Ca²⁺contents had little change.These results suggested that the transgene pyramiding plants have obviously enhanced the accumulation of Na⁺ in vacuoles to reduce the toxicity of excessive Na⁺ in cytoplasm to the cell.After that,the maize seedlings growing in Hoagland solution were examined for their tolerance to salt stress.The transgene pyramiding plants grew better than their parents and wild type control,and had lower cell membrane ion leakage and MDA contents.The decrease rate of chlorophyll contents were less than that of their parents and wild type control.The levels of transgene expression were higher than that of their parents under salt stress.We inferred that the two transgenes in the transgene pyramiding plants could co-expressed to produce a relatively better conditions in cells that was beneficial to the genes expression stably,accordingly the plant stress tolerance was improved.Above all,the betA and TsVP co-expression in plants could effectively improve the drought and salt tolerance in transgenic maize.Under drought stress,the transgene pyramiding plants could accumulate more solutes including organic and inorganic ions solute in cell,which would benefit the capability of water uptake at low soil solution water potential to maintain cell turgor.Under salt stress,the TsVP gene in transgene maize could enhance the accumulation of Na⁺ in vacuoles to reduce the toxicity of excessive Na⁺ to the cell,the betA gene could increase ability of stress tolerance,the cooperatioin of the two genes boosted up the plant salt resitence. Besides,compared with the wild type plants,the single gene transgenics had a higher extent stress tolerance.The transgene pyramiding plants showed a moderate improvement in drought and salt stress tolerance compared with the single gene transgenic plants,namely their parents.This results indicated that the improving of maize plant stress tolerance were also restricted by other metabolic pathways.This study has created some valuable maize materials for maize drought and salt tolerance breeding,that would make contribution to maize production and utilization of large areas of arid and saline-alkali soils in China.On the other hand,this study produces some important informations for better understanding the molecular mechanisms in maize drought and salt tolerance.更多还原