【作者】 刘晶；

【导师】 柏锡；

【作者基本信息】 东北农业大学 ， 植物学， 2012， 硕士

【摘要】 盐害是作物生长发育的主要限制因子之一,严重影响其产量和品质,是现代农业生产和生态环境所面临的一个全球性问题。培育耐盐碱的作物品种是推动盐碱化地区农业生产发展的有效措施。随着分子生物学与基因工程技术的不断发展,利用转基因技术向目标作物导入与抗逆相关的目的基因,已成为改良作物耐逆性、培育抗逆新品种的重要途径。特别是导入在信号转导网络中起调控作用的抗逆基因,如转录因子、蛋白激酶基因,可达到综合改良作物抗逆性的效果。紫花苜蓿(Medicago sativa L.)素有“牧草之王”的美誉,是世界上利用最早、栽培最广泛的优质豆科牧草,抗逆性强,具有耐寒、抗旱、耐盐碱等特性,在黑钙土、栗钙土和盐碱土上均能良好生长。然而苜蓿适宜在轻度盐碱地种植,在中、重度盐渍化土地上种植则受到很大限制。培育具有较强耐盐性的紫花苜蓿新品种进行盐碱地改良,是合理开发利用盐渍化土地资源的有效措施。传统的紫花苜蓿抗逆育种,周期长,并且其高耐盐碱种质资源匮乏,在耐盐性的提高上很难达到突破性进展。而通过基因工程手段,利用苜蓿近缘种质资源,将野生耐盐资源的耐盐基因导入栽培品种,培育具有较强耐盐性的转基因苜蓿新品种已成为紫花苜蓿抗逆育种的重要途径。野生大豆具有丰富的耐逆基因资源,实验室前期构建了耐盐东北野生大豆低温、干旱、高盐胁迫反应的基因表达谱。本研究选取在渗透胁迫早期应答网络中起调控作用的野生大豆耐盐基因GsbZIP33和GsCBRLK,转化肇东苜蓿(Medicago sativa L.cv.Zhaodong)。为了对获得的转基因苜蓿的耐盐性进行鉴定,本研究对转基因受体肇东苜蓿苗期的耐盐性进行了初步研究,确定了肇东苜蓿的耐盐程度。并以此为参考对转GsbZIP33和GsCBRLK基因肇东苜蓿进行了耐盐性分析。本研究获得的主要研究结果如下：1.获得转GsbZIP33和GsCBRLK基因肇东苜蓿采用农杆菌介导法对肇东苜蓿进行遗传转化,获得转GsbZIP33基因肇东苜蓿抗性植株101株,其中PCR阳性植株55株,PCR阳性率54%;获得转GsCBRLK基因肇东苜蓿抗性植株75株,PCR阳性植株36株,PCR阳性率48%。对部分PCR阳性植株进行RT-PCR检测,证明目的基因能够在转基因植株中转录并超量表达。2.确定了肇东苜蓿耐盐筛选浓度为了对获得的转基因苜蓿进行耐盐性分析,本研究对转基因受体肇东苜蓿苗期的耐盐性进行了初步研究,通过不同盐胁迫程度(0、50、100、200、300、400mmol/LNaCl)和胁迫时间(0、3、6、9、12、15、21d)下的表型观察及相关生理指标的分析,确定了肇东苜蓿的耐盐筛选浓度。低浓度盐胁迫对其生长无显著影响,肇东苜蓿具有较强的耐盐性,能够抵抗持续15d的200mmol/L的盐胁迫,但难以耐受300mmol/L的盐胁迫,特别是400mmol/L高盐胁迫。因此在对转基因肇东苜蓿进行耐盐性分析时,可以此为参考,选定300mmol/L作为胁迫浓度,400mmol/L作为致死浓度。3.获得耐盐性提高的转GsbZIP33和GsCBRLK基因肇东苜蓿对转GsbZIP33和GsCBRLK基因肇东苜蓿进行耐盐性分析,高浓度盐处理下的表型以及相关生理生化指标的分析结果均表明,目的基因在肇东苜蓿中的超量表达明显提高了转基因苜蓿的耐盐性。300mmol/L NaCl胁迫15d后,野生型苜蓿受害严重,生长发育受抑制,叶片逐渐变黄、卷曲、萎蔫,而转基因苜蓿只受到轻微影响,仍能正常生长。在400mmol/L NaCl处理下,大部分野生型苜蓿死亡,存活植株的生长也严重受抑制;而转基因苜蓿只有极少数植株死亡,存活植株的生长虽有所抑制,但表现明显好于野生型。此外,300mmol/L盐胁迫期间转基因苜蓿的相对电导率、MDA和叶绿素含量以及SOD活性均优于野生型；胁迫15d后,转基因苜蓿的相对电导率和MDA含量显著低于野生型,而Chl含量和SOD活性都显著高于野生型。这些结果表明GsbZIP33和GsCBRLK基因在转基因植株中的超量表达,有助于高盐胁迫下植株维持质膜的稳定性、减轻膜脂的过氧化作用、降低叶绿体的结构功能的损伤以及增强SOD活性,提高抗氧化能力,因而转基因苜蓿具有较高的耐盐性。更多还原

【Abstract】 Salinity stress is one of the major environmental factors that limit plant growth and development, adversely affecting crop production. With the rapid development of molecular biology and bioengineering, using the bioengineering strategies to improve stress tolerance in crops has been an important aspect of modern agricultural research. However, stress tolerance is a complex physiological process in which many genes are involved. Using the bioengineering strategies to transfer a key controlling gene (eg:kinase gene, responding and transducting the stress signal) is a more efficient way to slove the problem.Medicago sativa L. is an important leguminous forage crop worldwide, and it not only supplies abundance of forage for animals but also improves soil fertility. However, salinity problems in agriculture represent a major constraint in the productivity of crops and forage pastures. Therefore, breeding salt tolerant Medicago sativa cultivars is very necessary for this important forage crop adapting to saline soils. Salt tolerance of Medicago sativa can be improved by conventional breeding methods, but it takes a long time to select salt tolerant plants. With the rapid development of molecular biology and bioengineering, using the bioengineering strategies to improve stress tolerance in Medicago sativa has been an important aspect.Wild soybean (Glycine soja) is characteristic of better stress-resistance and adaptive capacity. It is an important gene resource in molecular breeding by means of transgenic technology. In this study,we use four Medicago sativa L.cv.Zhaodong as material, construct of plant expressive vectors of GsbZIP gene and GsCBRLK gene that regulated respectively by El2and CaMV35s promoters by Agrobacterium Tumefaciens. Many transgenic plants were obtained. The research work will provide the function for cultivating transgenic variety that resist to salt stress and studying salt stress resist mechanism. The main results were summarized as follows:1. Identification of transgenic Medicago sativa plantsWe transformed Medicago sativa Zhaodong with GsbZIP33and GsCBRLK gene by Agrobacterium-mediated method transformation. Including101resistant plants by transfomating GsbZIP33, there are55PCR positive plants, so positive rate is54%; Including75resistant plants by transfomating GsCBRLK, there are36PCR positive plants, so positive rate is48%. The results of PCR and RT-PCR showed that the purpose gene was integrated into Medicago sativa genome and overexpressed in transgenic plants.2. Establishment of salt tolerance identification system for Medicago sativa ZhaodongIn order to study the salt tolerance of Medicago sativa L.cv.Zhaodong, seedlings were cultured in1/2Hogland solution supplied with NaCl at different concentrations of0,50,100,200,300and400mmol/L, respectively. Salt injury was measured after the treatment. The content of malondialdehyde (MDA), chlorophyll, proline before and after3,6,9,12,15and21d of salt stress were measured and analyzed, respectively. The results showed that there was no significant effect on the growth of two Medicago sativa under low concentration of NaCl, they had certain salt tolerance ability. The content of MDA increased with the increase of NaCl concentration. While under the stress of200and300mmol/L, the content of MDA increased primary stage then decreased, and then increased with time prolonging. With the concentration and duration of NaCl stress increasing, salt injury was increased, the content of chlorophyll reduced, and proline content increased remarkably. But the accumulation of proline was not entirely consistent with their salt-tolerant performance.3. Over-expression of GsbZIP33, GsCBRLK increases salt tolerance of alfalfaTransgenic Medicago sativa plants overexpressing the GsbZIP33, GsCBRLK showed enhanced salt tolerance. Transgenic Medicago sativa grew well in the presence of300mM NaCl for15days, while wild-type plants exhibited severe chlorosis and growth retardation. Although transgenic Medicago sativa grew slowly and even appeared yellow leaves in400mM NaCl treatment, wild-type plants exhibited chlorosis and even most of wild-type plants died. In addition, Samples from transgenic and wild-type plants treated with300mM NaCl for0,3,6,9,12and15days were selected for physiological analysis. Lower membrane leakage and MDA content were observed in transgenic alfalfa by contrast with wild-type plants during salt treatment. Moreover, reduction of chlorophyll content in transgenic alfalfa was less than that in wild-type plants. Furthermore, the transgenic plants showed enhanced activities of SOD, when compared to wild-type plants. These results indicated that the expression of GsCBRLK confers enhanced tolerance to salt stress in transgenic alfalfa.更多还原