【作者】 张华；

【导师】 乔代蓉；

【作者基本信息】 四川大学 ， 微生物学， 2004， 硕士

【摘要】 盐碱化是导致我国环境危机的重要因素之一，利用基因工程培育抗旱耐盐品种已成为解决我国盐碱化日益严重问题的一条重要途径。盐生杜氏藻(Dunaliella salina)是一种真核单细胞绿藻，能在0.1-5.5mol／L的NaCl浓度中生存，是目前发现的唯一能进行渗透压调节的单细胞真核光合生物。盐生杜氏藻主要通过集聚甘油进行细胞膨压的调节，因此克隆甘油合成途径中的关键酶3-磷酸甘油脱氢酶(glycerol-3-phosphate dehydrogenase，简称为DsGPD)基因，确定其在植物抗旱耐盐中的作用十分必要。 本文是在克隆获得盐生杜氏藻具调渗功能的3-磷酸甘油脱氢酶基因(DsGPD)的基础上，应用DsGPD进行苜蓿抗旱耐盐基因工程改造，目的是选育抗旱耐盐苜蓿新品种，这将对于盐碱化土地的改造和治理，扩大优质豆科牧草苜蓿种植面积发挥极其重要的作用。取得的主要结果如下： 1．首先构建了植物高效表达载体pCAMBIA2301G-DsGPD，成功地将该质粒导入根癌农杆菌(Agrobacterium tumefaciens)EHA105中； 2．完成了苜蓿不同转化方法的优化。通过直接侵染、超声波震荡、真空负压法等农杆菌介导转化苜蓿的方法的优化，叶片经超声波和负压处理后，转化效率分别为27.7％和22.2％，比直接浸染分别提高转化效率150％和110％。愈伤组织电击法以电压650V／cm，电容50uF的转化条件为最佳，GUS染色获得的蓝色条带数目最多； 3．建立了苜蓿的转化体系。采用农杆菌介导的方法进行DsGPD基因的苜蓿转化。根癌农杆菌侵染苜蓿外植体后，三月左右胚状体萌发再生芽，通过诱导生根获得完整植株，生根率达到60％左右，最后将再生苗成功移过诱导生根获得完整植株，生根率达到60%左右，最后将再生苗成功移 栽到蛙石:沙(l:l)的培养介质中培养;4.比较了子叶、下胚轴、叶片的转化效率。子叶和叶片更容易被转化，分 化率为17%左右。当根癌农杆菌菌液OD二0.3一0.5时，愈伤组织形成率 为60%一80%，效果较好;5.完成了转基因植株的分子检测。获得了转DsGPD基因首稽植株102株， 经PCR检测有21%的再生苗为阳性。为进一步功能研究及提高首稽在盐 环境下的耐盐性奠定基础。关键词:盐生杜氏藻;3一磷酸甘油脱氢酶基因;叶盘转化法;紫花首蓓;GUS化学组织染色;根癌农杆菌;愈伤组织;更多还原

【Abstract】 Drought and salination are the key factor of environmental crisis. So the research on the halo-tolerant gene engineering will play extreme important function for the alteration to the salination of soil. Dunaliella salina is the most extreme halotolerat alga in the world , The eukaryotic unicellular green alga able to proliferate under saline condition ranging from 0.1 to 5.5 mol/L NaCl. It is a good model organism in the plant halotolerance research. The alga regulates its inside turgor to adapt the outside osmotic change by synthesizing a large amount of glycerol. It is important to study glycerol-3-phosphate dehydrogenase gene, the key enzyme gene of the glycerol metabolism to know how the species can sustain the hyperosmotic press by accumulating so much glycerol. We introduced DsGPD gene into Medicago sativa L by gene engineering and take the hereditary change of the alfalfa halo-tolerance into practice, it can enlarge the growth range of alfalfa, which play important role in improvement environment, prevention the loss of water and soil and enhancement the nutrition of soil.We have got the DsGPD gene from the cDNA library. Now, we constructed the vector pCAMBIA2301G-DsGPD, which was introduced into Agrobacterium tumefaciens EH105 using the freeze-thaw method. After infected by A. tumefaciens, the explants of Medicago sativa L were selected on platescontaining UM inducement medium supplemented with 50mg/L kanamycin. Soon, calli were inducted and form embryoid, after three month, the embryoid germinate .We cut the regeneration bud and insert into MS3 medium .The rootage rate of regenerated plants was 60%. Regenerated plants transplanted into vermiculite: sand (1:1) medium. We got regenerated plants 102 strains, distill the total DNA of regenerated plants as the template of PCR, PCR analysis showed that approximate 21% tested plants produced the target band. We constitute the system to transformation and regeneration, set up the basis for the further function search and enhance the salt tolerance of Medicago sativa L.Compare the efficiency of transform, cotyledon and lamina were transformed more easy than hypocotyls. When the OD value of Agrobacterium tumefaciens was 0.3 ~0.5 ,the rate of callue formation was 60%~80%.The rate of differentiation was 17%.We transfer DsGPD to alfalfa lamina under the circumstance of ultrasonic and vacuum. GUS stain to the lamina and callus, then we get the efficiency of gene-transferring. The rate of transformation under the circumstance of ultrasonic and vacuum was 27.7% and 22.2% respectively, it was enhanced clearly under the circumstance of ultrasonic and vacuum. In addition, DsGPD was transformed into the callus of medicago, the best condition of electric-transformation was voltage 650V/cm, capacitance 50 uF which was the best condition to transform the Medicago sativa L.更多还原