【作者】 程贵兰；

【导师】 姜静；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2004， 硕士

【摘要】 本研究以小黑杨（Populus simonii×P．nigra）花粉植株为试材，采用根癌农杆菌介导法进行胆碱脱氢酶基因（betA基因）的转化。betA基因能一步将胆碱合成为甘氨酸甜菜碱，甘氨酸甜菜碱是一种重要的调渗物质，盐胁迫下，甘氨酸甜菜碱的积累可以保护细胞内蛋白质的结构和功能，降低细胞水势，从而增强植物自身的耐盐能力。探讨了影响植物遗传转化的若干因素，初步建立了小黑杨花粉植株的遗传转化体系。经PCR检测、Southern斑点杂交及Southern印迹杂交，证明betA基因已整合到小黑杨花粉植株基因组中，在NaCl盐胁迫条件下，转基因植株的耐盐能力比对照植株有所提高。研究结果如下： （1） 确定了筛选小黑杨花粉植株转化体选择压为卡那霉素，其浓度为40mg·L^-1。 （2） 测试了三种头孢类抗生素有效的抑菌浓度及对小黑杨花粉植株叶片分化的影响。确定使用上海制药四厂和哈尔滨制药总厂生产的头孢唑林钠为脱菌抗生素，使用浓度为700 mg·L^-1。 （3） 建立了以小黑杨花粉植株叶片为外植体的转化体系： 预培养：在分化培养基(MH+6-BA0.5mg·L^-1+NAA0.05mg·L^-1+蔗糖25g·L^-1+琼脂6g·L^-1) 中预培养2d； 侵染：用OD₆₀₀为0.1的工程菌液进行侵染，时间为3～5min； 共培养：在分化培养基上置暗处共培养2～3d； 选择培养：置选择脱菌培养基(MH+6-BA0.5mg·L^-1+NAA0.05mg·L^-1+40mg·L^-1Km+700mg·L^-1头孢唑林钠+25g·L^-1蔗糖+琼脂6g·L^-1)上进行选择培养；共获得4个卡那霉素抗性芽（T1～T4），经PCR及Southern斑点杂交检测均为阳性，转化率为1.23％。 生根：在选择生根培养基(MH+0.2mg·L^-1IBA+20mg·L^-1Km+300mg·L^-1头孢唑林钠+20g·L^-1蔗糖+6g·L^-1琼脂)上，选择生根率为100％，将4个转基因株系共485株无菌苗移栽至温室，移栽成活率为100％。 （4） 耐盐实验结果表明，各转基因植株的耐盐性较对照均有所提高，T1、T2耐受NaCl最高限为0.75％，T3最高限为0.65％、T4最高限为0.70％，4个转化株系耐盐能力由强到弱依次为：T2、T1、T4、T3。更多还原

【Abstract】 In this study, salt tolerance gene- bet-A (choline dehydrogenase gene ) was transformed into Populus simonii X P. nigra plantlets medted by Agrobacterium tumefaciens ,which obtained from pollen. bet-A can compose betaine only in one step .In plant , betaine is an important osmotic regulator .Under salt stress , accumulation of betaine can protect the structure and function of protein in cell decreasing the flow of water ,and the plant can acquire high salt-tolerant ability .In this paper ,the factors that affect plant genetic transformation were discussed ,and the genetic transformation system of Populus simoniiX P. nigra plantlets was found also .The results of PCR amplification and Southern blotting hybridization show that the betA gene has been integrated into the genome of Populus simoniiX P. nigra .Under salt- stress condition , the transgenic plants have higher salt tolerance ability than those of control plants . The results it as follows:1. The select concentration of Kanamycin was 40mgL-1.2. The optimal concentration of antibiotics and its effects on leaves differentiationof Populus simonii X P. nigra were studied. The results showed that the antibiotics-cefazolin sodium which made in Shang Hai and Harbin were the best alternation and the optimal concentration was 700 mg-L-1.3. The transgenic system of Populus simoniiX P. nigra plantlets obtained from pollen was set up:Pre-culture: the leaves cultured in differentiation medium (MH+6-BA 0.5mg L-1+NAA 0.05 mgL-1+sucrose 25 g L-1+agar 6 g L-1) for 2days ;Dip: dipped the leaves on the Agrobacterium (OD600=0.1) ,the infection time was3~5 minutes ;Co-culture: Co-cultured in the differentiation medium for 2~3 days, Selective culture: the leaves in the select medium(MH+ 6-BA 0.5 mg L-1+ NAA 0.05 mg L-1+Km 40mg L-1+cefazolin sodium 700 mg L-1 + sucrose 25 gL-1+ agar 6 g L-1). We obtained 4 transgenic plants ,and analyzed by Southern blotting and PCR amplification .The results showed that the bet-A has been introduced into the genome of Populus simoniiX P. nigra ,the transformation rate was 1.23% . Rooting: On the select rooting medium (MH+0.2mg L-1 IBA+ Km 20mg L-1 +cefazolin sodium 300 mg L-1+ sucrose 20 g L-1 agar 6 g L-1), all the transgenic plants produce roots. The 485 transgenetic seeding survived in the green house.4. The salt tolerance test showed that all the transgenic plants have high in salt tolerance ability than those control plants .T1 andT2 can survive on the medium which contains 0.75% NaCl . T3 can survive in 0.65% ,T4 can survive in 0.70% NaCLThe sequence of salt tolerance ability from high to low was T2, T1, T4, T3.更多还原