guD基因和SOD2基因植物表达载体构建及gutD基因苜蓿转化体系的优化.docx

2．SOD2基因的植物表达载体的构建通过RT-PCR的方法从裂殖酵母中扩增出了SOD2基因的全长aDNA片段，将其克隆 2．SOD2基因的植物表达载体的构建 通过RT-PCR的方法从裂殖酵母中扩增出了SOD2基因的全长aDNA片段，将其克隆 到pMDl8T—vector中，序列分析表明克隆的片段长度为1404bp，包含了完整的S002 基因的编码序列，其它序列与所报道的序列完全一致。以Xba I和HindlII酶切限 制性内切酶从克隆载体上切取SOD2基因获得具粘性末端的目的片段(SOD2)与植 物表达载体pCAMBIA2300连接，重组质粒通过PCR鉴定正确后，采用直接转化法将 pCAMBIA2300一SOD2质粒导入根癌农杆菌GV3101，并用PCR方法进行了鉴定。 3．gutD基因转化苜蓿的研究 利用农杆菌(Agrobacterium tumefaciens)介导法，将gutD基因转入苜蓿。结果表 明：(1)适宜侵染浓度00600为0．3；(2)用液体分化培养基活化农杆菌，侵染120mira (3)最佳共培养时间3d、共培培养基pH为5．5；(4)400 mg／L头孢霉素可有效抑制 农杆菌；(5)22。C下暗培养60d，延迟lO15d选择时，Kan能对转化体有效筛选。 先用50mg／L Kan筛选两个月，再用去Kan筛选一个月，然后在50mg／L Kan生根培 养基上筛选。 关键词：6一磷酸山梨醇脱氢酶基因(gutD)；SOD2基因；克隆；序列分析；PCR 及酶切检测；苜蓿；遗传转化；根癌农杆菌 SummaryDrought，salinity Summary Drought，salinity and low temperature inhibited the growth of plants in nature， at salt stress，the balance of plant ion will be persecuted，especially in the cytoplasm excessive N式．Plant cells could choose two ways to adapt a coercive environment，one by accumulate soluble organic substances；the other hand through the Na+or separate from，and adjust the K+／Na+rate mechanism to eliminate the poison Na+．Sorbitol contained Six hydroxy has strong hydrophilic,it coulde mainten water effectionly．It is not only merely a middle product of metabolism，but also many other features，such as osmotic adjustment，reduce the cell water potential，carbon and energy storage material， regulation of isozyme and the elimination of hydroxyl radical，It may be stable under conditions of protein structure and function of small molecular weight or may be the molecular chaperone at stress；SOD2 gene is to control the outflow of Nd in yeast，it is important significance in Schizosaccharomyces pombe Pai Na+codingoplasma membrane Na+／H+reversal of transport,and keeping merozoite．balanced on the yeast cells． Alfalfa is also known as purple alfalfa(Medicaosativa)，and it is the legume perennial herbs．Alfalfa is a largest area of cultivation of high—quality forage in 01．117 country,．It is nutrient-rich and strong resist