【摘要】 采用RNAi抑制NHX1基因家族的表达,并观察其对拟南芥耐盐性和耐旱性的影响.根据从拟南芥(Ara-bidopsis thaliana)cDNA中扩增出编码Na+/H+反向转运蛋白基因AtNHX1长度为210 bp高度保守序列作为RNAi的靶标区,并正反2个方向插入载体pHANNIBAL中,2个片段用intron连接;将RNAi表达框连入具有NPTⅡ筛选标记基因的表达载体pART27中,构建以拟南芥NHX1基因家族为靶标的RNAi载体.采用农杆菌介导的真空渗透法转化拟南芥,得到T0代转基因拟南芥种子.对转基因阳性植株进行RT-PCR检测以及耐盐性和耐旱性分析.结果表明,利用本实验构建的NHX1基因家族RNAi载体,拟南芥NHX1基因家族表达被成功地抑制;耐盐和耐旱分析表明RNAi技术对基因表达沉默是有效的.更多还原

【Abstract】 To inhibit the expression of NHX1 gene family with RNAi and explore its effects on salinity resistance and drought resistance of Arabidopsis thaliana.A highly conserved sequence of 210 bp of AtNHX1 gene that encodes a Na+/H+ antiport protein was isolated from A.thaliana,the 210 bp fragment was amplified as RNAi target site and used in construction of RNAi vector,and was inserted into pHANNIBAL as reverted repeat with an intron.The expressional box was linked into the vector of pART27,NPTⅡgene is the screening marker in this vector.Using Agrobacterium tumefaciens-mediated genetic transformation,pART27-NHX1 was introduced into A.thaliana by vacuum infiltration method.T0 transgenic plantlets were confirmed by of analyses RT-PCR,salinity resistance and drought resistance.The results indicated that the RNAi technology could significantly suppress the expression of NHX1 genes.Salinity resistance and drought resistance of transgenic plants indicated that RNAi technology is effective for gene silencing in plants.更多还原