【摘要】 提取酿酒酵母总RNA进行RT-PCR反转录,根据HAL1基因(GeneID:856113)序列设计阅读框引物,对反转录产物进行PCR扩增,并进行全序列测定,构建筛选标记为带内含子的Km抗性基因的植物双元表达载体,经根癌农杆菌介导法将HAL1基因导入烟草品种龙江911和K326中,进行卡那霉素抗性筛选、GUS染色、目的基因扩增和HAL1基因mRNA荧光定量PCR检测,并对T1代各转化烟草材料烟叶含钾量进行化验分析。测序结果证实PCR扩增得到的DNA与酿酒酵母HAL1基因序列相同,获得抗卡那霉素、GUS阳性的转化植株12株。经PCR扩增检测证实HAL1基因已整合入转基因烟草的基因组,荧光定量PCR分析结果表明HAL1基因已在烟苗根系表达,烟叶含钾量化验结果证明HAL1基因可使烟叶含钾量提高30%。更多还原

【Abstract】 A fragment of size 885bp, whose sequence is the same as HAL1 gene (GeneID:856113), was amplified from Saccharomyces cerevisia with RT-PCR methods. HAL1 gene was cloned into the plant binary expression vector containing intron kanamycin gene, and introduced to tobacco varieties LJ911 and K326 by Agrobacterium mediated transformation. 12 transgenic tobacco plants were obtained and approved by PCR, GUS. Results of chamical content assays and realtime PCR analysis confirmed that HAL1 gene was effectively expressed in the modified tobacco root and potassium content in the leaf of transgenic T1 generation was increased by 30%.更多还原