AtNHX1基因对草木樨状黄芪的转化和耐盐性表达研究  

TRANSFORMATION OF ASTRAGALUS MELILOTOIDES PALL WITH AtNHX1 GENE AND THE EXPRESSION OF SALINITY TOLERANCE OF TRANSFORMANTS

作　　者：赵宇玮[1] 步怀宇[1] 郝建国[1] 王英娟[1] 贾敬芬[1] 

机构地区：[1]西北大学生命科学学院

出　　处：《分子细胞生物学报》2008年第3期213-221,共9页Journal of Molecular Cell Biology

基　　金：陕西省教育厅重点科研资助项目(05JS48);陕西省教育厅基金项目(JH06238);陕西省自然科学基金重点项目(2003C108)资助

摘　　要：应用RT-PCR技术从100mmol/LNaCl胁迫处理的拟南芥幼苗中克隆得到编码液泡膜Na+/H+逆向转运蛋白的AtNHX1基因cDNA编码ORF,并在该ORF上游分别插入CaMV 35启动子和TMV RNA5′UTR的Ω片段,而在下游插入NOS polyA构建真核表达盒,进而将该表达盒插入双元植物表达载体pNT质粒的T-DNA区构建了携带AtNHX1基因的植物表达载体质粒pNT-AtNHX1。将pNT-AtNHX1导入农杆菌LBA4404,用农杆菌介导法将AtNHX1基因导入豆科牧草草木樨状黄芪中,共获得103株Kan抗性再生植株。通过对农杆菌菌液浓度、侵染时间和乙酰丁香酮浓度等影响转化效率的因素进行优化,初步建立了稳定的草木樨状黄芪农杆菌转化体系。经过PCR检测、Southern杂交和RT-PCR检测表明,AtNHX1基因已被成功整合到草木樨状黄芪基因组中,并且能够正常转录。野生型和转基因株系诱发的愈伤组织进行耐盐生长实验,结果显示相同盐胁迫条件下,转基因愈伤组织的相对生长率显著高于野生型愈伤组织。施加梯度NaCl胁迫后,植株叶片K+,Na+含量和叶片相对电导率测定结果显示,转基因植物叶片比野生型积累更多的Na+和K+,维持较高的K+/Na+;而转基因株系叶片相对电导率显著低于野生型。上述结果表明,AtNHX1基因的导入和表达在提高草木樨状黄芪耐盐性的同时减轻了盐胁迫对植物细胞膜的伤害。ABSTRACT Using RT-PCR method, the open reading frame （ORF） of AtNHX1-cDNA, encoding the vacuolar Na＋/H＋ antiportor, was cloned from Arabidopsis thaliana seedlings pretreated with 100 mmol/L NaC1 for 24h. This ORF was inserted between CaMV35S promoter, a Ω fragment of TMV RNA 5＇UTR and NOS polyA terminator in the T-DNA region of a binary expression vector pNT （Fig1） . The recombinant plasmid, designated as pNT-AtNHX1, was then transformed into Agrobacterium tumefaciens LBA4404. Mediated by this engineering Agrobacterium, the AtNHX1 gene was transferred into To generation transgenic plant strains of A. melilotoides and 103 regenerated plants resistant to Kanamycin （Kan） were obtained. Some factors influencing the transformation efficiency, such as the concentration and infection duration of Agrobacterium, the concentration of Acetosyringone （AS）, were optimized to establish a stable Agrobacterium mediated gene transformation protocol of A. melilotoides. PCR analysis, Southern blot and RT-PCR detection of some To transgenic plants showed that the A tNHX1 gene was evidently integrated into the genome of transgenic plants and couldl be transcripted properly. Under the same salt stress conditions, the detection of NaC1 resistance revealed the difference between the wild-type calli and the transgenic calli that induced from the transgenic plants, i.e, the relative growth rates of the transgenic calli were remarkably higher than that of the wild-type calli. The K^＋ and Na^＋ contents and relative conductivity in the leaves of the transgenic plants and wild-type plants were estimated. It suggested that under the stress of different concentration of NaC1, K^＋/Na^＋ ratio in the transgenic plant cells were always higher than that in wild-type, however the situation of relative conductivity was on the opposite. From the facts above mentioned, the transformation of A tNHX1 gene not only enhanced the salt tolerance of transgenic A. melilotoides, but also reduced the cell membrane damage induced b

关 键 词：ATNHX1 草木樨状黄芪 农杆菌 遗传转化 耐盐性 

分 类 号：S541.9[农业科学—作物学]