根据NCBI中紫花苜蓿1型金属硫蛋白基因(MET1,登录号:AF189766.1)cDNA序列设计一对特异性引物,以紫花苜蓿品种“农菁1号”的cDNA为模板,利用PCR技术克隆出的一个基因,命名为MsMT1,测序发现该基因全长228 bp,编码75个氨基酸.通过碱基序列比对发现MsMT1与MET1的相似度为99％.通过氨基酸序列分析和进化树分析,发现与其他植物的1型金属硫蛋白基因具有较高的同源性.利用qRT-PCR对MsMT1在苜蓿不同器官中的表达进行分析,发现该基因在根和子叶中表达量较高.将苜蓿幼苗进行不同浓度NaCl、Na2CO3、NaHCO3及不同pH值胁迫处理后,观察MsMT1基因的表达,发现MsMT1的表达量随盐碱处理液浓度和pH值变化发生改变,说明MsMT1与植物的抗逆性相关.采用农杆菌介导法将MsMT1导入苜蓿植株体内,卡那抗性的筛选和Northern blot结果显示MsMT1基因能够在转基因苜蓿中高效表达.用不同浓度NaCl、NaHCO3处理野生型和转化MsMT1基因的苜蓿幼苗,观察幼苗受胁迫后的表型,发现转基因的苜蓿幼苗比未转基因的苜蓿幼苗抵抗力高.本研究表明MsMT1基因能够增加苜蓿对胁迫的抗性.%A gene sequence was obtained from Medicago sativa "Nongjing No.1" cDNA via PCR amplification using specific primers designed from the cDNA sequence of Type 1 Metallothionein gene (MET1,GenBank accession number:AF189766.1) in M.sativa.This gene is assigned as MsMT1.The total length of this gene fragment is 228 bp and it encodes 75 amino acids.The nucleotide sequence of MsMT1 has 99％ homology with MET1.Multiple sequence alignment and the phylogenetic tree showed a high similarity between the MsMT1 and the MT1 in other plants.MsMT1 gene expression was analyzed in different organs of M.sativa by qRT-PCR,with higher quantities found in roots and cotyledons.MsMT1 gene expression was examined in M.sativa seedlings under different concentrations of salt solution and pH values.The results revealed that the expression quantity of MsMT1 gene changed with the saline or alkaline solution concentration,indicating that MsMT1 is associated with stress tolerance.The MsMT1 gene was transformed into M.sativa plant through Agrobacterium tumefaciens-mediated method.The positive transformation was further confirmed by the kanamycin resistance test and the Northern blot test confirmed that MsMT1 was successfully expressed in the transgenic M.sativa.Wild type and transgenic M.sativa seedlings were treated with different concentrations of NaC1 or NaHCO3 which revealed that the transgenic plants had higher stress tolerance than wild type plants.The results of this study highlight the MsMT1 gene can increase plants tolerance to stress.
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