紫花苜蓿1型金属硫蛋白基因的表达及抗逆性分析

Expression of type 1 metallothionein gene from Medicago sativa and analysis of its function in stress tolerance

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228bp,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、Na_2CO_3、NaHCO_3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、NaHCO_3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(MET 1,GenBank accession number:AF189766.1)in M.sativa.This gene is assigned as MsMT 1.The total length of this gene fragment is 228 bp and it encodes 75 amino acids.The nucleotide sequence of MsMT 1 has 99%homology with MET 1.Multiple sequence alignment and the phylogenetic tree showed a high similarity between the MsMT 1 and the MT 1 in other plants.MsMT 1 gene expression was analyzed in different organs of M.sativa by qRT-PCR,with higher quantities found in roots and cotyledons.MsMT 1 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 MsMT 1 gene changed with the saline or alkaline solution concentration,indicating that MsMT 1 is associated with stress tolerance.The MsMT 1 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 MsMT 1 was successfully expressed in the transgenic M.sativa.Wild type and transgenic M.sativa seedlings were treated with different concentrations of NaCl or NaHCO 3 which revealed that the transgenic plants had higher stress tolerance than wild type plants.The results of this study highlight the MsMT 1 gene can increase plants tolerance to stress.