紫花苜蓿1型金属硫蛋白基因的表达及抗逆性分析

doi:10.11686/cyxb2017230

草业学报 ›› 2018, Vol. 27 ›› Issue (4): 89-97.DOI: 10.11686/cyxb2017230

紫花苜蓿1型金属硫蛋白基因的表达及抗逆性分析

徐畅¹,何好¹,李国良²,金淑梅^1,*

1.东北林业大学东北盐碱植被恢复与重建教育部重点实验室,黑龙江哈尔滨 150040;
2.黑龙江省农业科学院玉米研究所,黑龙江哈尔滨 150068

收稿日期:2017-05-08 修回日期:2017-06-14 出版日期:2018-04-20 发布日期:2018-04-20
通讯作者: *, E-mail:jinshumei1972@163.com
作者简介:徐畅(1993-),男,安徽安庆人,在读硕士。E-mail:allenxu1993@126.com
基金资助:
中央高校基本科研业务专项资金项目(2572016EAJ6),东北油田盐碱植被恢复与重建教育部重点实验室开放基金(SAVER1701),国家自然科学基金面上项目(31500317)和国家自然科学基金(31070616)资助

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

XU Chang¹, HE Hao¹, LI Guo-liang², JIN Shu-mei^{1, *}

1.Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, Northeast Forestry University, Harbin 150040, China;
2.Institute of Maize, Heilongjiang Academy of Agricultural Sciences, Harbin 150068, China

Received:2017-05-08 Revised:2017-06-14 Online:2018-04-20 Published:2018-04-20

摘要/Abstract

摘要： 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。

关键词: 紫花苜蓿, 金属硫蛋白, 表达特性, 抗逆性

Abstract: 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 NaCl 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.

Key words: Medicago sativa, metallothionein, expression characterization, stress tolerance

徐畅,何好,李国良,金淑梅. 紫花苜蓿1型金属硫蛋白基因的表达及抗逆性分析[J]. 草业学报, 2018, 27(4): 89-97.

XU Chang, HE Hao, LI Guo-liang, JIN Shu-mei. Expression of type 1 metallothionein gene from Medicago sativa and analysis of its function in stress tolerance[J]. Acta Prataculturae Sinica, 2018, 27(4): 89-97.

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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

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