华北农学报 ›› 2025, Vol. 40 ›› Issue (3): 19-26. doi: 10.7668/hbnxb.20195314

所属专题: 抗旱节水 生物技术

• 作物遗传育种·种质资源·生物技术 • 上一篇    下一篇

异源表达苎麻金属硫蛋白BnMLP2基因增强拟南芥对干旱胁迫的耐受性

王立琦, 张蕾, 李茜, 管圣, 卢建宁, 许明志, 崔国贤 , 佘玮   

  1. 湖南农业大学 农学院,苎麻研究所,湖南 长沙 410128
  • 收稿日期:2024-10-23 出版日期:2025-06-28
  • 通讯作者:
    佘 玮(1982—),女,湖南慈利人,教授,博士,主要从事作物抗逆栽培育种研究。
    佘 玮(1982—),女,湖南慈利人,教授,博士,主要从事作物抗逆栽培育种研究。
  • 作者简介:

    王立琦(1999—),男,江苏盐城人,在读硕士,主要从事苎麻分子遗传育种研究。

  • 基金资助:
    湖南省自然科学基金项目(2021JJ30341); 国家自然科学基金项目(31871673); 财政部和农业农村部国家现代农业产业技术体系-麻类产业技术体系(CARS-16-E11)

Heterologous Expression of Ramie Metallothionein BnMLP2 Gene Enhances Arabidopsis Tolerance to Drought Stress

WANG Liqi, ZHANG Lei, LI Xi, GUAN Sheng, LU Jianning, XU Mingzhi, CUI Guoxian , SHE Wei   

  1. College of Agriculture, Ramie Research Institute, Hunan Agricultural University, Changsha 410128, China
  • Received:2024-10-23 Published:2025-06-28

摘要:

为探究BnMLP2基因在苎麻抗旱过程中的作用,通过分析苎麻转录组数据,以中苎1号为材料得到了编码苎麻金属硫蛋白的BnMLP2基因。从苎麻转录组数据中筛选并克隆BnMLP2基因,进行生物信息学分析,包括序列比对、结构域预测及亚细胞定位预测。利用荧光定量PCR技术,测定BnMLP2基因在苎麻不同组织中的表达情况,并探究其在干旱胁迫下的表达变化。将BnMLP2基因导入拟南芥中,构建转基因植株。在干旱胁迫条件下,比较转基因拟南芥与野生型拟南芥的表型、生理生化指标差异,以及抗逆相关基因的表达情况。结果表明,苎麻BnMLP2基因开放阅读框全长共243 bp,编码80个氨基酸。BnMLP2与苹果MT或MLP的氨基酸序列亲缘关系最近,同属于金属硫蛋白家族成员;带有PFAM01439结构域,属于Ⅱ类金属硫蛋白;预测定位于细胞质。BnMLP2基因在苎麻各组织中均表达,并且BnMLP2的表达受干旱显著诱导,尤其在茎中的表达量最高。在干旱胁迫下,转BnMLP2拟南芥相较于野生型表现出更强的抗旱能力,具体表现为根长和鲜质量显著增加,表现出更强的抗氧化酶和γ-GCL活性,积累了更多的脯氨酸(Pro)、还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)、植物螯合态(PCs)来调节细胞内的离子稳态,转基因株系丙二醛(MDA)、过氧化氢(H2O2)的含量显著低于野生型,约为野生型的55%,80%。此外,转基因拟南芥在干旱条件下钠离子和钾离子的含量最高为野生型的4.4,1.4倍。BnMLP2的过表达能诱导3个抗逆相关基因AtMT1aAtNCED3AtWRKY40的表达量提高,最高分别是野生型的1.5,1.9,2.8倍。上述结果表明,BnMLP2基因能够提高拟南芥对干旱胁迫的耐受能力。

关键词: 苎麻, 金属硫蛋白, 干旱胁迫, 耐受性, 异源表达

Abstract:

To investigate the role of the BnMLP2 gene in drought tolerance in ramie,the BnMLP2 gene encoding a metallothionein-like protein in ramie was obtained by analyzing ramie transcriptome data using Zhongzhu-1 as the material.The BnMLP2 gene was screened and cloned from the ramie transcriptome data,and bioinformatics analyses were conducted,including sequence alignment,domain prediction,and subcellular localization prediction.Fluorescent Quantitative PCR was used to determine the expression profile of the BnMLP2 gene in different tissues of ramie and to explore its expression changes under drought stress.The BnMLP2 gene was introduced into Arabidopsis thaliana to construct transgenic plants.Under drought stress conditions,phenotypic,physiological,and biochemical differences between transgenic and wild-type Arabidopsis were compared,along with the expression of stress-related genes.The results showed that the open reading frame of the BnMLP2 gene in ramie was 243 bp in length,encoding 80 amino acids.BnMLP2 had the closest amino acid sequence homology to metallothionein(MT)or metallothionein-like protein (MLP) from Malus domestica,both belonging to the metallothionein family; it contained the PFAM01439 domain and belonged to class Ⅱ metallothionein,with a predicted subcellular localization in the cytoplasm.The BnMLP2 gene was expressed in all tissues of ramie,and its expression was significantly induced by drought,especially in stems.Under drought stress,transgenic Arabidopsis overexpressing BnMLP2 exhibited stronger drought tolerance compared to wild-type plants,as evidenced by significantly increased root length and fresh weight,enhanced antioxidant enzyme and γ-glutamylcysteine ligase (γ-GCL) activities,and accumulation of more proline (Pro),glutathione(GSH),glutathione disulfide (GSSG),and phytochelatins (PCs) to regulate intracellular ion homeostasis.The contents of malondialdehyde (MDA)and hydrogen peroxide (H2O2)in transgenic lines were significantly lower than in wild-type plants,at 55% and 80% of wild-type levels,respectively.In addition, the content of sodium and potassium ions in transgenic Arabidopsis under drought conditions was 4.4 times and 1.4 times higher than that of the wild type, respectively. Overexpression of BnMLP2 induced increased expression of three stress-related genes,AtMT1a,AtNCED3,and AtWRKY40,with maximum expression levels of 1.5,1.9,and 2.8 times those in wild-type plants,respectively.These results suggest that the BnMLP2 gene enhances the tolerance of Arabidopsis to drought stress.

Key words: Boehmeria nivea, Metallothionein, Drought stress, Tolerance, Heterologous expression

中图分类号: 

引用本文

王立琦, 张蕾, 李茜, 管圣, 卢建宁, 许明志, 崔国贤, 佘玮. 异源表达苎麻金属硫蛋白BnMLP2基因增强拟南芥对干旱胁迫的耐受性[J]. 华北农学报, 2025, 40(3): 19-26. doi: 10.7668/hbnxb.20195314.

WANG Liqi, ZHANG Lei, LI Xi, GUAN Sheng, LU Jianning, XU Mingzhi, CUI Guoxian, SHE Wei. Heterologous Expression of Ramie Metallothionein BnMLP2 Gene Enhances Arabidopsis Tolerance to Drought Stress[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 19-26. doi: 10.7668/hbnxb.20195314.