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

doi:10.7668/hbnxb.20195314

Abstract

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

CLC Number:

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.

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Figures/Tables 8

Tab.1 Primers used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	引物用途 Usage or purpose
HTP-F(855)	GGAGGGCGAAGAATCTCGTG	转基因拟南芥筛选
HTP-R(855)	ACTTCTACACAGCCATCGGT
AtMT1a-F	TCCTGCAAATGTGGTGACTCT	拟南芥qPCR
AtMT1a-R	ACCCACAGCTGCAGTTTGAT
AtMT2a-F	AATTTGGGAGCTTCCTCTATCATT
AtMT2a-R	CAGGGTACATTTTGCAACCTCC
AtMT3a-F	AATTTGGGAGCTTCCTCTATCATT
AtMT3a-R	CAGGGTACATTTTGCAACCTCC
AtNCED3-F	TGTCCTGTCTGAAATCCGCC
AtNCED3-R	TTACGGCCGAGCATGTTTCT
AtWRKY40-F	TGGCTTAAACCGCCACATCT
AtWRKY40-R	ATTCTTGACGTTGGGCTCGT
ACTIN8-F	TCAGCACTTTCCAGCAGATG	拟南芥内参基因
ACTIN8-R	CTGTGGACAATGCCTGGAC
BnMLP2-F	ACTCTCATCATCGGTGTCGC	苎麻qPCR
BnMLP2-R	GGGTCGCAGGAACAGCTATC
Actin-F	GCTCCGTTGAACCCTAAG	苎麻内参基因
Actin-R	GCTCCGATTGTGATGATTT
35S-BnMLP2-EcoR ⅠF	CAAGAGACAGGATCCGAATTCGAAAATGTCTTGCTGCGGAGG	BnMLP2克隆
35S-BnMLP2-Sal ⅠR	CATCGGTGCACTAGTGTCGACTTTACAAGTGCAGGGGTCGCA

Fig.1 Comparison of homology between BnMLP2 amino acid sequences in ramie and MT amino acid sequences in other plants Black.100% sequence homology;Pink.Sequence homology greater than 75%;Blue.Sequence homology greater than 50%.

Fig.2 Phylogenetic tree of BnMLP2 and other plant MLP proteins

Fig.3 Expression of BnMLP2 in different organs of ramie under drought stress Different lowercase letters within the same treatments indicate significant differences at the 0.05 level.The same as Fig.4—5,7.

Fig.4 Phenotypic and morphological indicators of wild-type and BnMLP2 transgenic Arabidopsis under drought stress

Fig.5 Antioxidant and stress-related physiological indicators of overexpressing BnMLP2 in Arabidopsis thaliana under drought stress

Fig.6 Contents of sodium and potassium in Arabidopsis with overexpression of BnMLP2 gene under drought stress Different lowercase letters indicate significant differences between different genotypes(P<0.05).

Fig.7 Expression levels of genes related to stress response in Arabidopsis with overexpression of BnMLP2 gene under drought stress

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