黄花苜蓿MfWRKY22的克隆及抗逆性功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0930

摘要/Abstract

摘要：

目的探究MfWRKY22转录因子在黄花苜蓿（Medicago falcata）响应盐、干旱和低温胁迫中的作用，为进一步解析MfWRKY22调节植物适应非生物胁迫的分子机理提供理论依据。方法基于课题组前期盐、干旱胁迫黄花苜蓿转录组数据，利用RT-PCR和基因组步移技术分别克隆MfWRKY22基因及其启动子，并进行生物信息学、基因表达、亚细胞定位和转录激活分析，同时创制OE-MfWRKY22转基因拟南芥植株，在盐、干旱和低温处理10 d时观测植株的鲜重、根长和侧根数。结果 MfWRKY22 CDS长度为1 017 bp，编码338个氨基酸，分子量为37.05 kD，理论等电点为5.65，定位于细胞核，具有转录激活活性，属于Group Ⅱ亚家族。获得MfWRKY22启动子区1 888 bp序列，其含有响应盐胁迫、干旱胁迫、渗透胁迫、激素应答以及W-box顺式作用元件。MfWRKY22在根、茎和叶中均有表达，但在叶中的表达量最高，且盐、干旱和低温胁迫均可诱导其表达。盐、干旱和低温胁迫下，OE-MfWRKY22转基因拟南芥植株幼苗的鲜重、根长和侧根数均显著高于野生型植株。结论 MfWRKY22属于WRKY转录因子Group Ⅱ亚家族成员，其表达受盐、干旱、低温胁迫诱导，且在盐、干旱以及低温等非生物胁迫下，超表达MfWRKY22可促进转基因幼苗生长。

关键词: 黄花苜蓿, MfWRKY22, 非生物胁迫, 功能分析

Abstract:

Objective To explore the role of MfWRKY22 transcription factor in response to salt, drought and low temperature stresses in Medicago falcata, and to provide a theoretical basis for further investigating the molecular mechanism of MfWRKY22 in M. falcata responding to the abiotic stresses. Method Based on the RNA-Seq data of M. falcata treated with salt and drought, MfWRKY22 gene and its promoter sequence were cloned respectively by RT-PCR and Genome Walker method, and the bioinformatics, gene expression, subcellular localization and transcriptional activation activity analysis were performed. Meanwhile, the OE-MfWRKY22 transgenic Arabidopsis lines were constructed, and the fresh weight, root length and lateral root number of transgenic Arabidopsis seedlings were determined respectively at the day 10 of salt, drought and low temperature stresses treatments. Result The CDS of MfWRKY22 is 1 017 bp, encoding a protein with 338 amino acids, with predicted molecular mass of 37.05 kD and an isoelectric point of 5.65. MfWRKY22, localized in the nucleus and with transcriptional activation activity, is a Group Ⅱ member. A 1 888 bp sequence of MfWRKY22 promoter was obtained, and the cis-acting elements, such as salt stress induction, drought responsive, osmotic stress responsive, ethylene responsive and W-box, were identified. Meanwhile, it was found that the MfWRKY22 gene had the highest expression in the leaves of M. falcata, and the expressions of MfWRKY22 gene in the roots, stems and leaves were up-regulated after salt, drought and low temperature treatment. Under salt, drought and low temperature stresses, the fresh weight, root length and lateral root number of transgenic Arabidopsis seedlings were higher than that of wild seedlings. Conclusion MfWRKY22, whose expression is up-regulated by salt, drought and low temperature stresses,is a Group Ⅱ WRKY transcription factor member, and the overexpressing MfWRKY22 could improve the growth of transgenic plants under abiotic stresses.

Key words: Medicago falcata, MfWRKY22, abiotic stresses, function analysis

赵静玮, 常娜, 金宵溦, 张立全. 黄花苜蓿MfWRKY22的克隆及抗逆性功能分析[J]. 生物技术通报, 2026, 42(7): 236-245.

ZHAO Jing-wei, CHANG Na, JIN Xiao-wei, ZHANG Li-quan. Cloning and Function Analysis of MfWRKY22 Gene in Medicago falcata[J]. Biotechnology Bulletin, 2026, 42(7): 236-245.

图/表 10

表1 MfWRKY22启动子顺式作用元件

Table 1 Cis-acting elements in MfWRKY22 promoter

顺式作用元件 Cis-acting element	序列 Sequence	位置 Site	功能 Function
ERE	TATGAAAT	-1 731；-1 299	乙烯应答元件 Ethylene response element
MYB	TAACCA	-1 364；-311；-252	参与干旱响应顺式作用元件 Cis-acting element involved in drought-responsiveness
TGACG-motif	TGACG	-840；-543	参与茉莉酸甲酯反应顺式作用元件 Cis-acting regulatory element involved in the MeJA-responsiveness
STRE	AGGGG	-623；-584	渗透压胁迫应答元件 Osmotic stress-responsive elements
GT1-motif	GGTTAA	-1 626	参与盐胁迫诱导 Involvement in salt-stress induction
W-box	TTGACC	-1 616；-599；-236	WRKY结合位点 Binding site of WRKY

图1 MfWRKY22蛋白序列分析A：MfWRKY22蛋白与不同物种WRKY22s序列比对分析；B：蛋白进化树分析；C：蛋白二级结构预测；D：蛋白保守结构域分析；E：蛋白三级结构预测

Fig. 1 Analysis of MfWRKY22 protein sequenceA： Sequence alignment and analysis of protein MfWRKY22 with WRKY22s from different plant species. B： Phylogenetic tree analysis of protein. C： Prediction of the secondary structure. D： Prediction of protein conserved domain. E： Prediction of the tertiary structure

图2 MfWRKY22启动子序列

Fig. 2 Promoter sequence of MfWRKY22

图3 MfWRKY22的组织表达模式分析

Fig. 3 Tissue-specific expression pattern analysis of MfWRKY22

图4 MfWRKY22的表达分析A：盐胁迫条件下的相对表达量； B：干旱胁迫条件下的相对表达量； C：4 ℃低温胁迫条件下的相对表达量。*，**和***分别代表在P<0.05， P<0.01， P<0.001水平差异显著

Fig. 4 Expression analysis of MfWRKY22A: Relative expression under salt stress. B: Relative expression under drought stress. C: Relative expression under 4 ℃. *, ** and *** indicate significant difference at P<0.05, P<0.01, and P<0.001, respectively

图5 MfWRKY22亚细胞定位分析Bar=20 µm

Fig. 5 Subcellular localization analysis of MfWRKY22

图6 MfWRKY22转录激活分析

Fig. 6 Transcriptional activation analysis of MfWRKY22

图7 超表达MfWRKY22植株鉴定不同字母表示在P<0.01水平差异显著

Fig. 7 Identification of transgenic lines overexpressing MfWRKY22Different letters indicate significant difference at P<0.01

图8 胁迫条件下，超表达MfWRKY22植株的生长情况

Fig. 8 Growth situation of overexpressing MfWRKY22 lines under stresses

图9 胁迫对超表达MfWRKY22植株的生长影响不同字母表示在P<0.01水平差异显著

Fig. 9 Effects of stresses on the growths of overexpressing MfWRKY22 linesDifferent letters indicate significant difference at P<0.01

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