紫花苜蓿MsDWF4的表达特性及耐盐性效应

doi:10.3864/j.issn.0578-1752.2020.18.003

Abstract

Abstract:

【Objective】 Cloning the Brassinolide (BRS) synthetase gene MsDWF4 of alfalfa (Medicago sativa L.), analyzing the gene characteristics and gene expression pattern, performed the salt-tolerance analysis, therefore to clarify the function of MsDWF4 in abiotic stress resistance and provide a reference for revealing the molecular mechanism of MsDWF4 in regulating abiotic stress resistance in alfalfa. 【Method】 The homologous gene of DWF4 in M. sativa was cloned by using homology-based cloning according to the known DWF4 gene sequence of Arabidopsis. Sequence characteristics were analyzed by bioinformatics tools. Quantitative Real-time PCR (qRT-PCR) was applied for detecting the gene expression in different alfalfa tissues and the expression patterns under multiple abiotic stress (high temperature, cold damage, drought and high salt) and hormones treatment (auxin, brassinolide, abscisic acid and jasmonate). The MsDWF4 overexpression vector was constructed and transformed into alfalfa by Agrobacterium mediated transformation, and MsDWF4 overexpression transgenic alfalfa plants were obtained. The transgenic alfalfa lines were treated with high salt (200 mmol·L ^-1 NaCl) and the activities of antioxidant enzymes were analyzed to study whether MsDWF4 could improve the salt tolerance of alfalfa. 【Result】 The length of MsDWF4 CDS was 1 470 bp, which encoded a protein of 489 amino acids, belonging to the single oxygenases of cytochrome P450 family. The MsDWF4 had 67 kinase phosphorylation sites. Sequence and phylogenetic tree analysis show that MsDWF4 protein was most closely related to DWF4 protein of legume plant M. truncatula and had the farthest relationship with Gramineaeis. Tissue-specific expression analysis illustrated that MsDWF4 had the highest expression in the root tip, followed by flowers and leaves. High temperature, cold, PEG, NaCl, ABA and IAA all positively induced the expression of MsDWF4 gene in the alfalfa. After BR treatment, the expression of MsDWF4 was down regulated in shoot and root. The expression of MsDWF4 was inhibited by JA treatment. A 35S∷MsDWF4 overexpression vector was constructed and transformed into alfalfa by Agrobacterium mediated method. PCR identification showed that MsDWF4 gene has been transferred into alfalfa and 6 positive transgenic lines were obtained. Under salt stress, the expression of MsDWF4 gene and the activity of antioxidant enzymes of alfalfa overexpression MsDWF4 gene are higher than that of the control plants. 【Conclusion】 The cDNA sequence of alfalfa brassinolide synthetase gene MsDWF4 was obtained. It was found that the gene expressed the highest in root tip and its expression is responded to a variety of abiotic stress and exogenous hormone treatment. MsDWF4 gene enhanced the resistance to salt stress in transgenic alfalfa. The results showed that MsDWF4 may participate in many kinds of stress response processes and positively regulate the salt tolerance of alfalfa.

Key words: Medicago sativa, brassinosteroids, MsDWF4, abiotic stress, salt resistance

CUI MiaoMiao,MA Lin,ZHANG JinJin,WANG Xiao,PANG YongZhen,WANG XueMin. Gene Expression and Salt-Tolerance Analysis of MsDWF4 Gene from Alfalfa[J].Scientia Agricultura Sinica, 2020, 53(18): 3650-3664.

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References 53

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生物信息学工具 Bioinformatics tools	网址 Website	用途 Purpose
DNAMAN	https://www.lynnon.com/qa.html	多序列比对Multiple sequence alignment
MEGAX	https://www.megasoftware.net/	系统发育进化树的建Establishment of phylogenetic tree
Evolview	https://www.evolgenius.info/evolview	系统发育进化树的美Beautification of phylogenetic tree
ExPASy	https://web.expasy.org/protparam/	蛋白质一级结构分析Protein primary structure analysis
TMHMM2.0Server	http://www.cbs.dtu.dk/services/TMHMM	蛋白跨膜结构域分析Analysis of protein transmembrane domain
ExPASy-Protscale	https://web.expasy.org/protscale/	蛋白亲疏水性分析Protein hydrophobicity analysis
ExPASy-Swiss-model	https://swissmodel.expasy.org/interactive	蛋白三级结构预测Protein tertiary structure prediction
CDD	https://www.ncbi.nlm.nih.gov/Structure/cdd	蛋白保守结构域预测Protein conserved domain prediction
NetPhos3.1Server	http://www.cbs.dtu.dk/services/NetPhos/	蛋白激酶磷酸化修饰位点预测 Prediction of protein kinase phosphorylation modification sites

引物名称 Primer name	引物序列 Primer sequence (5′-3′)
ORF-F	CACCCTATTGGCTTCACA
ORF-R	GAAAGTGGTCCTATTGACA
qDF	AACAAAACATGCCAAACCCAA
qDR	CCATTTCCCAAGTATTCCACCA
Msactin-F	CAAAAGATGGCAGATGCTGAGGAT
Msactin-R	CATGACACCAGTATGACGAGGTCG
pMsDWF4-F	ACGGGGGACGAGCTCGGTACCATGTCTGACTCAGATGTAACTT
pMsDWF4-R	CTTGCTCACCATGTCGACTCTAGATATTATAGAGTGGGCTTGGACTCTAATTTGTAGG
MDF	CCACTGACGTAAGGGATGACG
MDR	TGTCGAAACCGATGATACGAACGAA

Gene Expression and Salt-Tolerance Analysis of MsDWF4 Gene from Alfalfa

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[3]	LIU RuiDa, GE ChangWei, WANG MinXuan, SHEN YanHui, LI PengZhen, CUI ZiQian, LIU RuiHua, SHEN Qian, ZHANG SiPing, LIU ShaoDong, MA HuiJuan, CHEN Jing, ZHANG GuiYin, PANG ChaoYou. Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum [J]. Scientia Agricultura Sinica, 2022, 55(10): 1877-1890.
[4]	ZHAO JingJing,ZHOU Nong,CAO MingYu. Advance on the Methylglyoxal Metabolism in Plants Under Abiotic Stress [J]. Scientia Agricultura Sinica, 2021, 54(8): 1627-1637.
[5]	WANG Xiao,CAI Jian,ZHOU Qin,DAI TingBo,JIANG Dong. Physiological Mechanisms of Abiotic Stress Priming Induced the Crops Stress Tolerance: A Review [J]. Scientia Agricultura Sinica, 2021, 54(11): 2287-2301.
[6]	LIU JiaoJiao,WANG XueMin,MA Lin,CUI MiaoMiao,CAO XiaoYu,ZHAO Wei. Isolation, Identification, and Response to Abiotic Stress of MsWRKY42 Gene from Medicago sativa L. [J]. Scientia Agricultura Sinica, 2020, 53(17): 3455-3466.
[7]	ZHAO JinFeng,DU YanWei,WANG GaoHong,LI YanFang,ZHAO GenYou,WANG ZhenHua,CHENG Kai,WANG YuWen,YU AiLi. Identification NADP-ME Gene of Foxtail Millet and Its Response to Stress [J]. Scientia Agricultura Sinica, 2019, 52(22): 3950-3963.
[8]	DING Lan,GU Bao,LI PeiYing,SHU Xin,ZHANG JianXia. Genome-Wide Identification and Expression Analysis of SAP Family in Grape [J]. Scientia Agricultura Sinica, 2019, 52(14): 2500-2514.
[9]	SONG Xi, PU DingFu, TIAN LuShen, YU QingQing, YANG YuHeng, Dai BingBing, ZHAO ChangBin, HUANG ChengYun, DENG WuMing. Genetic Analysis and Characterization of Hormone Response of Semi-Dwarf Mutant dw-1 in Brasscia napus L. [J]. Scientia Agricultura Sinica, 2019, 52(10): 1667-1677.
[10]	GUO YangDong, ZHANG Lei, LI ShuangTao, CAO YunYun, QI ChuanDong, WANG JinFang. Progresses in Research on Molecular Biology of Abiotic Stress Responses in Vegetable Crops [J]. Scientia Agricultura Sinica, 2018, 51(6): 1167-1181.
[11]	ZHAO QiuFang, MA HaiYang, JIA LiQiang, CHEN Shu, CHEN HongLiang. Genome-Wide Identification and Expression Analysis of SRO Genes Family in Maize [J]. Scientia Agricultura Sinica, 2018, 51(15): 3009-3019.
[12]	DU Hai, RAN Feng, LIU Jing, WEN Jing, MA Shan-shan, KE Yun-zhuo, SUN Li-ping, LI Jia-na. Genome-Wide Expression Analysis of Glucosinolate Biosynthetic Genes in Arabidopsis Across Diverse Tissues and Stresses Induction [J]. Scientia Agricultura Sinica, 2016, 49(15): 2879-2897.
[13]	LIU Zi-cheng, MIAO Li-li, WANG Jing-yi, YANG De-long, MAO Xin-guo, JING Rui-lian. Cloning and Characterization of Transcription Factor TaWRKY35 in Wheat (Triticum aestivum) [J]. Scientia Agricultura Sinica, 2016, 49(12): 2245-2254.
[14]	XUAN Ning, LIU Xu, ZHANG Hua, CHEN Gao, LIU Guo-xia, BIAN Fei, YAO Fang-yin. The Response of a Putative Maize Zinc-Finger Protein Gene ZmAN14 in Transgenic Tabacco to Abiotic Stress [J]. Scientia Agricultura Sinica, 2015, 48(5): 841-850.
[15]	ZHANG Hai-li, YOU Shi-dong, ZHANG Hao, GAO Jing, LI Sheng-hui, ZHANG Li-hui, XING Ji-hong, WANG Feng-ru, DONG Jin-gao. Structure Analysis of SSMP and Its Function in Salt Tolerance in Arabidopsis thaliana [J]. Scientia Agricultura Sinica, 2015, 48(4): 804-812.