过量表达紫花苜蓿MsHB7基因对拟南芥耐旱性的影响

doi:10.11686/cyxb2020181

草业学报 ›› 2021, Vol. 30 ›› Issue (4): 170-179.DOI: 10.11686/cyxb2020181

过量表达紫花苜蓿MsHB7基因对拟南芥耐旱性的影响

候怡谣¹^,²(), 李霄², 龙瑞才², 杨青川¹^,², 康俊梅², 郭长虹¹()

^1.哈尔滨师范大学生命科学与技术学院，黑龙江省分子细胞与遗传育种重点实验室，黑龙江哈尔滨 150000
^2.中国农业科学院北京畜牧兽医研究所，北京 100193

收稿日期:2020-04-21 修回日期:2020-05-25 出版日期:2021-04-20 发布日期:2021-03-16
通讯作者: 郭长虹
作者简介:Corresponding author. E-mail: kaku3008@126.com
候怡谣（1994-），女，山西临汾人，硕士。E-mail: yyhou66@163.com
基金资助:
国家牧草产业技术体系项目(CARS-34);农业农村部牧草种质资源与育种重点实验室开放运行费项目(2019-YWF-ZX-08)

Effect of overexpression of the alfalfa MsHB7 gene on drought tolerance of Arabidopsis

Yi-yao HOU¹^,²(), Xiao LI², Rui-cai LONG², Qing-chuan YANG¹^,², Jun-mei KANG², Chang-hong GUO¹()

^1.Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province，College of Life Science and Technology，Harbin Normal University，Harbin 150000，China
^2.Institute of Animal Science，Chinese Academic of Agriculture Science，Beijing 100193，China

Received:2020-04-21 Revised:2020-05-25 Online:2021-04-20 Published:2021-03-16
Contact: Chang-hong GUO

摘要/Abstract

摘要：

同源异型域-亮氨酸拉链蛋白（HD-Zip）第I类亚家族在植物非生物胁迫调控过程中起着重要作用，已在多个物种中进行了克隆鉴定，但关于紫花苜蓿该家族基因的研究还鲜有报道。本研究旨在研究紫花苜蓿HD-Zip第I类亚家族基因MsHB7对拟南芥抗旱性的调控功能。通过克隆得到大小为738 bp、编码245个氨基酸的MsHB7基因的开放阅读框。多重序列比对和系统进化树分析结果显示，MsHB7蛋白属于HD-Zip I亚家族，且与拟南芥中ATHB7和ATHB12亲缘关系较近。实时荧光定量分析表明，MsHB7基因受干旱诱导。将MsHB7基因转化拟南芥并获得了阳性植株。干旱处理后，转基因拟南芥比野生型拟南芥萎蔫程度更明显，转基因植株相对含水量显著低于野生型拟南芥，并积累了更多的脯氨酸和丙二醛。qRT-PCR检测发现处理之后逆境胁迫指示基因ATCAT1、 ATDREB2A和ATRD29A在转基因拟南芥中的表达量显著升高，而ATLEA3的表达量显著下降。上述结果表明MsHB7基因的过表达可降低转基因拟南芥的耐旱性，为进一步开发利用该基因提供理论依据。

关键词: 紫花苜蓿, 同源异型域-亮氨酸拉链蛋白, 干旱胁迫, 转基因拟南芥

Abstract:

The homeodomain-leucine zipper （HD-Zip） protein subfamily I plays an important role in the regulation of plant abiotic stress. There are few reports of HD-Zip I genes in alfalfa， although they have been cloned and identified in many species. The purpose of this study was to investigate the role of MsHB7， a HD-Zip I gene from alfalfa， in the regulation of Arabidopsis thaliana drought resistance. The MsHB7 gene with an open reading frame of 738 bp and encoding 245 amino acids was obtained by cloning. Multiple sequence alignment and phylogenetic tree analysis showed that MsHB7 belonged to the HD-Zip I subfamily and was closely related to ATHB7 and ATHB12 in A. thaliana. Real-time fluorescence quantitative analysis showed that the MsHB7 gene was induced by drought. The MsHB7 gene was transformed into A. thaliana and positive plants were obtained. After drought treatment， the transgenic A. thaliana wilted more obviously. The relative water content of transgenic plants was significantly lower than that of wild-type A. thaliana， and the proline and malondialdehyde levels accumulated in transgenic plants were more than wild-type. qRT-PCR results showed that the expression levels of stress response genes ATCAT1， ATDREB2A and ATRD29A in the transgenic A. thaliana were significantly increased， while the expression levels of ATLEA3 were significantly decreased after stress. The above results indicate that the overexpression of MsHB7 gene can reduce the drought tolerance of transgenic A. thaliana， which provides theoretical information as a basis for the further development and utilization of this gene.

Key words: alfalfa, homeodomain-leucine zipper, drought stress, transgenic Arabidopsis

候怡谣, 李霄, 龙瑞才, 杨青川, 康俊梅, 郭长虹. 过量表达紫花苜蓿MsHB7基因对拟南芥耐旱性的影响[J]. 草业学报, 2021, 30(4): 170-179.

Yi-yao HOU, Xiao LI, Rui-cai LONG, Qing-chuan YANG, Jun-mei KANG, Chang-hong GUO. Effect of overexpression of the alfalfa MsHB7 gene on drought tolerance of Arabidopsis[J]. Acta Prataculturae Sinica, 2021, 30(4): 170-179.

图/表 9

表1 引物列表

Table 1 Primer list

引物Primer	引物Sequence (5'-3')
MsHB7F	CAAAACTTAGGCCTTAGCCATATAT
MsHB7R	GCAACATAGAAGAACATGGTGCA
qMsHB7F	ATGAGGGTTTGGAGGATAAAATCGT
qMsHB7R	CAAGTCCAAAAATCCAACCATTGAG
qMsactin2F	CAAAAGATGGCAGATGCTGAGGAT
qMsactin2R	CATGCACCAGTATGACGAGGTCG
pMsHB7F	TGCTCTAGAATGATGGAGGAAGAAGAG
pMsHB7R	ACGGGATCCTCAAGTCCAAAAATCC
MsHB7F1	CGACACACTTGTCTACTCCAAAAAT
MsHB7R1	TTCAAGTCCAAAAATCCAACCATTG
qMsHB7F1	TGGAGCCAAGGAAGAAGATGC
qMsHB7R1	CCATCATGCGATGTTTCCACC
qATactin7F	AGCTAGAGACAGCCAAGAGC
qATactin7R	GCTTCCATTCCGATGAGCGA
qATCAT1F	CGCCATGCCGAAAAATACCC
qATCAT1R	CTTGCCTGTCTGAATCCCAGGAC
qATDREB2AF	CTGGAGAATGGTGCGGAAGA
qATDREB2AR	CAGATAGCGAATCCTGCTGTTGT
qATLEA3F	GATTGACCCGGCTGAGCTACGA
qATLEA3R	AGATGGGATTCACCACAAAAGA
qATRD29AF	GATATCGACAAGGATGTGCCG
qATRD29AR	GTATCCAGGTCTTCCCTTCGC

图 1 MsHB7与拟南芥中一些同源异型域-亮氨酸拉链蛋白进化树分析ATHB7: NP_182191.1; ATHB12: NP_191748.1; ATHB20: NP_186771.1; ATHB5: NP_201334.1; ATHB6: NP_565536.1; PHV: NP_174337.1; ATHB8: NP_195014.1; ATHB15: NP_849795.1; PHB: NP_181018.1; REV: NP_200877.1; ATHB4: NP_182018.1; HAT3: NP_191598.1; HAT1: NP_193476.1; ATHB2: NP_193411.1; ATHB17: NP_178252.2; FWA: NP_567722.1; GL2: NP_001185443.1; PDF2: NP_567274.1; ANL2: NP_567183.2; HDG1: NP_191674.1.

Fig.1 Phylogenetic tree analysis of MsHB7 and some homeodomain-leucine zipper proteins from A. thaliana

图 2 MsHB7与其他植物中同源异型域-亮氨酸拉链蛋白的氨基酸序列比对分析A: 同源异型框结构域序列比对 Sequence alignment of homeobox domain; B: 亮氨酸拉链结构域序列比对，6个保守的亮氨酸残基用“L”表示 Sequence alignment of leucine zipper domain, the six conserved leucine residues are represented by “L”. XP_003602321.2: 蒺藜苜蓿 M. truncatula ATHB-12; XP_004502719.1: 鹰嘴豆 Cicer arietinum ATHB-12-like; XP_017420762.1: 小豆 Vigna angularis ATHB-12-like; XP_016695089.1: 陆地棉 Gossypium hirsutum ATHB-12-like; XP_018842791.1: 核桃 Juglans regia ATHB-12-like; XP_002320889.1: 毛果杨Populus trichocarpa ATHB-7; XP_018729630.1: 巨桉 Eucalyptus grandis ATHB-12-like; XP_007218213.1: 桃树 Prunus persica ATHB-12; XP_012088620.1: 麻疯树 Jatropha curcas ATHB-12; NP_001281285.1: 苹果 Malus domestica ATHB-12-like; XP_016538252.1: 辣椒 Capsicum annuum ATHB-12-like; NP_001311821.1 ATHB-12-like: 烟草 Nicotiana tabacum ATHB-12-like; XP_006491341.1: 橙 Citrus sinensis ATHB-7; XP_003548207.2: 大豆 Glycine max ATHB-12; XP_004230017.1: 番茄 Solanum lycopersicum ATHB-12; NP_191748.1: 拟南芥 A. thaliana ATHB-12.

Fig.2 The amino acid sequence alignment analysis between MsHB7 and homeodomain-leucine zipper proteins from other plants

图3 紫花苜蓿在PEG处理下MsHB7基因的相对表达量分析*和**分别表示同一组织中不同时间点与0 h相比差异显著（P<0.05）和极显著（P<0.01）。* and ** respectively indicate that the difference between different time points in the same organization and 0 h is significant (P<0.05) and extremely significant (P<0.01).

Fig.3 Analysis of relative expression of MsHB7 gene in alfalfa treated with PEG

图4 MsHB7转基因拟南芥的PCR检测M: DNA分子量标准DNA marker; 0: 水ddH2O; -: 阴性对照Negative control; +: 阳性对照Positive control.

Fig.4 PCR identification of transgenic A. thaliana with MsHB7

图5 转基因拟南芥中MsHB7的相对表达水平*表示转基因拟南芥与WT相比差异显著（P<0.05）。* indicates that the difference between transgenic A. thaliana and WT is significant (P<0.05).

Fig.5 The relative expression level of MsHB7 in transgenic A. thaliana

图6 干旱胁迫下拟南芥表型分析

Fig.6 Phenotypic analysis of A. thaliana under drought stress

图7 干旱胁迫下拟南芥生理指标分析*表示转基因拟南芥与WT相比差异显著（P<0.05），**表示转基因拟南芥与WT相比差异极显著（P<0.01）。下同。* indicates that the difference between transgenic A. thaliana and WT is significant (P<0.05); ** indicates that the difference between transgenic A. thaliana and WT is extremely significant (P<0.01). The same below.

Fig.7 Analysis of physiological indexes of A. thaliana under drought stress

图 8 干旱胁迫下拟南芥抗性相关基因的表达分析

Fig.8 Expression analysis of resistance related genes in A. thaliana under drought stress

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过量表达紫花苜蓿MsHB7基因对拟南芥耐旱性的影响

Effect of overexpression of the alfalfa MsHB7 gene on drought tolerance of Arabidopsis

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