玉米生长素响应因子基因<i>ZmARF10</i>在干旱胁迫中的功能分析

doi:10.7668/hbnxb.20194560

摘要/Abstract

摘要：

生长素响应因子(ARF)是一类具有B3结构域的转录因子,是调节生长素应答反应、控制基因表达的直接分子。前期通过分析转录组数据,在玉米中筛选到1个编码ARF蛋白并积极参与干旱-复水胁迫响应的基因ZmARF10。为进一步研究该基因调控玉米抗旱性的分子机制及抗旱分子育种提供新的思路,首先通过系列生物信息学分析软件对该基因进行生物信息学分析,之后利用实时荧光定量PCR(qRT-PCR)检测ZmARF10在玉米不同组织中的表达模式,分析在高温、干旱、高盐、ABA胁迫和解除胁迫处理下的表达情况,以及在不同玉米自交系间的表达差异,最后利用CRISPR/Cas9技术分析ZmARF10的功能。结果表明,ZmARF10位于玉米的第3号染色体,编码区全长2 127 bp,编码708个氨基酸,具有典型的B3结构域;该基因ATG上游2 kb区域内含有茉莉酸甲酯、生长素、脱落酸、低温响应等应答元件;系统发育树显示,ZmARF10基因编码的蛋白质与高粱同源蛋白的亲缘关系最近。qRT-PCR结果表明,ZmARF10是组成型表达基因,在玉米成熟根中的表达量最高;在高温、干旱、高盐以及ABA 等4种胁迫处理下,ZmARF10基因的表达量均显著上调,干旱胁迫后上调倍数最高达8.2倍;干旱胁迫后,ZmARF10基因在抗旱自交系郑36中的表达量上升幅度显著高于干旱敏感自交系B73。观察拟南芥野生型与ARF10缺失型突变体发现,与野生型相比,干旱条件下突变体植株出现叶片萎蔫甚至干死的现象,根系发生卷曲,根系分支数减少,侧根生长发育受阻;测定生理生化指标发现,干旱胁迫后缺失型突变体的相对含水量、叶绿素含量、净光合速率显著低于野生型植株,说明敲除ARF10基因后拟南芥抗旱能力下降。

关键词: 玉米, 生长素响应因子, 非生物胁迫, 抗旱性, 表达模式

Abstract:

Auxin response factor(ARF)is a class of transcription factors with B3 domain,which is a direct molecule regulating auxin response and controlling gene expression.A gene,ZmARF10,which encoded ARF protein and actively participated in drought-rehydration stress response,was previously screened in maize by analyzed transcriptome data.To further research the molecular mechanism of ZmARF10 regulating drought resistance of maize,and also provide a new idea for molecular breeding of drought resistance,the gene was firstly analyzed by bioinformatics software.Secondly,Quantitative Real-time polymerase chain reaction(qRT-PCR)was used to detect the expression pattern of ZmARF10 in different tissues,under high temperature,drought,high salt,ABA and restoring treatments,and in different inbred lines.Finally,the function of ZmARF10 was analyzed using CRISPR/Cas9 technology.The results showed that ZmARF10 was located on chromosome 3 of maize,with a total length of 2 127 bp,and encoded 708 amino acids with a typical B3 domain.The upstream 2 kb region of ATG of this gene contained response elements related to methyl jasmonate,auxin,abscisic acid and low temperature.The phylogenetic tree showed that the protein encoded by the ZmARF10 gene was closely related to sorghum.qRT-PCR results showed that ZmARF10 was a constitutive expression gene,and the expression level of ZmARF10 was the highest in mature corn roots of maize.Under high temperature,drought,high salt and ABA treatments,the expression of this gene was significantly up-regulated,and the up-regulation ratio was up to 8.2 times after drought stress.After drought stress,the expression level of ZmARF10 gene was significantly higher in the drought-resistant inbred line Zheng 36 than that in the drought-sensitive inbred line B73.Investigation of Arabidopsis wild type and ARF10-deficient mutants showed that,compared with wild type,the mutant plants showed leaf wilting and even dry death,roots curled,root branch number decreased,and lateral root growth and development were hindered under drought conditions.Determination of physiological and biochemical indexes showed that the relative water content,chlorophyll content and net photosynthetic rate of the deficient mutants were significantly lower than those of the wild-type plants after drought stress,indicating that the drought resistance of Arabidopsis decreased after ARF10 gene was knocked out.

Key words: Maize, Auxin response factor, Abiotic stress, Drought tolerance, Expression pattern

曹丽茹, 叶飞宇, 李伟亚, 马晨晨, 庞芸芸, 梁小菡, 张新, 鲁晓民. 玉米生长素响应因子基因ZmARF10在干旱胁迫中的功能分析[J]. 华北农学报, 2024, 39(2): 1-10. doi: 10.7668/hbnxb.20194560.

CAO Liru, YE Feiyu, LI Weiya, MA Chenchen, PANG Yunyun, LIANG Xiaohan, ZHANG Xin, LU Xiaomin. Functional Analysis of Maize Auxin Response Factor Gene ZmARF10 under Drought Stress[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 1-10. doi: 10.7668/hbnxb.20194560.

http://www.hbnxb.net/CN/Y2024/V39/I2/1

图/表 11

图1 干旱-复水后ZmARF10基因表达水平的RNA-seq和qRT-PCR分析不同小写字母表示差异显著(P<0.05)。图4—7同。

Fig.1 Expression of ZmARF10 gene analyzed by RNA-seq and qRT-PCR after drought and rehydration Different letters indicate significant difference(P<0.05).The same as Fig.4—7.

图2 ZmARF10基因结构(A)与编码蛋白质的保守结构域(B)示意 A.方块代表外显子区域,折线代表内含子区域;B.方块代表保守结构域。

Fig.2 Diagram of ZmARF10 gene structure(A) and conserved domain(B)of the encoded protein A.The squares represent the exon regions,and the broken lines represent the intron regions;B.The squares represents the conservative domain.

表1 ZmARF10蛋白质的氨基酸组成

Tab.1 Amino acid composition of ZmARF10 protein

氨基酸类型 Amino acid type	类型比例/% Type percentage	氨基酸 Amino acid	氨基酸比例/% Amino acid percentage
疏水性氨基酸	38.9	Ala (A)	6.5
Hydrophobic		Ile (I)	2.8
amino acids		Leu (L)	5.8
		Met (M)	3.0
		Phe (F)	4.7
		Pro (P)	7.9
		Trp (W)	1.8
		Val (V)	6.4
亲水性氨基酸	48.4	Asp (D)	5.2
Hydrophilic		Cys (C)	1.1
amino acids		Gln (Q)	7.1
		Glu (E)	6.2
		Gly (G)	6.1
		Ser (S)	10.6
		Thr (T)	6.1
		Tyr (Y)	2.0
		Asn (N)	4.0

表2 ZmARF10蛋白质的亚细胞定位预测

Tab.2 Subcellular localization prediction of ZmARF10 protein

亚细胞结构 Destination of cell compartment	可信度 Certainty
细胞核 Nucleus	0.500
细胞质 Cytosol	0.214
叶绿体Chloroplast	0.051
线粒体Mitochondrion	0.071
液胞膜Vacuolar membrane	0.063
过氧化物酶体Peroxisome	0.071

图3 ZmARF10蛋白质进化树(A)及保守基序(B)分析不同数字的方块代表不同motif,方块的大小代表motif的长度。

Fig.3 Evolutionary tree(A)and conserved motifs(B)analysis of ZmARF10 protein The squares of different number represent different motifs,and the size of squares represents the length of the motifs.

表3 ZmARF10基因启动子顺式作用元件分析

Tab.3 Cis-element analysis of ZmARF10 gene promoter

核心元件 Core element	位点个数 Number of site	来源 Source	功能注释 Function annotation
TGACG-motif	6	大麦	参与MeJA反应的顺式作用调控元件
CGTCA-motif	6	大麦	参与MeJA反应的顺式作用调控元件
G-box	1	拟南芥、豌豆	参与光响应的顺式调节元件
ABRE	1	拟南芥	参与脱落酸反应的顺式元件
TGA-element	1	甘蓝	生长素响应元件
TCA-element	1	烟草	参与水杨酸反应的顺式作用元件
LTR	1	大麦	参与低温反应的顺式元件

图4 ZmARF10基因在玉米不同组织中的表达水平

Fig.4 The expression of ZmARF10 gene in different tissues of maize

图5 ZmARF10基因响应非生物胁迫的表达模式分析 R.解除胁迫。图6同。

Fig.5 Expression pattern of ZmARF10 gene in response to abiotic stress R.Restoring.The same as Fig.6.

图6 干旱胁迫下ZmARF10基因在不同自交系间的表达模式分析

Fig.6 Expression pattern of ZmARF10 gene in different inbred lines under drought stress

图7 拟南芥arf10突变体在干旱胁迫下的表型(A、B)和生理指标(C—E)

Fig.7 Seedling phenotypes(A,B)and physiological indices(C—E)of Arabidopsis arf10 mutants under drought stress

图8 ZmARF10的亚细胞定位分析

Fig.8 Subcellular localization of ZmARF10

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玉米生长素响应因子基因ZmARF10在干旱胁迫中的功能分析

Functional Analysis of Maize Auxin Response Factor Gene ZmARF10 under Drought Stress

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玉米生长素响应因子基因ZmARF10在干旱胁迫中的功能分析

Functional Analysis of Maize Auxin Response Factor Gene ZmARF10 under Drought Stress

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