苹果MdWRKY74的克隆和功能分析

doi:10.16420/j.issn.0513-353x.2021-0244

园艺学报 ›› 2022, Vol. 49 ›› Issue (3): 482-492.doi: 10.16420/j.issn.0513-353x.2021-0244

苹果MdWRKY74的克隆和功能分析

相立¹, 赵蕾¹, 王玫¹, 吕毅², 王艳芳³, 沈向¹, 陈学森¹, 尹承苗¹^,^**(), 毛志泉¹^,^**()

¹山东农业大学园艺科学与工程学院,作物生物学国家重点实验室,山东泰安 271018
²威海市农业科学院,山东威海 264200
³山东农业大学化学与材料学院,山东泰安 271018

收稿日期:2021-04-26 修回日期:2021-05-10 出版日期:2022-03-25 发布日期:2022-03-25
通讯作者: 尹承苗,毛志泉 E-mail:yinchengmiao@163.com;mzhiquan@sdau.edu.cn
基金资助:
国家自然科学基金项目(32072510);国家现代农业产业技术体系建设专项资金项目(CARS-27);山东省自然科学基金项目(ZR2020MC131);山东省农业重大应用技术创新项目(SD2019ZZ008);泰山学者项目(ts20190923);山东省高等学校青创科技支持计划项目(2019KJF020);山东省水果创新团队项目(SDAIT-06-07)

Cloning and Functional Analysis of MdWRKY74 in Apple

XIANG Li¹, ZHAO Lei¹, WANG Mei¹, LÜ Yi², WANG Yanfang³, SHEN Xiang¹, CHEN Xuesen¹, YIN Chengmiao¹^,^**(), MAO Zhiquan¹^,^**()

¹State Key Laboratory of Crop Biology,College of Horticulture Science and Engineering,Shandong Agricultural University,Tai’an,Shandong 271018,China
²Weihai Acedemy of Agricultural Sciences,Weihai,Shandong 264200,China
³College of Chemistry and Material Science,Shandong Agricultural University,Tai’an,Shandong 271018,China

Received:2021-04-26 Revised:2021-05-10 Online:2022-03-25 Published:2022-03-25
Contact: YIN Chengmiao,MAO Zhiquan E-mail:yinchengmiao@163.com;mzhiquan@sdau.edu.cn

摘要/Abstract

摘要：

以苹果砧木M9T337（Malling 9 NAKBT337）为试材,克隆了1个WRKY转录因子基因,命名为MdWRKY74（XM_029090147.1）。其开放阅读框为939 bp,编码312个氨基酸,含有1个典型的WRKY结构域。氨基酸序列比对和进化树分析发现MdWRKY74与梨PyWRKY74同源性最高。亚细胞定位结果显示MdWRKY74定位于细胞核。荧光定量PCR分析表明,MdWRKY74在苹果的不同组织中均有表达。在拟南芥中过表达MdWRKY74可提高植株的抗盐性,并能促进SOS1和NHX1的表达。结果表明MdWRKY74可被盐胁迫诱导,可能参与苹果抗盐调控。

关键词: 苹果, MdWRKY74, 亚细胞定位, 盐胁迫, 功能分析

Abstract:

In this study,a WRKY transcription factor gene,named MdWRKY74（XM_029090147.1）was obtained from apple rootstock M9T337（Malling 9 NAKBT337）. The open reading frame of MdWRKY74 was 939 bp,which encoded 312 amino acids,and contained a typical WRKY domain. Amino acid sequence alignment and phylogenetic tree analysis showed that MdWRKY74 had the highest homology with Pyrus bretschneideri WRKY74. Subcellular localization assays showed that MdWRKY74 was located in the nucleus. Quantitative real-time PCR analysis showed that MdWRKY74 was expressed in different apple tissues. Overexpression of MdWRKY74 in Arabidopsis could improve the salt resistance of plants,and promote the expression of SOS1 and NHX1. The results showed that MdWRKY74 can be induced by salt stress,so it may participate in the regulation of salt resistance in apple.

Key words: apple, MdWRKY74, subcellular localization, salt stress, function analysis

中图分类号:

S661.1

相立, 赵蕾, 王玫, 吕毅, 王艳芳, 沈向, 陈学森, 尹承苗, 毛志泉. 苹果MdWRKY74的克隆和功能分析[J]. 园艺学报, 2022, 49(3): 482-492.

XIANG Li, ZHAO Lei, WANG Mei, LÜ Yi, WANG Yanfang, SHEN Xiang, CHEN Xuesen, YIN Chengmiao, MAO Zhiquan. Cloning and Functional Analysis of MdWRKY74 in Apple[J]. Acta Horticulturae Sinica, 2022, 49(3): 482-492.

图/表 10

表1 基因克隆和实时荧光定量所用引物序列

Table 1 Primers used for cloning and qRT-PCR analysis in this study

用途 Use	引物名称 Primer name	序列（5′-3′） Sequence
CDs	MdWRKY74	F：ATGGAGGAGGTTGAGGCAGC;R：CCAGGCCGATTGTGAATGC
qRT-PCR	MdWRKY74	F：GGAAGAGGAAGAGTGAGA;R：GGAAAGTAGGAGGGAGTAA
qRT-PCR	MdActin	F：TGACCGAATGAGCAAGGAAATTACT;R：TACTCAGCTTTGGCAATCCACATC
qRT-PCR	AtSOS1	F：GGAAGAGGAAGAGTGAGA;R：GGAAAGTAGGAGGGAGTAA
qRT-PCR	AtNHX1	F：TCTTGCTATTGGTGCCATAT;R：AGGTGTCTCGTCTTGATTC
qRT-PCR	AtActin	F：CGCTCTTTCTTTCCAAGCTC;R：AACAGCCCTGGGAGCATC
亚细胞定位 Subcellular localization	MdWRKY74	F：CAAGCTTGCATGCCTGCAGGTCGACATGGAGGAGGTTGAGGCAGC; R：CTCGCCCTTGCTCACCATGGATCCCCAGGCCGATTGTGAATGC

图1 苹果砧木‘M9T337’MdWRKY74的PCR扩增

Fig. 1 PCR amplification results of the MdWRKY74 gene in apple rootstock‘M9T337’

图2 MdWRKY74蛋白结构分析

Fig. 2 Protein structure analysis of MdWRKY74

图3 苹果MdWRKY74与其他物种WRKY74蛋白系统进化树分析节点处数字代表亲缘关系的远近。

Fig. 3 Phylogenetic tree analysis of apple MdWRKY74 and other species WRKY74 proteins The numbers at node represent the distance of the kinship.

表2 MdWRKY74基因上游调控序列顺式作用元件分析

Table 2 Analysis of cis-acting regulatory elements in the upstream regulatory sequences of MdWRKY74 gene

作用元件 Functional element	序列 Sequence	位点功能 Function of site	位置 Location
ABRE	ACGTG	参与ABA响应 cis-acting element involved in the abscisic acid responsiveness	+ 1 530
CGTCA-motif	CGTCA	参与MeJA响应 cis-acting regulatory element involved in the MeJA-responsiveness	-215
LTR	CCGAAA	低温响应 cis-acting element involved in low-temperature responsiveness	+ 485
MBS	CAACTG	与干旱诱导相关的MYB绑定位点 MYB binding site involved in drought-inducibility	-175
TC-rich repeats	ATTTTCTTCA	参与防御反应和胁迫响应 cis-acting element involved in defense and stress responsiveness	+ 927

图4 MdWRKY74亚细胞定位

Fig. 4 The subcellular localization of MdWRKY74

图5 MdWRKY74的组织表达（A）和在NaCl处理下根系中的表达（B） ** α = 0.01.

Fig. 5 Expression of MdWRKY74 in different tissues（A）and expression in root under NaCl treated（B）

图6 NaCl处理条件下野生型（WT）和过表达MdWRKY74（OE）拟南芥形态（A）和qRT-PCR检测基因的表达量（B） **α = 0.01.

Fig. 6 The Arabidopsis thaliana that overexpressing MdWRKY74 treated with NaCl（A）,and qRT-PCR detection of MdWRKY74 expression in transgenic Arabidopsis（B）

图7 NaCl处理条件下野生型（WT）和过表达MdWRKY74（OE）拟南芥相关生理指标 *α = 0.05;**α = 0.01.

Fig. 7 Related physiological indexes of overexpressing MdWRKY74（OE）Arabidopsis and wile type（WT）treated with NaCl

图8 野生型（WT）和过表达MdWRKY74（OE）拟南芥相关基因表达水平分析 **α = 0.01.

Fig. 8 The expression level analysis of related gene of overexpressing MdWRKY74（OE）Arabidopsis and wile type（WT）

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苹果MdWRKY74的克隆和功能分析

Cloning and Functional Analysis of MdWRKY74 in Apple

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苹果MdWRKY74的克隆和功能分析

Cloning and Functional Analysis of MdWRKY74 in Apple

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