猕猴桃AcWRKY94基因的克隆及其在盐胁迫下的功能分析

doi:10.3969/j.issn.1004-1524.20240485

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (11): 2501-2509.DOI: 10.3969/j.issn.1004-1524.20240485

猕猴桃AcWRKY94基因的克隆及其在盐胁迫下的功能分析

高憬¹^,²(), 陆玲鸿², 古咸彬², 范飞², 宋根华², 张慧琴²^,^*()

1.浙江师范大学生命科学学院,浙江金华 321004
2.浙江省农业科学院园艺研究所,浙江杭州310021

收稿日期:2024-06-05 出版日期:2024-11-25 发布日期:2024-11-27
作者简介:高憬(1998—),女,江西上饶人,硕士研究生,研究方向为植物学。E-mail: gaojing202406@163.com
通讯作者: *张慧琴,E-mail:zhqzaas@163.com
基金资助:
国家自然科学基金青年科学基金(31801836);浙江省自然科学基金(LY24C150008);浙江省农业(果品)新品种选育重大科技专项(2021C02066-8)

Cloning of AcWRKY94 gene from kiwifruit and its functional analysis under salt stress

GAO Jing¹^,²(), LU Linghong², GU Xianbin², FAN Fei², SONG Genhua², ZHANG Huiqin²^,^*()

1. College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
2. Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2024-06-05 Online:2024-11-25 Published:2024-11-27

摘要/Abstract

摘要：

为明确AcWRKY94在盐胁迫下的功能,从红阳猕猴桃中克隆得到WRKY转录因子AcWRKY94,对AcWRKY94进行生物信息学分析,并分析其在盐处理下的表达水平;利用农杆菌介导的转基因方法在本氏烟中过表达AcWRKY94,并分析转基因烟草的耐盐性。结果显示,AcWRKY94基因的开放阅读框长906 bp,编码301个氨基酸。启动子序列分析显示,AcWRKY94可能响应脱落酸、水杨酸、茉莉酸甲酯、赤霉素、干旱和厌氧等多种信号。多序列比对和系统进化树分析显示,AcWRKY94与多个物种中的同源序列均包含1个WRKYGQK保守结构域和1个C₂HC型锌指结构,属于WRKY Group Ⅲ成员,其中猕猴桃AcWRKY94与茶树中CsWRKY70-like蛋白亲缘关系最近。实时荧光定量PCR(qRT-PCR)分析表明,猕猴桃根中AcWRKY94的表达量受盐处理显著诱导。盐处理后,与野生型烟草相比,过表达AcWRKY94烟草对盐的抗性显著增强,丙二醛(MDA)和H₂O₂含量显著降低,脯氨酸(Pro)含量显著升高,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性显著升高。上述结果表明,AcWRKY94可受盐胁迫诱导,可能参与猕猴桃抗盐调控。

关键词: 猕猴桃, WRKY, 盐胁迫, 功能

Abstract:

In order to clarify the function of AcWRKY94 under salt stress, the WRKY transcription factor AcWRKY94 was cloned from ‘Hongyang’ kiwifruit. Bioinformatics analysis of AcWRKY94 was carried out using relevant websites and biological software. The expression of AcWRKY94 under salt treatment was analyzed. AcWRKY94 was overexpressed in Nicotiana benthamiana by Agrobacterium-mediated transgenic technology, and the salt tolerance of transgenic tobacco plants were analyzed. The results showed that the open reading frame of the AcWRKY94 was 906 bp and encoded 301 amino acids. Promoter sequence analysis revealed that AcWRKY94 might respond to a variety of signals such as abscisic acid, salicylic acid, methyl jasmonate, gibberellin, drought, and anaerobic. Multiple sequence alignment and phylogenetic tree analysis showed that the homologous sequences of AcWRKY94 and several species contain a WRKYGQK conserved domain and a C₂HC zinc finger structure, which belong to WRKY Group III. Among them, the kiwifruit AcWRKY94 was closely related to the CsWRKY70-like proteins of tea plant. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of AcWRKY94 in roots of kiwifruit was significantly induced by salt treatment. After salt treatment, compared with wild type tobacco, the resistance of AcWRKY94-overexpressed line to salt was significantly enhanced, the content of malondialdehyde (MDA) and H₂O₂ decreased significantly, the content of proline (Pro) increased significantly, and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) increased significantly. The above results suggested that AcWRKY94 could be induced by salt stress and might be involved in the regulation of salt resistance in kiwifruit.

Key words: kiwifruit, WRKY, salt stress, function

中图分类号:

S663.4

高憬, 陆玲鸿, 古咸彬, 范飞, 宋根华, 张慧琴. 猕猴桃AcWRKY94基因的克隆及其在盐胁迫下的功能分析[J]. 浙江农业学报, 2024, 36(11): 2501-2509.

GAO Jing, LU Linghong, GU Xianbin, FAN Fei, SONG Genhua, ZHANG Huiqin. Cloning of AcWRKY94 gene from kiwifruit and its functional analysis under salt stress[J]. Acta Agriculturae Zhejiangensis, 2024, 36(11): 2501-2509.

图/表 9

表1 引物序列

Table 1 Sequences of primers

引物名称 Primer name	正向引物序列(5'→3') Forward primer sequence (5'→3')	反向引物序列(5'→3') Reverse primer sequence (5'→3')	用途 Application
AcWRKY94-CDS	CAGTGGTCTCACAACATGGGCATCCTTCGGCCTGA	CAGTGGTCTCATACACTCACCCTCACCAAAGCAAA	基因克隆 Gene cloning
AcWRKY94-qPCR	AAGACCTTGGACGCATGGAT	ATTTGGTGGGGCTCCTCTTG	实时荧光定量PCR qRT-PCR
AcActin	TGGAATGGAAGCTGCAGGA	CACCACTGAGCACAATGTTGC	内参基因Reference gene
AcWRKY94-check	GAAGTGTGCGTGTGATTCGTGT	TGAGATTTTGAGGGTGTTTGTG	转基因苗鉴定
			Identification of transgenic seedling

图1 红阳猕猴桃AcWRKY94基因的PCR扩增

Fig.1 PCR amplification results of AcWRKY94 gene from ‘Hongyang’ kiwifruit

表2 AcWRKY94 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 AcWRKY94

作用元件 Functional element	序列 Sequence	位置 Position	位点功能 Function of site
ABRE	ACGTG	-160、-430、-734、-1005、-1613	参与ABA响应
			Cis-acting element involved in the abscisic acid responsiveness
TCA-element	CCATCTTTTT	-1170	参与SA响应
			Cis-acting element involved in salicylic acid responsiveness
CGTCA-motif	CGTCA	-171、-174、-185	参与MeJA响应
			Cis-acting regulatory element involved in the MeJA-responsiveness
GARE-motif	TCTGTTG	-463	参与赤霉素响应
			Cis-acting element involved in gibberellin-responsiveness
TATC-box	TATCCCA	-490、-1293、-1471	参与赤霉素响应
			Cis-acting element involved in gibberellin-responsiveness
MBS	CAACTG	-526	参与干旱诱导的MYB结合位点
			MYB binding site involved in drought-inducibility
ARE	AAACCA	-790、-1550	参与厌氧诱导
			Cis-acting regulatory element essential for the anaerobic induction
W box	TTGACC	-382	WRKY转录因子结合位点
			WRKY transcription factor binding site

图2 猕猴桃AcWRKY94与其他物种同源蛋白的保守结构域分析

Fig.2 Conserved domain analysis of kiwifruit AcWRKY94 and homologous proteins of other species

图3 猕猴桃AcWRKY94及其他物种同源蛋白的进化树

Fig.3 Phylogenetic tree of kiwifruit AcWRKY94 and homologous proteins from other species

图4 盐处理下猕猴桃叶(A)和根(B)中AcWRKY94的表达模式分析 *和****分别表示在P<0.05和P<0.0001水平差异显著。

Fig.4 Expression pattern analysis of AcWRKY94 in kiwifruit leaves (A) and roots (B) under salt treatment * and **** meant significant differences at the levels of P<0.05 and P<0.0001,respectively.

图5 过表达AcWRKY94烟草的PCR(A)和qRT-PCR(B)鉴定 +表示质粒阳性对照,-表示阴性对照,数字1~4、6、7表示6个转基因阳性株系。WT表示野生型,OE表示过表达株系。***表示在P<0.001水平差异显著。下同。

Fig.5 PCR (A) and qRT-PCR (B) identification of AcWRKY94 overexpressing tobacco +represented a positive plasmid control,-represented a negative control, and the numbers 1 to 4, 6 and 7 represented 6 transgenic positive lines. WT represented the wild type and OE represented the overexpressed line. *** meant significant differences at the level of P<0.001, The same as below.

图6 野生型和过表达AcWRKY94烟草在盐处理下的表型(A)和鲜重(B) *、**分别表示在P<0.05和P<0.01水平差异显著。下同。

Fig.6 Phenotype (A) and fresh weight (B) of wild-type and AcWRKY94-overexpressed tobacco under salt stress * and ** meant significant differences at the levels of P<0.05 and P<0.01, respectively. The same as below.

图7 盐胁迫条件下野生型和过表达AcWRKY94烟草的生理指标分析数据以鲜重计。柱上无相同小写字母表示差异显著(P<0.05)。

Fig.7 Analysis of physiological indices in the wild-type and AcWRKY94-overexpressed tobacco under salt stress Data was detected based on fresh weight. The bars marked without the same lowercase letter indicated significant differences at P<0.05.

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猕猴桃AcWRKY94基因的克隆及其在盐胁迫下的功能分析

Cloning of AcWRKY94 gene from kiwifruit and its functional analysis under salt stress

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