‘红地球’葡萄VvARF18功能分析

doi:10.3864/j.issn.0578-1752.2024.07.012

摘要/Abstract

摘要：

【目的】生长素响应因子ARF是生长素信号转导途径中的重要调控因子，在植物生长发育和各类生理过程中发挥着重要作用。分析‘红地球’葡萄VvARF18启动子、异源表达、内源激素含量及其对激素响应的表达，以探究VvARF18在‘红地球’葡萄生长素（IAA）信号转导途径及花芽分化进程中的作用机理。【方法】以设施‘红地球’葡萄花芽为试验材料，通过同源克隆获得VvARF18序列，利用在线数据库PLACE分析启动子的顺式作用元件。以pCAMBIAI2300植物表达载体为基础，通过双酶切和同源重组法构建植物超表达载体pC2300-VvARF18。采用电击法将重组载体pC2300-VvARF18转化至根癌农杆菌GV3101菌株中，以本氏烟草叶片为外植体，通过农杆菌介导的愈伤组织转化法转入烟草中，经PCR检测获得阳性转基因幼苗。利用实时荧光定量PCR（qRT-PCR）对转VvARF18烟草株系表达水平进行分析，筛选出高表达量的转基因株系培养至T₃代，并分别进行IAA和GA₃处理，以分析VvARF18的表达情况。通过酶联免疫法测定转基因烟草花芽和叶片中的IAA、GA、ABA、CTK含量。【结果】‘红地球’葡萄VvARF18位于第13条染色体，含有3个外显子和2个内含子。VvARF18启动子区域存在多种光响应、植物激素响应和逆境响应的顺式作用元件。表型分析发现，转基因烟草的花芽分化进程快于野生型烟草。qRT-PCR结果显示，VvARF18在转基因烟草花芽发育的4个时期中呈先上升后下降的表达趋势，且S3时期表达量达到最高。转基因烟草植株花芽和叶片中IAA、CTK、GA和ABA测定结果表明，转基因烟草花芽和叶片中4种植物激素的含量均高于野生型植株，其中GA/IAA在转基因烟草花芽发育的4个时期中变化趋势与VvARF18表达趋势相一致。转基因烟草植株经IAA和GA₃处理后，VvARF18的表达量随IAA处理浓度的增高而降低，也随GA₃处理时间的延长而降低。【结论】葡萄VvARF18负调控生长素参与植物花芽分化进程，可能与赤霉素信号转导途径中的关键因子相互作用协同调控植物花芽中的激素水平，对植物花芽分化具有促进作用。

关键词: ‘红地球’葡萄, VvARF18, 转基因植株, 花芽分化, 植物激素

Abstract:

【Objective】Auxin response factor (ARF) is a significant regulatory factor in the auxin signaling pathway and plays an important role in plant growth and development as well as various physiological processes. Analysis of the Red Globe grape VvARF18 promoter, heterologous expression, endogenous hormone content and its expression in response to hormones was made in order to explore the mechanism of VvARF18 gene in the auxin (IAA) signaling pathway and flower bud differentiation process in Red Globe grapes. 【Method】The VvARF18 gene sequence was obtained by homologous cloning by using facility Red Globe grape flower buds as experimental materials. The cis-acting elements of the promoter were analyzed using the online database PLACE. The plant overexpression vector pC2300-VvARF18 was constructed based on the pCAMBIAI2300 plant expression vector by double enzyme digestion and homologous recombination method. The recombinant vector pC2300-VvARF18 was transformed into Agrobacterium tumefaciens strain GV3101 by using electrical shock method. The tobacco leaves were used as explants and transferred into tobacco by Agrobacterium-mediated callus transformation method, and positive transgenic seedlings were obtained by PCR. The quantitative real-time PCR (qRT-PCR) was used to analyze the expression level of VvARF18 transgenic tobacco lines, and the transgenic lines with high expression level were screened and cultured to T3 generation, and treated with IAA and GA₃ to analyze the expression level of VvARF18. The content of IAA, GA, ABA and CTK in flower buds and leaves of transgenic tobacco were determined by enzyme-linked immunosorbent assay. 【Result】VvARF18 of Red Globe grape located on chromosome 13, and contained 3 exons and 2 introns. There are multiple cis-acting elements in the VvARF18 promoter region that respond to light, plant hormones, and stress. The phenotypic analysis found that the process of flower bud differentiation was faster in transgenic tobacco than in wild-type tobacco. The qRT-PCR results showed that the expression of VvARF18 showed an increasing and then decreasing trend during the four periods of flower bud development in transgenic tobacco, and the highest expression level was reached in the S3 stage. The results of IAA, CTK, GA and ABA determination in flower buds and leaves of transgenic tobacco plants showed that the content of four plant hormones in flower buds and leaves of transgenic tobacco plants were higher than those of wild-type plants. The change trend of GA/IAA during the four periods of transgenic tobacco flower bud development were consistent with the expression trend of VvARF18. The expression level of VvARF18 in transgenic tobacco plants treated with IAA and GA₃ decreased with the increase of IAA treatment concentration and also decreased with the extension of GA₃ treatment time. 【Conclusion】Grape VvARF18 negatively regulated auxin to participate in the process of plant flower bud differentiation, which could interact with key factors in the gibberellin signaling pathway to synergistically regulate hormone levels in plant flower buds and had a facilitative effect on plant flower bud differentiation.

Key words: Red Globe grape, VvARF18, transgenic plants, flower bud differentiation, phytohormone

袁苗, 周娟, 党仕卓, 汤学燊, 张亚红. ‘红地球’葡萄VvARF18功能分析[J]. 中国农业科学, 2024, 57(7): 1363-1376.

YUAN Miao, ZHOU Juan, DANG ShiZhuo, TANG XueShen, ZHANG YaHong. Functional Analysis of VvARF18 Gene in Red Globe Grape[J]. Scientia Agricultura Sinica, 2024, 57(7): 1363-1376.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.07.012

https://www.chinaagrisci.com/CN/Y2024/V57/I7/1363

图/表 10

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表2

VvARF18启动子顺式作用元件分析"

顺式作用元件 Cis-element	序列 Sequence (5'-3')	功能 Function	数量 Number
TGACG-motif	TGACG	参与MeJA反应的顺式作用调节元件Cis-acting regulatory element involved in the MeJA-responsiveness	1
LTR	CCGAAA	参与低温反应的顺式作用元件 Cis-acting element involved in low-temperature responsiveness	2
CGTCA-motif	CGTCA	参与MeJA反应的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	1
G-Box	CACGTT	参与光响应的顺式调节元件 Cis-acting regulatory element involved in light responsiveness	1
G-box	CACGTC	参与光响应的顺式调节元件 Cis-acting regulatory element involved in light responsiveness	1
MBS	CAACTG	参与干旱诱导MYB结合位点 MYB binding site involved in drought-inducibility	2
CAAT-box	CAAAT	在启动子和增强子区域的共同顺式作用元件 Common cis-acting element in promoter and enhancer regions	12
AE-box	AGAAACAA	光响应模块的一部分 Part of a module for light response	2
TATA-box	TATA	在转录起始的-30 附近的核心启动子元件 Core promoter element around -30 of transcription start	10
AuxRR-core	GGTCCAT	参与生长素反应性的顺式调控元件 Cis-acting regulatory element involved in auxin responsiveness	1
ABRE	ACGTG	参与脱落酸反应的顺式元件 Cis-acting element involved in the abscisic acid responsiveness	2
CCAAT-box	CAACGG	MYBHv1结合位点 MYBHv1 binding site	1
Box 4	ATTAAT	参与光反应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness	7
P-box	CCTTTTG	赤霉素反应元件 Gibberellin responsive element	1
TCCC-motif	TCTCCCT	部分光响应元件 Part of a light responsive element	2
GT1-motif	GGTTAAT	光响应元件 Light responsive element	4
ARE	AAACCA	厌氧诱导所必需的顺式调节元件 Cis-acting regulatory element essential for the anaerobic induction	4

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引物名称 Primer name	引物序列 Primer Sequence (5′-3′)	引物用途 Primer use
Q-VvARF18-F	ATGCTGAACACGCCTATGGGAATG	实时荧光定量 qRT-PCR
Q-VvARF18-R	CACAGTAACGAGGGACGGAGAAAC	实时荧光定量 qRT-PCR
pC2300-VvARF18-F	GAGAACACGGGGGACGAGCTCATGGATCCCATGAAGGAGCTG	过表达载体构建 Overexpression vector construction
pC2300-VvARF18-R	CTTGCTCACCATGGTGTCGACGGGTGTTGCTGCCGAATCG	过表达载体构建 Overexpression vector construction
CaMV35S-F	GCTCCTACAAATGCCATCA	农杆菌检测 Agrobacterium testing
CaMV35S-R	GATAGTGGGATTGTGCGTCA	农杆菌检测 Agrobacterium testing
ACTIN-F	GGCTTACATTGCTCTTGACTATGAAC	烟草的内参基因 Internal reference gene of tobacco
ACTIN-R	ATCAGGCAGCTCGTAGCTCTTCT	烟草的内参基因 Internal reference gene of tobacco