山葡萄VaJAZ9和VaCOI1基因的抗寒功能研究

doi:10.16420/j.issn.0513-353x.2023-0910

摘要/Abstract

摘要：

以山葡萄（Vitis amurensis Rupr.）‘左山-1’为试材，克隆了VaCOI1基因并对其功能进行分析。结果表明，VaCOI1基因位于13号染色体，其ORF全长1 797 bp，编码598个氨基酸，属于F-box家族基因；聚类分析表明VaCOI1蛋白与锦葵HuCOI1和可可TcCOI1同源性较高。双分子荧光互补实验表明VaJAZ9与VaCOI1蛋白具有互作关系。对异源过表达VaJAZ9和VaCOI1拟南芥株系进行低温处理，过表达VaCOI1拟南芥的成活率高于野生型，电导率低于野生型，而过表达VaJAZ9拟南芥的成活率低于野生型，电导率高于野生型。实时荧光定量PCR分析表明，过表达VaCOI1拟南芥中与低温相关的基因AtKIN1、AtCOR15A及AtRD29A表达量上调，而过表达VaJAZ9植株中AtKIN1、AtCOR15A及AtRD29A表达量下调。综上所述，VaCOI1在低温胁迫反应中起正调控的作用，而VaJAZ9起负调控的作用。

关键词: 山葡萄, 茉莉酸信号途径, JAZ9, COI1, 抗寒

Abstract:

In this study，the VaCOI1 gene from Vitis amurensis Rupr.‘Zuoshan-1’were cloned，and the gene function was analysed. The results show that the VaCOI1 gene is located on chromosome 13，and its ORF has a full-length of 1 797 bp，encoding 598 amino acids，and belongs to the F-box family. Clustering analysis indicates that the VaCOI1 protein performed a high homology with HuCOI1（Herrania umbratica）and TcCOI1（Theobroma cacao）. Bimolecular fluorescence complementation（BiFC）assays indicate that VaJAZ9 can interact with the VaCOI1 protein. The heterologously overexpressing of VaJAZ9 and VaCOI1 in Arabidopsis thaliana shows that at the low temperature，the VaCOI1 overexpressing plants had a higher survival rate and a lower conductivity than the wild type plants，whereas the VaJAZ9 overexpressing plants had a lower survival rate and a higher conductivity than the wild type plants. Quantitative real-time PCR（qRT-PCR）analysis show that the low temperature related genes including AtKIN1，AtCOR15A and AtRD29A were up-regulated in the VaCOI1 overexpressing A. thaliana，whereas these genes were down-regulated in overexpressing VaJAZ9 plants. In conclusion，the results show that the VaCOI1 acts as a positive regulator in response to low temperature stress，whereas the VaJAZ9 acts as a negative regulator.

Key words: Vitis amurensis, JA signaling pathway, JAZ9, COI1, cold resistance

孙雨桐, 刘德帅, 黄栩筝, 迟敬楠, 齐迅, 姚文孔. 山葡萄VaJAZ9和VaCOI1基因的抗寒功能研究[J]. 园艺学报, 2024, 51(10): 2243-2254.

SUN Yutong, LIU Deshuai, HUANG Xuzheng, CHI Jingnan, QI Xun, YAO Wenkong. Study on the Cold Resistance Function of VaJAZ9 and VaCOI1 Genes in Vitis amurensis[J]. Acta Horticulturae Sinica, 2024, 51(10): 2243-2254.

图/表 8

表1 各种载体构建及RT-PCR的相关引物

Table 1 PCR primers for vector construction and RT-PCR

用途 Application	引物 Primer	引物序列（5′-3′） Primer sequence
构建载体	COI1-2300-F	AGAACACGGGGGACGAGCTCATGGAGGATGGAAACGAGAGAAAG
Vector construction	COI1-2300-R	ACCATGGTGTCGACTCTAGACAACGTAAGAAAAGATGCAGGGTC
	JAZ9-2300-F	ACGAGCTCGGTACCCGGGGATCCATGTCGAGCTCCTCGGATATTGC
	JAZ9-2300-R	CCCTTGCTCACCATGGTGTCGACCTTTGGAGATTTGGCAGCCAAGC
BiFC验证	221-CE-JAZ9-XbaI-F	GAGAACACGGGGGACTCTAGAATGTCGAGCTCCTCGGATATTGC
BiFC verification	221-CE-JAZ9-XhoI-R	GTACATGGTACCCCGCTCGAGCTTTGGAGATTTGGCAGCCAAGC
	221-NE-COI1-BamHI-F	GGCCCAGGCCTACTAGTGGATCCATGGAGGATGGAAACGAGAGAAAG
	221-NE-COI1-XhoI-R	TACCCGGGAGCGGTACCCTCGAGCTACAACGTAAGAAAAGATGCAGGGTC
过表达植株检测	COI1-F	ATGGAGGATGGAAACGAGAGAAAG
Detection of	COI1-R	CTGCTCATCAGCACCTCTCTC
overexpressed plant	JAZ9--F	ATGTCGAGCTCCTCGGATATTGC
	JAZ9-R	CACGATCACTTGGCCACCGTAG
实时荧光定量PCR	RT-COI1-F	GCTTGCTTGACACAGAGGACCA
Real-time	RT-COI1-R	CCAATCACGTTCCTGGCCTCAA
Quantitative PCR	RT-AtActin-F	CTTGCACCAAGCAGCATGAA
	RT-AtActin-R	CCGATCCAGACACTGTACTTCCTT
	AtRT-CBF1-F	ACTTCGCTGACTCGGCTTGG
	AtRT-CBF1-R	ACGCACCTTCGCTCTGTTCC
	AtRT-CBF2-F	GGAATCAACCTGTGCCAAGGAA
	AtRT-CBF2-R	CCAACATCGCCTCTTCATCCATAT
	AtRT-CBF3-F	TTCCTCAGGCGGTGATTATATTCC
	AtRT-CBF3-R	CTCCGACGAACTCCTCTGTATATTG
	RT-RD29A-F	CAGAGGAACCACCACTCAACACA
	RT-RD29A-R	CTCTAGGTTTACCTGTTACGCCTG
	RT-RD29B-F	ATGGAGTCACAGTTGACACGTCCT
	RT-RD29B-R	CTTCTGGGTCTTGCTCGTCATACT
	RT-KIN1-F	ATGTCAGAGACCAACAAGAATGCC
	RT-KIN1-R	CTACTTGTTCAGGCCGGTCTTG
	RT-AtCOR15A-F	AGTCGTTGATCTACGCCGCTAA
	RT-AtCOR15A-R	TCTCACCATCTGCTAATGCCTCTT

图1 山葡萄‘左山-1’和欧洲葡萄‘霞多丽’COI1基因受0 ℃低温诱导表达比较 *和**表示同期品种间差异显著（* P < 0.05，** P < 0.01）。

Fig. 1 Comparisons of low temperature induced expression of‘Zuoshan-1’and‘Chardonnay’COI1 genes at 0 ℃ The asterisk（* and **）represent significant differences compared to the control for the same period（* P < 0.05，** P < 0.01）.

图2 VaCOI1基因生物信息分析 A：VaCOI1染色体定位；B：聚类分析。

Fig. 2 Bioinformatics analysis of the VaCOI1 A：VaCOI1 chromosome localization analysis；B：Clustering analysis.

图3 BiFC验证VaCOI1和VaJAZ9互作

Fig. 3 BiFC assay validates the interaction between VaCOI1 and VaJAZ9

图4 VaCOI1（A）与VaJAZ9（B）过表达拟南芥株系的鉴定 M：DNA marker。泳道1：阴性对照，泳道2：阳性对照，其余为拟南芥阳性幼苗的PCR条带。

Fig. 4 Identification of heterologous expression Arabidopsis thaliana of VaCOI1（A）and VaJAZ9（B） M：DNA marker. Lane 1：Negative control，Lane 2：Positive control，the rests are PCR bands of positive seedlings of Arabidopsis thaliana.

图5 VaCOI1过表达拟南芥的耐寒性分析 A：在基质中过表达拟南芥播种14 d后-6 ℃处理6 h，室温恢复4 d；B：在培养基中-6和-8 ℃处理植株；C、D：-6和-8 ℃处理幼苗的电导率和存活率。WT：野生型。OE：过表达拟南芥株系。** P < 0.01。下同。

Fig. 5 Analysis of cold overexpression VaCOI1 in Arabidopsis thaliana A：Heterologous expression of Arabidopsis sown in substrate for 14 d after-6 ℃ treatment for 6 h；B：In medium，plants treated with -6 and-8 ℃；C and D：Conductivity and survival of plants with medium at-6 and-8 ℃. WT：Wild type. OE：Overexpression of Arabidopsis thaliana. ** P < 0.01. Tge same below.

图6 VaJAZ9过表达拟南芥的耐寒性分析

Fig. 6 Analysis the cold resistance of the VaJAZ29 gene in Arabidopsis thaliana

图7 过表达VaJAZ9和VaCOI1拟南芥中抗寒相关基因的表达量分析

Fig. 7 The qRT-PCR analysis of cold resistance related genes in heterologously expressing Arabidopsis thaliana * P < 0.05；** P < 0.01.

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