VvmiR164s-VvNAC100作用模块鉴定及其在葡萄子房发育过程中响应赤霉素的表达分析

doi:10.3864/j.issn.0578-1752.2023.10.012

摘要/Abstract

摘要：

【目的】鉴定葡萄miR164s（VvmiR164a/b/c/d）及其靶基因，明确VvmiR164s及其靶基因应答外源赤霉素在葡萄单性结实过程中的调控作用。【方法】以‘魏可’葡萄（Vitis vinifera L. Wink）为试材，利用miR-RACE、PCR、RLM-RACE与PPM-RACE、qRT-PCR及生物信息学等技术，分析VvmiR164s-VvNAC100模块应答外源赤霉素在葡萄单性结实过程中的时空表达特征及其潜在功能。【结果】赤霉素在花前处理‘魏可’葡萄，可诱导其单性结实，导致葡萄的无核。克隆鉴定了VvmiR164a/b/c/d的精确序列，预测到其4条靶基因VvNAC100-1、VvNAC100-2、VvNAC098、VvNAC021，结合匹配程度与前期数据，在此重点分析靶基因VvNAC100-2，并将其命名为VvNAC100。克隆鉴定靶基因VvNAC100，验证其裂解位点，并对其开展蛋白进化、染色体定位及结构分析。VvNAC100裂解位点位于miRNA 5′端的第9位与第11位，裂解频度为17/20与11/20，定位于葡萄的Chr14上，编码363个氨基酸，含有一个NAM结构域，且其定位于细胞核。VvNAC100蛋白与其他物种氨基酸序列保守性较高，功能相似，其中与辣椒、烟草等物种亲缘关系较近。VvMIR164a/b/c/d的启动子与其靶基因VvNAC100的启动子均包含多种激素作用元件，表明其可能通过响应不同的激素来参与葡萄生长发育的调控。RT-qPCR结果显示，随着葡萄子房的发育，VvmiR164b的表达水平呈下降趋势，其靶基因VvNAC100在子房发育前期呈上升的表达趋势，具有一定的负相关，而VvmiR164a/c/d与VvNAC100表达模式相似，呈现一定的正相关；GA处理后，VvmiR164a/c/d在葡萄子房单性结实过程中的表达极显著地上升，进而也显著抑制了VvNAC100在这一时期的表达，从而促进了VvmiR164a/c/d-VvNAC100表达水平的负相关；但VvmiR164b在GA处理后表现出下降的趋势，且其与VvNAC100的表达水平呈现一定的正相关，表明GA处理增强了VvmiR164a/c/d对VvNAC100的负调控，减弱了VvmiR164b对VvNAC100的负调控作用。【结论】VvNAC100是VvmiR164 家族VvmiR164a/b/c/d的真实靶基因；在葡萄单性结实过程中，赤霉素诱导了VvmiR164a/c/d对靶基因VvNAC100的负调控作用，抑制了VvmiR164b对靶基因VvNAC100的负调控作用，VvmiR164a/c/d是VvmiR164家族参与赤霉素诱导葡萄单性结实的主效因子。

关键词: 葡萄, 赤霉素, 单性结实, VvmiR164s, VvNAC

Abstract:

【Objective】The aim of this study was to identify grapevine miR164s (VvmiR164a/b/c/d) and their target genes, and to elucidate the regulatory roles of VvmiR164s and their target genes during exogenous GA-induced grape parthenocarpic process.【Method】Using ‘Wink’ grapes (Vitis vinifera L. Wink) as the test material, miR-RACE, PCR, RLM-RACE and PPM-RACE, qRT-PCR and bioinformatics were used to analyze the spatio-temporal expression of VvmiR164s-VvNAC100 module in response to exogenous GA and its potential functions during grape parthenocarpic process.【Result】Gibberellin application on ‘Wink’ grapes before flowering induced parthenocarpy, leading to seedless berries. The precise sequence of VvmiR164a/b/c/d was cloned and identified, and four of its target genes were predicted, including VvNAC100-1, VvNAC100-2, VvNAC098, and VvNAC021. Combining the degree of match with the comprehensive analysis of the previous data, this work focused on the analysis of the target gene VvNAC100-2, which was named as VvNAC100. The VvNAC100 cleavage site was located at position 9 and position 11 of the 5′ end of miRNA, with a cleavage frequency of 17/20 and 11/20, respectively, and was localized on Chr14, encoding 363 amino acids and containing a NAM. The VvNAC100 protein is highly conserved in amino acid sequence and functionally similar to other species, among which it is more closely related to species such as pepper and tobacco. The promoters of VvMIR164a/b/c/d and its target gene VvNAC100 both contain multiple hormone-acting elements, suggesting that it might be involved in the regulation of grape growth and development by responding to mult-hormones. The RT-qPCR results showed that the expression level of VvmiR164b tended to decrease as the grape ovary developed, while its target gene VvNAC100 showed an increasing expression trend in the early stage of ovary development with a certain negative correlation. On the other hand, VvmiR164a/c/d showed a similar expression pattern with VvNAC100 with a certain positive correlation. However, after GA treatment, the expression of VvmiR164a/c/d was highly significantly increased during parthenocarpy in the grape ovary, which also significantly suppressed the expression of VvNAC100 during this period, promoting a negative correlation between VvmiR164a/c/d-VvNAC100 their expression levels. VvmiR164b showed a decreasing trend after GA treatment, and it’s the expression levels showed some positive correlation with those of VvNAC100, indicating that GA treatment promoted the negative regulation of VvmiR164a/c/d on VvNAC100, but repressed the negative regulation of VvmiR164b on VvNAC100.【Conclusion】VvNAC100 was the true target gene of VvmiR164 family. During grape parthenocarpy, GA induced the negative regulation of VvmiR164a/c/d on the target gene VvNAC100 and suppressed that of VvmiR164b on the target gene VvNAC100. VvmiR164a/c/d were the main effectors of the VvmiR164 family involved in modulation of GA-induced grape parthenocarpy.

Key words: grape, gibberellin, parthenocarpy, VvmiR164, VvNAC

王霏, 肖迎珂, 宣旭娴, 张晓雯, 刘菲, 查紫仙, 代梦瞳, 王西成, 吴伟民, 房经贵, 王晨. VvmiR164s-VvNAC100作用模块鉴定及其在葡萄子房发育过程中响应赤霉素的表达分析[J]. 中国农业科学, 2023, 56(10): 1966-1981.

WANG Fei, XIAO YingKe, XUAN XuXian, ZHANG XiaoWen, LIU Fei, ZHA ZiXian, DAI MengTong, WANG XiCheng, WU WeiMin, FANG JingGui, WANG Chen. Identification of the VvmiR164s-VvNAC100 Action Module and Analysis of Their Expressions Responsive to Exogenous GA During Grape Ovary Development[J]. Scientia Agricultura Sinica, 2023, 56(10): 1966-1981.

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基因名称 Gene name	正向引物序列 Forward primer sequence	反向引物序列 Reverse primer sequence	用途 Application
VvmiR164aQT	GATGAATTGCCTACAGTTACCAG	CCAAGGCCTCCTGCAATTT	VvmiR164a前体序列的扩增 Amplification of VvmiR164a precursor
VvmiR164bQT	GATGGTCAGCTGCCTAATATTG	ACACGCATCTTGGATTTGCT	VvmiR164b前体序列的扩增 Amplification of VvmiR164b precursor
VvmiR164cQT	CTTCATTTGCCGCTTTCG	CTCCACCAAGAAAATGATAGGC	VvmiR164c前体序列的扩增 Amplification of VvmiR164c precursor
VvmiR164dQT	GCAATCACTTGGGAGTTGG	ATAGCCAATAGAGGGGAAAAGG	VvmiR164d前体序列的扩增 Amplification of VvmiR164d precursor
VvNAC100	ATGGAAAAGGCTCCTGACCA	GGAATGTTTCCCTTCTAATCTGT	VvNAC100靶全长序列的扩增 Amplification of full-length sequences of VvNAC100
VvNAC100p	ATGGAAAAGGCTCCTGACC	GTAATTCCAGAGGCAATCAATG	VvNAC100靶片段序列的扩增 Amplification of VvNAC100 target fragment sequences
VvmiR164a-qRT	TCAAAACCCGAAACAAGTCC	ACCCACCCTCAACCATACAA	VvmiR164a定量RT-PCR引物 Real-time PCR of VvmiR164a
VvmiR164b-qRT	CGAGTGTTGAGCAAGATGGA	CTCCGTGATAATTGGGGAGA	VvmiR164b定量RT-PCR引物 Real-time PCR of VvmiR164b
VvmiR164c-qRT	TTGGGGGTGTGAAGAAGAAG	GAGGAACCCATGTTGGAGAA	VvmiR164c 定量RT-PCR引物 Real-time PCR of VvmiR164c
VvmiR164d-qRT	TTGGGGGTGTGAAGAAGAAG	GAGGAACCCATGTTGGAGAA	VvmiR164d定量RT-PCR引物 Real-time PCR of VvmiR164d
VvNAC100-qRT	TTCTCATCCCTGACCAGTCC	TCTACAGGCCCAGCTGAAGT	VvNAC100定量RT-PCR引物 Real-time PCR of VvNAC100
Vvactin	GCTCGCTGTTTTGCAGTTCTAC	AACATAGGTGAGGCCGCACTT	葡萄actin内参基因 Actin internal reference primer

miRNAs	MiRNA 成熟体长度 Length of miRNA	靶基因编号 Target_Acc.	错配率Expectation	靶区 Target region	靶基因匹配初始位置 Target_start	靶基因匹配初始位置 Target_end	切割方式 Interaction mode inhibition
VvmiR164a/c/d (UGGAGAAGCAGGGCACGUGCA)	21	VIT_217s0000g06400 (VvNAC100-1)	0.5	gene=VIT_217s0000g06400 CDS=732-1814	1375	1395	裂解 Cleavage
	21	VIT_219s0014g02200 (VvNAC098)	1.5	gene=VIT_219s0014g02200 CDS=1105-2196	1826	1846	裂解 Cleavage
	21	VIT_219s0027g00230 (VvNAC021)	1.8	gene=VIT_219s0027g00230 CDS=317-1219	939	959	裂解 Cleavage
	21	VIT_214s0108g01070(VvNAC100-2)	1.5	gene=VIT_214s0108g01070 CDS=348-1439	988	1008	裂解 Cleavage
VvmiR164b (UGGAGAAGCAGGGCACAUGCU)	21	VIT_217s0000g06400(VvNAC100-1)	1.0	gene=VIT_217s0000g06400 CDS=732-1814	1375	1395	裂解 Cleavage
	21	VIT_219s0014g02200 (VvNAC098)	2.5	gene=VIT_219s0014g02200 CDS=1105-2196	1826	1846	裂解 Cleavage
	21	VIT_219s0027g00230 (VvNAC021)	1.8	gene=VIT_219s0027g00230 CDS=317-1219	939	959	裂解 Cleavage
	21	VIT_214s0108g01070(VvNAC100-2)	2.5	gene=VIT_214s0108g01070 CDS=348-1439	988	1008	裂解 Cleavage