GA3介导miR171s及其靶基因VvSCLs调控葡萄种子发育的作用分析

doi:10.3864/j.issn.0578-1752.2021.02.011

摘要/Abstract

摘要：

【目的】鉴定vvi-miR171s成员及其靶基因,明确vvi-miR171s成员及其靶基因在葡萄种子发育中的重要作用及其响应赤霉素（Gibberellin,GA）调控种子发育的表达模式。【方法】以‘魏可’葡萄（‘Wink’）果实为试材,利用miR-RACE技术鉴定vvi-miR171a/b/e/f/g/h的成熟体序列;通过PsRNATarget软件预测vvi-miR171s的靶基因,利用生物信息学软件对其进行染色体定位、系统进化树、基因结构、保守结构域及motif分析;通过启动子作用元件分析预测vvi-miR171s及其靶基因的潜在功能;采用RLM-RACE和PPM-RACE验证vvi-miR171s对靶基因的裂解作用;利用qRT-PCR鉴定其应答外源GA₃在葡萄种子区中的表达模式。【结果】从‘魏可’葡萄果实中鉴定并克隆了6条葡萄vvi-miR171s成熟体序列,并用RLM-RACE和PPM-RACE鉴定到3条靶基因VvSCL6/15/22。靶基因的生物信息学分析显示,VvSCL6/22与苹果、桃子和樱桃同源基因遗传距离较近,而VvSCL15则与拟南芥、苹果、桃子和樱桃中的同源基因遗传距离相同,且VvSCLs的基因结构也与进化树中亲缘关系较近的基因相似;SCLs蛋白均具有GRAS结构域,鉴定到5个与GRAS结构域对应的保守motif,且它们含有的motif元件类型及排列顺序均相似,表明SCL家族结构较为保守。vvi-miR171s及靶基因的启动子均具有大量的GA、水杨酸（Salicylic acid,SA）和种子发育相关作用元件,表明它们可能参与响应GA、SA和种子发育过程;qRT-PCR表达分析显示,GA₃强烈抑制了种子区vvi-miR171a的表达,但同时显著上调种子区的VvSCL15和VvSCL22表达;vvi-miR171a和VvSCL15在对照组和GA₃处理后的种子/种子区均呈强烈的负相关,表明GA₃可能在葡萄种子/种子区显著增强vvi-miR171a对VvSCL15的负调控作用,从而介导葡萄种子发育。【结论】在‘魏可’葡萄中鉴定到vvi-miR171a/b/e/f/g/h 6个成员;均可裂解VvSCl6/15/22 3个靶基因;vvi-miR171a-VvSCL15可能作为主要的调控途径响应赤霉素并参与调控葡萄种子发育。

关键词: 葡萄, vvi-miR171s, VvSCLs, 种子发育, 赤霉素

Abstract:

【Objective】The main objective of the present research was to identify the vvi-miR171s and their target genes VvSCL6/15/22 from grapevine genome, and to confirm the role of vvi-miR171s and VvSCL6/15/22 in grape seed development process and their expression pattern in response to exogenous GA₃. 【Method】The mature sequences of vvi-miR171a/b/e/f/g/h were validated by miR-RACE technique from grape (Vitis vinifera L.) cv ‘Wink’. The potential target genes of vvi-miR171s were predicted by PsRNATarget software, and the chromosome localization, phylogenetic, gene structure, conserved domain and motifs analysis were performed by bioinformatics tools. The potential functions of vvi-miR171s and their target genes were predicted by promoters cis-elements analysis. RLM-RACE and PPM-RACE verified the cleavage roles of three target genes by vvi-miR171a/b/e/f/g/h, and the qRT-PCR method was used to detect their temporal expression patterns in grape seed and seedless grape induced by exogenous GA₃. 【Result】Six vvi-miR171s mature sequences in grape were determined and cloned, which were consistent of the sequences of miRBase. Based on this result, the target genes of them, including VvSCL6, VvSCL15 and VvSCL22, were prophesied. The target genes bioinformatics analysis showed evolutionary genetic distance between VvSCL6/22 genes and homologous genes in apple (Monodelphis domestica), peach (Prunus persica), and cherry (Prunus avium) is relatively closed, while VvSCL15 had the same genetic distance from the homologous genes in Arabidopsis, apple, peach and cherry. Meanwhile, the gene structure of VvSCLs was similar to their close genes in phylogenetic tree. All SCL protein sequences had GRAS domain, both element types, arrangement order of motifs. And 5 same motifs for GRAS domain were identified, indicating that the SCL protein structure was relatively conservative, with the conserved functionality. Subsequently, an array of gibberellin and Salicylic acid responsive cis-acting elements were identified in vvi-miR171s and target genes VvSCLs promoter sequences, indicating their possible involvement in the regulation of GA-responsive and SA-responsive grape growth and development. Moreover, the qRT-PCR analysis revealed that the expression of vvi-miR171a was significantly repressed by GA₃ in seed, while its target gene VvSCLs showed reciprocal expression pattern, particularly. There was a strong negative correlation between vvi-miR171a -VvSCL15 in the grape seed of CK and seed region of GA₃treatment. Taken together, vvi-miR171a might the major member of vvi-miR171s in response to GA₃, additionally, it might involve in the grape berry seed development through negative regulating of VvSCL15 gene developmental in response to GA₃. 【Conclusion】vvi-miR171a/b/e/f/g/h were identified from grapevine ‘Wink’, and all of members could cleave three target genes VvSCL6/15/22. The vvi-miR171a-VvSCL15 might be the major member in response to gibberellin and involve in regulating grape seed development.

Key words: grape, vvi-miR171s, VvSCLs, seed development, gibberellin

王文然,解振强,诸葛雅贤,白云赫,管乐,吴伟民,张培安,郑婷,房经贵,王晨. GA₃介导miR171s及其靶基因VvSCLs调控葡萄种子发育的作用分析[J]. 中国农业科学, 2021, 54(2): 357-369.

WANG WenRan,XIE ZhenQiang,ZHUGE YaXian,BAI YunHe,GUAN Le,WU WeiMin,ZHANG PeiAn,ZHENG Ting,FANG JingGui,WANG Chen. Function Analysis of GA₃ Mediate miR171s and Its Target Genes VvSCLs in Grape Seed Development[J]. Scientia Agricultura Sinica, 2021, 54(2): 357-369.

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miRNA成员 miRNA_Acc.	miRNA 成熟体序列 miRNA_mature sequence	靶基因基因编号 Target_Acc.	期望值 Expectation	未配对时的能量UPE	裂解区域 Target region	抑制作用 Inhibition	多重性 Multiplicity
vvi-miR171a	TGATTGAGCCGTGC CAATATC	VIT_204s0023g01380.1(VvSCL15)	0.5	-1	CDS:656-676	裂解 Cleavage	1
		VIT_215s0048g00270.1 (VvSCL6)	0.5	-1	CDS:1594-1614	裂解 Cleavage	1
		VIT_202s0154g00400.1 (VvSCL22)	0.5	-1	CDS:1724-1744	裂解 Cleavage	1
vvi-miR171b	TGATTGAGCCGCGT CAATATC	VIT_204s0023g01380.1 (VvSCL15)	0.5	-1	CDS:656-676	裂解 Cleavage	1
		VIT_215s0048g00270.1 (VvSCL6)	0.5	-1	CDS:1594-1614	裂解 Cleavage	1
		VIT_202s0154g00400.1 (VvSCL22)	0.5	-1	CDS:1724-1744	裂解 Cleavage	1
vvi-miR171e	TGATTGAGCCGCGC CAATATC	VIT_204s0023g01380.1 (VvSCL15)	0	-1	CDS:656-676	裂解 Cleavage	1
		VIT_215s0048g00270.1 (VvSCL6)	0	-1	CDS:1594-1614	裂解 Cleavage	1
		VIT_202s0154g00400.1 (VvSCL22)	0	-1	CDS:1724-1744	裂解 Cleavage	1
vvi-miR171f	TTGAGCCGCGCCAA TATCACT	VIT_215s0048g00270.1 (VvSCL6)	1	-1	CDS:1591-1611	裂解 Cleavage	1
		VIT_202s0154g00400.1 (VvSCL22)	1	-1	CDS:1721-1741	裂解 Cleavage	1
		VIT_204s0023g01380.1 (VvSCL15)	1	-1	CDS:653-673	裂解 Cleavage	1
vvi-miR171g	TTGAGCCGAACCAA TATCACC	VIT_204s0023g01380.1 (VvSCL15)	4	-1	CDS:653-673	翻译 Translation	1
		VIT_215s0048g00270.1 (VvSCL6)	4	-1	CDS:1591-1611	翻译 Translation	1
		VIT_202s0154g00400.1 (VvSCL22)	4	-1	CDS:1721-1741	翻译 Translation	1
vvi-miR171h	TGGTTGAGCCGCGC CAATATC	VIT_204s0023g01380.1 (VvSCL15)	0.5	-1	CDS:656-676	裂解 Cleavage	1
		VIT_215s0048g00270.1 (VvSCL6)	0.5	-1	CDS:1594-1614	裂解 Cleavage	1
		VIT_202s0154g00400.1 (VvSCL22)	0.5	-1	CDS:1724-1744	裂解 Cleavage	1

GA₃介导miR171s及其靶基因VvSCLs调控葡萄种子发育的作用分析

Function Analysis of GA₃ Mediate miR171s and Its Target Genes VvSCLs in Grape Seed Development

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