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VvSCl6/15/223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。
【Objective】The main objective of the present research was to identify the vvi-miR171 s and their target genes VvSCL6/15/22 from grapevine genome,and to confirm the role of vvi-miR171 s and VvSCL6/15/22 in grape seed development process and their expression pattern in response to exogenous GA3.【Method】The mature sequences of vvi-miR171 a/b/e/f/g/h were validated by mi R-RACE technique from grape(Vitis vinifera L.)cv’Wink’.The potential target genes of vvi-miR171 s 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-miR171 s 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-miR171 a/b/e/f/g/h,and the q RT-PCR method was used to detect their temporal expression patterns in grape seed and seedless grape induced by exogenous GA3.【Result】Six vvi-miR171 s 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,Vv SCL15 and Vv SCL22,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-miR171 s 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-miR171 a 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-miR171 a-Vv SCL15 in the grape seed of CK and seed region of GA3 treatment.Taken together,vvi-miR171 a might the major member of vvi-miR171 s 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-miR171 a/b/e/f/g/h were identified from grapevine’Wink’,and all of members could cleave three target genes VvSCL6/15/22.The vvi-miR171 a-VvSCL15 might be the major member in response to gibberellin and involve in regulating grape seed development.
作者
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
WANG WenRan;XIE ZhenQiang;ZHUGE YaXian;BAI YunHe;GUAN Le;WU WeiMin;ZHANG PeiAn;ZHENG Ting;FANG JingGui;WANG Chen(College of Horticulture,Nanjing Agricultural University,Nanjing 210095;Jiangsu Vocational College of Agriculture and Forestry,Zhenjiang 212499,Jiangsu;Insititute of Horticulture,Jiangsu Academy of Agricultural Sciences,Nanjing 210014)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第2期357-369,共13页
Scientia Agricultura Sinica
基金
国家自然科学基金(31972373)
江苏省自然科学基金(BK20181318)
句容市农业课题(JRNW[2018]03号)。
