VvmiR397a及其靶基因VvLACs在葡萄果实发育中的作用分析

doi:10.16420/j.issn.0513-353x.2018-0059

摘要/Abstract

摘要： 以‘魏可’葡萄（Vitis vinifera‘Wink’）为试材，克隆了葡萄VvmiR397a前体基因（594 bp），其可折叠成典型的茎环结构；同时鉴定了其成熟体序列（21 bp），并为其预测到5条靶基因VvLAC4、VvLAC7、VvLAC11、VvLAC14和VvLAC17，它们属于木质素合成的关键酶漆酶家族（Laccase，LACs）。靶基因的domains、motif序列分析及三级结构预测显示，漆酶蛋白序列均具有3个相同的Cuoxidase结构域，其motif元件类型及排列顺序、三级结构均相似，表明漆酶结构较为保守。推测其功能比较保守，74个漆酶基因的进化分析显示葡萄漆酶基因与杨树漆酶基因亲缘关系近于拟南芥，葡萄漆酶基因在木本植物中的保守性高于草本植物。VvmiR397a及VvLAC4、VvLAC11、VvLAC14和VvLAC17的启动子均具有赤霉素响应顺式作用元件（TATC-box、GARE、P-box），表明它们可能响应GA参与葡萄生长发育的调控。荧光定量PCR表达分析显示，VvmiR397a能被GA诱导显著上调，且在幼果、花和成熟叶中具有高的表达，其他组织中表达相对较低，尤其在幼果中表达量最高，硬核期表达量最低；而其靶基因VvLACs表现出相反的表达模式，在硬核期有最高表达，而该期是葡萄果核木质素合成量最大的时期，表明VvmiR397a可能通过负调控VvLACs参与葡萄果核发育的调控。利用RLM-RACE和PPM-RACE验证了VvmiR397a对VvLACs的裂解作用，且在幼果中裂解作用最强，而在硬核期果实中裂解作用最弱，与VvmiR397a的表达趋势相似。

关键词: 葡萄, VvmiR397a, VvLAC, 核发育, 木质素

Abstract: Using grape（Vitis vinifera）cultivar‘Wink’as experimental materials，we determined VvmiR397a precursor（VvmiR397a）as 594 bp by clone and sequencing，which was further folded into a typical hairpin structure，representing the authenticity of VvmiR397a；meanwhile，the mature sequence of VvmiR397a was identified with 21 bp and based on this，we prophesied its target genes including VvLAC4，VvLAC7，VvLAC11，VvLAC14，and VvLAC17，which belong to LAC family of lignin synthesis key enzymes. The target domains，motif sequence analysis，and tertiary structure prediction showed that all LAC protein sequences have three same Cuoxidase domains，both element types，arrangement order of motifs，and tertiary structure，indicating that the LAC protein structure is relatively conservative，suggesting the conserved functionality. The phylogenetic analysis of 74 LAC genes from diverse plant species depicted that the genetic relationship between grape LAC genes and Populus trichocarpa LAC genes was closer than those of Arabidopsis thaliana，intimating that the grape LAC genes might have a high degree of conservation contrary to the woody plants. Subsequently，gibberellin responsive cis-acting elements（TATC-box，GARE，P-box）were identified in VvmiR397a，VvLAC4，VvLAC11，VvLAC14，and VvLAC17 promoter sequences，indicating their possible involvement in the regulation of GA-responsive grape growth and development. Moreover，the qRT-PCR analysis revealed that the expression of VvmiR397a was significantly hoisted by GA and expressed highly in young berries during development as compared to other tissues，while its target gene VvLACs showed reciprocal expression pattern，particularly，the highest expression at the stone hardening stage，elucidating that VvmiR397a may participate in the grape berry stone development through negative regulation of VvLACs gene. RLM-RACE and PPM-RACE verified the cleavage roles of various VvLACs by VvmiR397a，and their cleavages were the sturdiest in young grape berries and the weakest in stone hardening stage of berries，similar to the expression tendency of VvmiR397a.

Key words: grape, VvmiR397a, VvLAC, stone development, lignin

中图分类号:

S 663.1

王文然1，王晨1,*，解振强2，贾海锋1，汤崴1，崔梦杰1，房经贵1. VvmiR397a及其靶基因VvLACs在葡萄果实发育中的作用分析[J]. 园艺学报, 2018, 45(8): 1441-1455.

WANG Wenran1，WANG Chen1,*，XIE Zhenqiang2，JIA Haifeng1，TANG Wei1，CUI Mengjie1，and FANG Jinggui1. Function Analysis of VvmiR397a and Its Target Genes VvLACs in Grape Berry Development[J]. ACTA HORTICULTURAE SINICA, 2018, 45(8): 1441-1455.

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