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ACTA HORTICULTURAE SINICA ›› 2018, Vol. 45 ›› Issue (8): 1441-1455.doi: 10.16420/j.issn.0513-353x.2018-0059

• Research Papers • Previous Articles     Next Articles

Function Analysis of VvmiR397a and Its Target Genes VvLACs in Grape Berry Development

WANG Wenran1,WANG Chen1,*,XIE Zhenqiang2,JIA Haifeng1,TANG Wei1,CUI Mengjie1,and FANG Jinggui1   

  1. 1College of Horticulture,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China;2Jiangsu Vocational College of Agriculture and Forestry,Jurong,Jiangsu 212400,China
  • Online:2018-08-25 Published:2018-08-25

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

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