| As a kind of early-maturing bud,grapevine prompt buds always germinate for several times in the same year to form secondary shoots,which not only consumes a lot of tree nutrients,but also increases the cost of vineyard management in summer.In addition,the wound caused by the elimination of secondary shoots is also likely to cause pathogen invasion and cause the spread of disease,which adversely affecting grape growth and quality.Therefore,studying the physiological factors and molecular mechanisms affecting the germination of grapevine buds is of great significance for the application of artificial approaches to regulate the germination of grape buds,thereby reducing the cost of grape production.In this study,one-year-old Vitis vinifera L.cv.’Cabernet Sauvignon’ cuttings were used as materials,and the synthetic analogue of strigolactone,GR24 was smeared to the non-germinated prompt buds after decapitation.Then,the effects of GR24 on phenotypic changes,hormone levels,and transcriptomic levels of grapevine prompt buds were studied,in the purpose of uncovering the effects of strigolactones on grapevine prompt bud outgrowth and the key genes and pathways involved in.On the basis of this,the key transcription factor VviBRCs in SL signal transduction pathway and their promoters were cloned,and their expression pattern,subcellular localization,interacting proteins were screened and identified,and then the responding transgenic Arabidopsis were generated for functional verification.The results of this study would provide abundant genetic resources for grape breeding,and provide theoretical and technical support for reducing the production of grapevine secondary shoots and creating excellent grape germplasm resources with few secondary shoots.The main results of this study were as follows:(1)The outgrowth of grapevine prompt buds was inhibited by GR24 to some extent.Upon decapitation,the apical dominance was relieved,grapevine prompt buds sprouted rapidly and their length increased continuously,while GR24 treatment significantly reduced the elongation of prompt buds,and its effect presented a concentration-dependent trends.However,the growth,dormancy and dormancy-release of latent buds were not affected.GR24 treatment caused changes in endogenous hormone content in prompt buds,which slowed down the decline of ABA and IAA and reduced the increase of ZR and GA3 caused by decapitation.(2)Transcriptome analysis of GR24-treated grapevine prompt buds.RNA-sequencing was performed in grapevine prompt buds of GR24 treatment and CK group on 0 d,1 d,3 d and 5 d using the Illumina Hiseq-4000 platform.Differentially expressed genes(DEGs)were screened using 0 d as a reference,and then control group and GR24 treatment group on 1 d,3 d,5 d were compared.In total,6303,8891 and 10268 DEGs were found in GR1 d vs CK1 d,GR3d vs CK3 d,GR 5d vs CK 5d,respectively,and there were 3221 common DEGs in the three comparison combinations;GO Enrichment analysis revealed that DEGs were significantly enriched in nucleic acid-binding transcription factor activity,ribosomes and cellular ribonucleoprotein complexes.In the KEGG pathway enrichment analysis,the photosynthetic-antennaoprotein pathway was significantly enriched.Ribosome,endoplasmic reticulum protein processing,plant hormone signaling,carbon metabolism and MAPK signal transduction were also highly enriched.Among them,the ratio of up-regulated genes in the KEGG pathways related to signal transduction and cell activity was reduced by GR24 treatment.Many genes involved in plant hormone signal transduction pathways were differentially expressed.In addition,some transcription factors that could respond to GR24 signal were also identified,especially two members belonging to the TCP transcription factor family(BRANCHED,BRC),whose expression can be strongly induced by GR24.(3)Cloning and expression analysis of grape branching gene VviBRCs.In total,three VviBRC genes were identified in the grape genome,with open reading frames of 1377,1167 and 1131 bp,encoding 458,388 and 376 amino acids.Real-time PCR analysis showed that all of them were highly expressed in latent buds,prompt buds,young leaves of the grapevine.In addition,with the germination of prompt buds,the expression levels of the three genes all showed a decreasing trend.Furthermore,treatments accelerating germination like decapitation,cytokinin,gibberellin all reduced the expression of VviBRCs.(4)Cloning and expression analysis of the promoters of VviBRCs.The sequences of 2.0kb upstream in the three VviBRC gene promoters were cloned and ligated with the GUS reporter gene to construct a plant expression vector,and then transformed into Arabidopsis thaliana to obtain a transgenic homozygous line.Through GUS histochemical staining analysis,the three genes were highly expressed in the organs with rapid growth and development,such as in the petiole of the rhizome,the cotyledon and the true leaf of the seedling.Through promoter activity analysis,we found that GR24 treatment could induce GUS activity,while dark treatment can reduce GUS activity,which may be attributed to the relevant cis-acting elements in the promoter.(5)Subcellular localization,transcriptional activity analysis of VviBRCs,and the screening and identification of proteins interacting with VviBRCs.All the three VviBRCs were nuclear localized proteins.No transcriptional activation activity was detected for VviBRC1 and VviBRC2,while VviBRC3 had transcriptional activation activity,which was located at C-terminal 177-376 aa.By constructing yeast two-hybrid c DNA library,a number of candidate proteins were screened,including proteins involved in cell cycle,plant hormone signal transduction and ribosomal proteins,and the interations of VviBRC and proteins like Sn RK1、Histone H4 or SDA1 were preliminarily verified using yeast two-hybrid technology.(6)The functional verification of the grape branching gene VviBRCs in Arabidopsis thaliana.Plant overexpression vectors were constructed and transformed into wild type and BRC1-deleted mutant of Arabidopsis thaliana by Agrobacterium tumefaciens.After observation and statistics,VviBRC3 was found to negatively regulate the shoot branching of transgenic seedlings.Moreover,VviBRCs can partially restore the high branch number of the mutant,indicating that VviBRCs and its homologous genes in model plants were functionally conserved during the regulation of axillary bud germination.Real-time PCR of transgenic seedlings showed that with the overexpression of VviBRC3,the expressions of ABA-related genes like ABF3、ABI5、HB21、HB40 and NCED were enhanced,while cell division-related genes like Histone H4 and CYCD3 were down-regulated. |
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