Screening And Functional Identification Of Genes Related To Red And Blue Light-regulated Grapevine Bud Differentiation Based On Transcriptome Analysis

The low light environment in the production of facility ’red globe’ grapes in cultivation leads to poor differentiation of grape buds,resulting in reduced fruit yield and quality,which becomes a prominent problem in the production of facility viticulture.In order to improve the low light environment in the facility,the group found that red and blue light can promote flower bud differentiation through different red and blue light supplementation studies,and screened the optimal red and blue light ratio(R4B1).Based on this,the transcriptome sequencing analysis was carried out to screen differentially expressed genes during bud differentiation by using the optimal ratio of red and blue light to supplement light treatment in facility grapes,without supplemental light as a control.The candidate transcription factor VvARF18 was screened by differentially expressed gene interaction network analysis,the candidate key gene was cloned and bioinformatically analyzed.Preliminary exploration of the relative expression level of VvARF18 in grape tissues and flower buds at different developmental stages through real-time fluorescence quantitative PCR technology.Preliminary gene characterization by yeast self-activation,subcellular localization analysis.The function of the VvARF18 gene was further characterized by transgenic transfer into the model plant Nicotiana benthamiana.The results of the study were as follows:(1)To understand the regulatory mechanism of grape bud differentiation under red and blue compound light,transcriptome sequencing was performed and analyzed for buds at four stages of differentiation under optimal supplemental light.KEGG enrichment analysis of four comparative groups showed that differentially expressed genes were significantly enriched in the plant hormone signaling pathway.A total of 47 DEGs were enriched in the phytohormone signaling pathway,among which the most DEGs were involved in the growth hormone pathway with 16 genes,among which IAA17,ARF18,ARF19 and TIR1 genes were significantly up-regulated in expression during S2 and S3,and IAA17 and ARF18 genes were also significantly up-regulated in expression during S4;followed by the abscisic acid pathway with 9 differentially expressed The PP2C and ABF1 genes were significantly up-regulated in S2,S3 and S4 compared to CK;PYL9、ABF3 and ABF5 were most significantly up-regulated in S3.(2)A protein interaction network analysis of differentially expressed genes in the hormone pathway revealed that ARF18 is an important transcription factor in the hormone pathway and interacts with a variety of proteins in the hormone pathway,including abscisic acid pathway-related proteins ABF2,jasmonic acid pathway-related protein JAZ,growth hormone receptor protein TIR1,IAA17 and IAA1.ARF18 is an important transcription factor in the growth hormone pathway,is able to bind to downstream genes to co-regulate a variety of biological processes in plants.Therefore,we suggest that ARF18 is a key transcription factor in the hormone transduction pathway in response to red and blue compound light involved in grape flower bud differentiation process.(3)ARF is an important class of transcription factors in plants,which are widely involved in plant growth and development and various physiological processes.The VvARF18 gene was homologously cloned from ’Red Globe’ grapes by PCR,which is 2040 bp long,encodes 679 amino acids,has a molecular mass of 60.399 kDa and a theoretical isoelectric point of 8.81.binding domain and Auxin_resp structural domain.The phylogenetic tree showed that the VvARF1 8 protein was most closely related to the CsARF1 8 protein of Camellia sinensis,and the results of subcellular localization and transcriptional self-activation showed that VvARF18 was a transcription factor localized in the nucleus with transcriptional activation activity.The results of real-time fluorescence quantitative PCR showed that the expression of VvARF18 gene in the treated groups was higher than that in the control group during bud differentiation,and it was speculated that VvARF18 might be involved in the bud differentiation process in response to 4:1 induction of expression by red and blue light,but its specific regulatory mechanism needs to be further investigated.(4)To investigate the function of the VvARF18 gene in floral bud differentiation,a study on the transgenic function of the VvARF18 gene was conducted.The VvARF18 gene is located on chromosome 13 and contains three exons and two introns;analysis of the promoter revealed that the VvARF18 promoter contains multiple light-responsive and multiple hormone-and stress-responsive elements.Observation of the phenotype of transgenic tobacco revealed that the floral bud differentiation of transgenic tobacco was earlier than that of wild-type tobacco,indicating that the VvARF18 gene could promote the process of floral bud differentiation.Quantitative qRT-PCR analysis and endogenous hormone content analysis showed that VvARF18 gene expression showed an opposite trend of action with changes in IAA and CTK content The correlation between VvARF18 gene expression and endogenous hormone content showed that VvARF1 8 gene expression showed an opposite trend to the changes of IAA and CTK content,and the same trend as GA and ABA.The ratios of GA/IAA and ABA/IAA in transgenic tobacco were higher than those in wild-type tobacco.This indicates that the VvARF18 gene,as a transcription factor for growth hormone signaling,may be involved in the regulation of hormone homeostasis through some pathway.