Cloning,Expression Analysis And Functional Study Of HvGRF4 Gene In Barley

Growth-regulating factor(GRF)is a class of transcription factors that is unique to plants and is involved in a variety of processes including plant growth and development,morphogenesis,and resistance to adversity stress.GRF interacts with GRF-interacting factor(GIF)and is negatively regulated by micro RNA 396(mi R396),thus forming a regulatory network among the three,which plays an important role in plant growth and development and stress response.At present,the functional studies of GRF are mainly focused on model plants such as Arabidopsis and rice,but the functional studies of GRF in barley have not been reported.Therefore,it is necessary to carry out the study of HvGRF gene family in barley,and the study of this family has important theoretical and practical applications for barley breeding.In this study,we systematically analyzed the gene structure,protein motifs,evolutionary relationships,and potential phosphorylation sites of the HvGRF gene family and HvGIF gene family around the cloning,functional analysis,and application of HvGRF4 in breeding,studied the effect of HvGRF4 gene on grain type by transgenic technology,and verified the regulatory relationships of GRF,GIF,and mi R396 This study laid the foundation for an in-depth understanding of the regulatory mechanism of barley grain size and provided clues for further studies on the function of HvGRF4 gene.The results of the study are as follows:1.The identification and analysis of the GRF and GIF gene families were conducted using bioinformatics methods in Barley.We identified 14 HvGRF genes and3 HvGIF genes in barley.The HvGRF genes were uniformly distributed across 5chromosomes,and all members had high similarity in gene structure,conserved protein motifs,and protein structure.Multi-species evolutionary analysis divided the GRF members into 6 subgroups.Spatial and temporal expression pattern analysis showed that HvGRF4 and HvGRF6 had higher expression levels in the spike.Transmembrane structure domain prediction indicated that the family proteins were non-secretory proteins.The HvGIF genes were distributed on two chromosomes,and all members contained the SNH characteristic structural domain(SSXT),with highly similar protein structures.Prediction of potential phosphorylation sites showed differences in phosphorylation sites among the family members.2.Analysis of sequence polymorphism of HvGRF4 in different barley varieties was conducted.The GRF4 gene in different barley varieties was cloned,and the sequencing results were compared.Based on the differences in grain type,the positions with significant differences in the sequence were identified,and the polymorphism of HvGRF4 in different barley varieties was analyzed.In combination with the analysis of barley lemma shape,it was found that longer grains were mostly six-row barley,while shorter grains were mostly two-row barley.3.The expression analysis of the HvGRF4 gene in barley was conducted.Realtime fluorescence quantitative analysis was used to study the spatial and temporal expression pattern of HvGRF4 in barley,and the highest expression level of HvGRF4 was found in the spike.The subcellular localization of HvGRF4 was determined by transferring the HvGRF4 gene into tobacco,and the results showed that HvGRF4 was localized in the nucleus.4.Identification and functional analysis of HvGRF and HvGIF gene families in barley.Using bioinformatics methods,we identified 14 HvGRF genes and 3 HvGIF genes in barley.The HvGRF genes are evenly distributed on 5 chromosomes,and all members have high similarity in gene structure,protein conserved domains,and protein structure.Multiple species evolutionary analysis divided the GRF members into 6 subgroups.Spatial and temporal expression pattern analysis indicated that HvGRF4 and HvGRF6 have higher expression levels in the spike.Transmembrane domain prediction showed that the proteins in this family are non-secretory proteins.The HvGIF genes are distributed on 2 chromosomes,and all members contain the SNH structural domain(SSXT),with highly similar protein structures.Predictions of potential phosphorylation sites showed differences in phosphorylation sites among family members.5.Validation of the regulatory network involving GRF,GIF,and miR396.We validated the self-activation of HvGRF family members and HvGIF family members,and demonstrated the interaction between HvGRF4 and HvGIF2 using yeast twohybrid assays.Under stress treatment,we observed changes in the expression levels of HvGRF4 and mi R396 d in barley seedlings.Real-time fluorescence quantification showed that the changes in HvGRF4 and mi R396 d after stress treatment were in opposite trends,indicating that HvGRF4 is negatively regulated by miR396 d.