Preliminary Study On The Interaction And Function Of Arabidopsis Ovate Family Protein AtOFP1 And KNAT5

Arabidopsis thaliana Ovate Family Proteins(AtOFPs)are a new type of plant-specific transcription factor,including 19 members,which are divided into 3 sub-categories according to their phenotypic characteristics.AtOFP1 is divided into subgroup I,which is also a transcription repressor that has been studied more frequently.It has been proved that it can directly regulate the downstream GA synthetase key gene AtGA20 ox,inhibiting its transcription activity and inhibiting cell elongation.The roots,stems,leaves,floral organs,pods,etc.of the Arabidopsis thaliana show diminished traits.At the same time,AtOFP1 itself has no predictable DNA binding domain,so its transcriptional regulation about the target gene needs to be achieved by interacting with other protein factors having a DNA binding domain.The Three-aa Loop Extension homeodomain protein plays a central role in plant development.Studies have shown that the homology domains of the 9 OFPs and TALE proteins have tight functional relationships,among which there is AtOFP1.This article tentatively determined that AtOFP1 can interact with 13 transcription factors of TALE proteins through a wide screening method.Contain KNAT5,which is expressed in the lateral root primordia and may be involved in the formation of lateral roots.In addition,KNAT5 gene and OFP1 gene can affect the synthesis of gibberellin,what is the relationship between them,whether it is related to plant growth,and whether exogenous gibberellin has influence on the expression of OFP1 and KNAT5 gene is the main content of this experiment.In order to determine whether AtOFP1 interacts with KNAT5 and its function,this study first used directed yeast two-hybrid and two-molecule fluorescence complementary methods to determine whether OFP1 interacts with KNAT5 in vivo.Wild type,atofp1 mutant,35S:HA-OFP1 transgenic plant,knat5 mutant and 35S:HA-KNAT5 transgenic plant were used as materials to analyze the phenotypes of plants,and the OFP1 and KNAT5 genes were quantified analyze by qRT-PCR.To analyze the effects of exogenous GA3 on OFP1 and KNAT5 genes,GA was added to the culture medium at different concentrations,and the expression of OFP1 and KNAT5 genes was analyzed by qRT-PCR.The main methods and results are as follows:(1)Using the yeast two-hybrid technique,full-length OFP1 gene was constructed into the pGADT7 vector.The full-length KNAT5 gene was constructed into the pGBKT7 vector and co-transfected into the Y2 HGold yeast strain.Observations showed that AtOFP1 can interact with KNAT5;(2)AtOFP1 and KNAT5 were subcloned into pSAT6-nEGFP-N1 and pSAT6-cEGFP-N1 using the bimolecular fluorescence complementary technique,respectively,and then they were co-transformed into Arabidopsis protoplasts using polyethylene glycol transformation method.Proved that AtOFP1 can interact with KNAT5 in vivo;(3)Screening homozygous mutants of knat5 and constructing KNAT5 overexpressors to observe the changes in the roots.It was found that the apparent number of lateral roots increased in the ofp1 mutant and knat5 mutant plants,and the number of lateral roots of the HAOFP1 overexpressing plant was decreased,obviously shorter.(4)Analysis of expression of OFP1 and KNAT5 genes in Arabidopsis roots and leaves by wild-type,atofp1 mutant,35S:HA-OFP1 transgenic plants,knat5 mutants and 35S:HA-KNAT5 transgenic plants by pRT-PCR It was found that there is a synergistic expression of OFP1 and KNAT5 during plant growth.(5)By allowing Arabidopsis seeds to grow in different concentrations of GA media,OFP1 gene and KNAT5 gene expression,it was found that OFP1 gene and KNAT5 gene still existed in a synergistic expression,and exogenous gibberellins made OFP1 and KNAT5 genes The significant decrease in the expression level indicated that OFP1 may have KNAT5 involvement in inhibiting the transcription activity of gibberellin synthesis key enzyme genes.They may have formed the AtOFP1-KNAT5 complex to regulate the transcriptional activity of AtGA20 ox.In summary,this study demonstrated the interaction of AtOFP1 and KNAT5 in vivo based on yeast two-hybrid results and BiFC results.In Arabidopsis thaliana roots and leaves,AtOFP1 gene and KNAT5 gene have co-expression,and they The phenotype analysis of the number of lateral roots and root length of the overexpressor plants and the mutant plants are also similar,so AtOFP1 and KNAT5 may interact to form the OFP1-KNAT5 complex,affecting the number of lateral roots and root length,and affecting plant growth and development has an important influence.In addition,qRT-PCR data showed that the AtOFP1 and KNAT5 genes were co-expressed under exogenous GA treatment,and their gene expression was up-regulated under exogenous hormone treatment.AtOFP1 gene upregulation inevitably led to a decrease in the expression of AtGA20 ox,a key gene for GA synthesis.It is possible that the synthesis of endogenous GA is reduced,but the plant phenotype is restored by the treatment of exogenous hormones.