A Basic Study On Effect Of AtHD2D Gene On Root Development In Arabidopsis Thaliana

Root is an essential part of the growth of terrestrial plants,and its growth and development are an important part of plant growth,and are jointly affected and regulated by a variety of genetic signals and pathways.In this process,chromatin modification plays an important regulatory role.Among them,histone acetylation and deacetylation modification are the key mechanisms of gene transcription regulation,which are closely related to the activity and silence of gene expression.Histone deacetylases(HDACs),as a supergene family widely present in eukaryotes(including yeast,mammals and plants),play a key role in regulating plant growth,organ development and stress response.Previous studies have shown that the overexpression of histone deacetylase AtHD2D can effectively enhance the resistance of Arabidopsis thaliana to abiotic stress,but the function of AtHD2D in Arabidopsis thaliana root development has not yet been reported.In order to explore the role and function of AtHD2D in root development and elucidate its regulation mechanism on root development in Arabidopsis thaliana,we constructed AtHD2D overexpressed lines,pHD2D::GUS lines and screened AtHD2D T-DNA insert the homozygous mutant lines.And using these three Arabidopsis with different genetic backgrounds as research materials,preliminary exploration of the function of AtHD2D in roots will lay a foundation for further research on the molecular mechanism of AtHD2D's biological functions.The main research results are as follows:1.The bioinformatics prediction and analysis of the promoter region of AtHD2D showed that the promoter region of the AtHD2D gene has an auxin effect cis-acting element TGA-element(Brassica oleracea),and also contains I-box(Zea mays),TCT-motif(Arabidopsis thaliana),GATA-motif(Solanum tuberosum)and other light-responsive elements.2.Using GUS chemical tissue staining technology to analyze the expression patterns of pHD2D::GUS,the results showed that GUS was constitutively expressed in Arabidopsis tissues(roots,stems,leaves,flowers)in the pHD2D::GUS transgenic lines,and q RT-PCR analysis also proved this result;the staining results of the pHD2D::GUS lines showed that the expression sites in the seedling roots were mainly concentrated in the central pillar and lateral root primordia in the mature zone of the main root.3.The pHD2D::GUS transgenic lines were treated with 0.1?mol/L auxin IAA,and GUS chemical tissue staining analysis showed that the expression of GUS in roots increased,indicating that the expression of GUS initiated by pAtHD2D was induced by auxin;the wild-type Arabidopsis thaliana was treated with 0.1?mol/L IAA,q RT-PCR analysis showed that the expression of AtHD2D gene increased at the transcription level,and the results showed that AtHD2D gene expression was induced by auxin at both the initial transcription level and RNA level.In addition,AtHD2D transcriptional expression was also increased under stress treatment(100 mmol/L Na Cl,100 mmol/L mannitol).4.By analyzing the root phenotypes of three Arabidopsis lines with different genetic backgrounds,it is found that AtHD2D overexpression can increase the number of lateral roots and the number of lateral root primordia,the lateral roots of hd2d mutant were significantly less than those of wild type.After treatment with different concentrations of Na Cl,mannitol,and IAA,it was found that the number of lateral roots and lateral root primordia of AtHD2D overexpression transgenic lines were significantly increased compared with wild-type and T-DNA insert mutant lines.5.Lugol staining showed that AtHD2D gene up-regulated or down-regulated could lead to different degree of arrangement disorder of apical columella cells.q RT-PCR analysis showed that the transcriptional expression ofkey genes WOX5,PLT1,PLT2 required for maintainingroottip quiescent center(QC)activity in AtHD2D overexpression lines decreased,and the transcriptional expression of SCR and SHR increased.It was proved that AtHD2D overexpression affected the stability of root tip microenvironment.6.q RT-PCR analysis of genes related to lateral root development in AtHD2D overexpressed transgenic lines and wild type lines showed that AtHD2D overexpression could promote the transcriptional expression of key genes ARF7,ARF8,ARF19,LBD16,LBD29,IAA13,IAA14 and IAA28 in auxin signaling pathway.Compared with the wild type,overexpressed plant auxin transport related genes showed that PIN1 was down-regulated,PIN3 and AUX1 were up-regulated,indicating that AtHD2D may be involved in regulating auxin transport in roots.7.Analysis of ROS distribution in the roots of AtHD2D overexpression transgenic lines and wild-type Arabidopsis using NBT chemical tissue staining and DAB chemical tissue staining showed that AtHD2D overexpression can promote the accumulation of O₂^·-in the roots.q RT-PCR analysis showed that the overexpression of AtHD2D under adversity stress can affect the expression of the transcription level of reactive oxygen metabolism pathway-related genes.Among them,At ROBHD anAt ROBHF genes of NADPH oxidase,which are related to the productionof reactiveoxygen species,were increased at the transcriptional level,while CSD and CAT,which are related to the clearance of reactive oxygen species,were expressed differently at the transcriptional level under different stresses.8.The AtHD2D and At FIB1 fragments were constructed on Bi FC expression vector and yeast expression vector respectively to obtain expression vectors of YFP^N-AtHD2D,YFP^C-At FIB1,p GBKT7-AtHD2D and p GADT7-At FIB1.The interaction between AtHD2D and At FIB1 was verified by Bimolecular Fluorescence Complementary technology and Yeast two-hybrid technology.The above results indicated that AtHD2D affected the root tip microenvironment homeostasis by affecting the expression of gene transcription level required to maintain the root tip microenvironment;AtHD2D could regulate the expression of the transcription level of genes related to lateral root development and promote the occurrence and growth of lateral roots;it could also regulate the expression of auxin export vectors PIN1,PIN3,and AUX1,indicating that AtHD2D may be involved in root development by changing the gradient distribution of auxin in roots.AtHD2D could also promote the accumulation of reactive oxygen species in the root,indicating that AtHD2D can also participate in the reactive oxygen-mediated lateral root development process.Protein interaction experiments showed that there is a certain relationship between AtHD2D and methylation.