Bioinformatics Analysis Of RALF Polypeptide Family In Arabidopsis And Construction Of Transgenic Plants

Rapid Alkalinization Factor(RALF),as a Receptor-mediated signal carriers,plays a key role in the regulation of plant defense and development;Meanwhile,it is important peptide hormone of a class of inhibiting hydrogen pumps that are conserved in the plant kingdom to regulate cell elongation through its activity.The RALF protein family is present in plants prior to classification into monocots and dicots to perform different functions.Thirty-nine RALF genes can be retrieved from the Arabidopsis database,but the structural and biological functions of most of them are not clear,except for some RALFs whose functions and mechanisms are relatively well studied.In order to compare the biological functions of different RALFs,this paper proposes to conduct a preliminary analysis of the physicochemical properties and protein structures of different RALFs in Arabidopsis thaliana by bioinformatics.Then by construction of an overexpression vector for the RALF gene,establishment of Arabidopsis deletion mutants and overexpression transgenic plant material,observation of the phenotype in Arabidopsis mutants,and studying of the function on different RALF genes in Arabidopsis.From this thesis,the following main experimental results can be obtained:(1)Predicted inductive analysis of the physicochemical properties of the RALF family in Arabidopsis thaliana: The results showed that the molecular weight,hydrophilicity,positive/negative charge residue number,p I value,instability coefficient,and fat coefficient of most Arabidopsis RALF proteins were similar,but RALF23 and RALF34 had larger relative molecular masses.In addition to the hydrophobic AT2G32788 protein,the other 38 RALF proteins are hydrophilic proteins and 27 proteins are unstable proteins.Among them,AT2G32788,RALF14,and RALF25 had large lipid indices,which indicating that the RALF family has the function of inducing alkalinization among plant tissues and some proteins have unique physiological and biochemical functions.(2)The analysis of sequence similarities and affinities of Arabidopsis RALF families: The results show that 39 members of the Arabidopsis RALF family were grouped into six categories by a phylogenetic tree,and the gene dot plot test demonstrated that this classification is strongly correlated with function.At the same time,conservative base sequence analysis indicated that motif 1 and motif 2 were in the 6 RALF categories conserved in all.Logo analysis suggests that cysteine may play an important role in the maintenance of RALF biological function.Signal P 4.1analysis exhibited 90% of signal peptides present.(3)Identification of Arabidopsis RALF gene T-DNA insertion mutants and construction and identification of partial RALF overexpression plants: Thirteen pure mutants,including ralf1,ralf3,ralf4,ralf6,ralf7,ralf12,ralf17,ralf20,ralf21,ralf23,ralf27,ralf30 and ralf33,were identified by 3-priming method in 328 Arabidopsis insertion mutants purchased from the ABRC website,and the quantitative PCR results showed that the expression of 13 RALFs of the pure species were significantly reduced.(4)Preliminary analysis of the Arabidopsis partial RALF deletion mutant phenotype: Preliminary analysis of the phenotypes of the 13 homozygous mutants identified indicated reduced root hairs,elongated root length and early mossing in ralf1,ralf23 and ralf33 deletion mutants;the rosette leaves of the Arabidopsis ralf17 deletion mutant were small,the plants were dwarf and slow growing.In addition to ralf17,the other 12 mutant species WRKY53,PDF1.2and FRK1,which are resistant marker bases of the due to varying degrees of upward adjustment in expression levels.After root inoculation with endophytic fungi,the fungal load of both Arabidopsis ralf1 and ralf33 deletion mutants was reduced to varying degrees.Treatment of wild-type Arabidopsis with RALF1 resulted in a significant down-regulation of gene expression of RALF1 and RALF31.In summary,most of the 39 Arabidopsis RALF gene families have similar physicochemical properties and are closely related,but different RALF genes perform different functions.Some RALF genes regulate plant cell development and growth,other RALF genes regulate plant immunity and stress tolerance,but all RALF genes affect plant phenotype and plant resistance to stress to varying degrees.Also,there is an association between different RALF gene expressions.