Gene Cloning And Functional Study Of S01-17,the Suppressor Of Arabidopsis Thaliana Var6-1 Mutant

Photosynthesis is one of the most important chemical reactions on earth.Chloroplast is the main site of photosynthesis.Therefore,it is very important to study the function of genes involved in chloroplast development and photosynthesis regulation.Although PAC is an essential gene for chloroplast development,little is known about the biological function of PAC.In this project,PAC gene low expression mutant var6-1 was used as experimental material to study the mechanism of PAC in regulating chloroplast development from the perspective of suppressor mutants.In this paper,S01-17,the suppressor of var6-1,was used as the test material,and the following experiments were carried out.The following results were obtained.1.The laboratory screened the suppressor of var6-1 in the early stage,and obtained S01-17.We continued to backcross the Col-0 wild type with S01-17 to clear the background mutation.It was observed that the first pair of true leaves of the S01-17 mutant turned green,while the S01-17 and S01-17 single mutants were sharper than the Col-0,and the leaf area became smaller and the root became shorter.Plants with S01-17 phenotype in S01-17×Ler F2 generation were used as genetic mapping populations,and coarse mapping and fine mapping of mutant genes were carried out by map-based cloning.It was preliminarily determined that the mutant gene of S01-17 was AT5G60340（AAK6,Arabidopsis adenylate kinase）.The results of DNA sequencing indicated that a point mutation in G to A occurs at the68th base of the two exons of the AAK6,resulting in the glycine₂₃ of the gene coding product to aspartate.2.Comparing the amino acid sequences of AAK6 proteins from different species,it was found that AAK6 proteins were very conservative among species.The mutant amino acid is located in the walker A domain,and the walker A domain is the functional domain of the protein.The mutation of amino acid might cause the phenotype of the S01-17 mutant.The overexpression vector p BI-intron-AAK6 CDS was constructed and the phenotype of the S01-17 mutant was restored to var6-1,and the mutant gene was confirmed to be AAK6.The binary expression vector p Cambia1300-p AAK6-AAK6 g DNA-GFP was constructed,and then the vector containing the AAK6 endogenous promoter was used to restore the phenotype of S01-17 to var6-1 phenotype and the phenotype of S01-17single mutant to the wild type phenotype.The AAK6-GFP fusion protein was observed to localize to the cytoplasm and nucleus.AAK6-GFP fusion protein was detected in S01-17 transgenic plants by GFP antibody.Co-Immunoprecipitation（Co-IP）combined with mass spectrometry results showed that AAK6co-precipitated with RPS14 and other ribosomal proteins.The results of bimolecular fluorescence complementation（Bi FC）test confirmed the interaction between Arabidopsis thaliana AAK6 and RPS14 protein.Fluorescence signal observation results showed that the interaction place of AAK6 and RPS14 protein is cytoplasm and nucleus.3.An overexpression vector p BI-intron-FAP7 CDS containing FAP7,the yeast homologous protein encoding gene was constructed.It restored the short-root phenotype of the S01-17 mutant to a wild-type phenotype.FAP7 is functionally similar to AAK6 protein.