Generation And Primary Phenotypic Analysis Of Chloroplast PPIase Deficiency Mutants In Arabidopsis Thaliana

Peptidyl prolyl cis/trans isomerase?PPIase?exist in various organisms.Its principal function is to participate in protein folding by catalysing the cis-trans isomerisation of the X-Pro peptide bond in polypeptide chains.And it is the rate-limiting enzyme for protein folding.The chloroplast of plants have a number of PPIases.Nevertheless,their physiological function is poorly understood.To increase understanding for chloroplast PPIase,we create null mutants of many uncharacterized chloroplast PPIases in Arabidopsis thaliana by CRISPR/Cas9 gene editing technology and T-DNA mutant screening method in this study,then we analyzed the growth phenotype of these mutants and their functions in photosynthesis.There are three PPIases proteins in the chloroplast stroma of Arabidopsis thaliana,which are CYP20-3,TIG and PIN3.In this study,cyp20-3?SALK₀01615?,tig?SALK₀37730?,and pin3?GK₀68C12?were obtained by identifying T-DNA insertional mutants,then we had obtained cyp20-3×tig homozygous double mutant through hybridization.Phenotypic analysis experiments showed that there was no significant difference in growth and development between the mutants?cyp20-3,tig,pin3,cyp20-3×tig?and the wild type,we speculated that the stroma PPIase proteins may be redundant with other proteins.It was found that the stroma PPIases have the same effect on PSII supercomplexes in the BN-PAGE experiments and their deletion resulted in reduction of the PSII supercomplexes for varying degree.Interestingly,the BN-PAGE band of cyp20-3×tig is similar to cyp20-3,but different from tig,we propose that the function of TIG is dependent on CYP20-3.There are sixteen PPIases in the thylakoid lumen of Arabidopsis thaliana.We mainly carried out the creation and preliminary phenotypic analysis of seven PPIases which have not been reported.One T-DNA insertional mutant for FKBP16-4 were obtained,that is fkbp16-4?GABI₉05D10?;We had obtained four homozygous knockout mutants by CRISPR/Cas9,they are two different fkbp13 lines?fkbp13-38# and fkbp13-43#?,one fkbp17-1 line?fkbp17-1? and one fkbp18 line?fkbp18?.The homozygous knockout mutants of fkbp16-1,fkbp19 and fkbp16-3 have not yet obtained.In the phenotypic analysis experiment for these mutants,we found that fkbp16-4 was later bolting about 1.5 days than the wild type,it can be preliminarily determined that FKBP16-4 is involved in regulating the flowering of Arabidopsis thaliana from this result;In the growth and development,the phenotypes of fkbp13-38#,fkbp13-43#,fkbp17-1,fkbp18 were not significantly different from the wild type,we need deeper research and analysis to confirm their functions.In this study,we are committed to the creation and screening of chloroplast PPIase mutants in Arabidopsis thaliana,which provides materials for subsequent experiments.We also performed the preliminary phenotypic analysis of these mutants,it was found that the deletion of some PPIase affects plants,so they maybe play an important role in the plant's life process.Our study points the way for further research on chloroplast PPIase.