Exploring Physiological Roles Of CYP26-2 And A Sugar-sensitive Mutant Gene Mapping And Phenotype Identification In Arabidopsis Thaliana

Immunophilins,as receptors for immunosuppressive drugs,are originally found in animals.They are classified into two major families(according to their immunosuppressant ligand partners): the FK-506 binding proteins(FKBPs)and the cyclosporin A binding proteins(cyclophilins [CYPs]).The Immunophilins are widely identified in various organisms,including bacteria,fungi,animals and plants.Current studies have shown that immunophilins are the most abundant in plants.In Arabidopsis,52 genes encoding Immunophilins have been identified,17 are located in chloroplasts.According to literature,only one of the 17 Immunophilins in Arabidopsis chloroplasts is present in the stroma,the other 16 are located in the thylakoid lumen.In green plants thylakoid membrane is the place where photosynthetic macromolecules are assembled,so we wonder whether most of chloroplast immunophilins also involved in the formation of photosynthetic macromolecules? Current studies have shown that CYP38,an immunophilin in thylakoid lumen of Arabidopsis thaliana,plays an important role in the correct assembly and stability maintenance of PSII.This study provides a basis for the participation of the immunophilin in photosynthesis.CYP26-2 is also one of the cyclophilins in the thylakoid lumen,its molecular weight is about 26 k D,whether the protein is also involved in the process of photosynthesis? At present,there is no definite research about this aspect.In this study,we focused on the question whether CYP26-2 is involved in the basic process of photosynthesis to design experiments.In order to study whether the protein participates in the assembly of macromolecular complexes.Firstly,we plan to separate the thylakoid membrane of CYP26-2-HA transgenic plants by blue active gel and sucrose density gradient technique,and the corresponding position of CYP26-2 was detected by Western Blotting.Further,to obtain the deletion mutant of CYP26-2 by screening T-DNA insertion line and CRISPR-Cas9 system,Thus the he physiological function of CYP26-2 will further researched by observation the phenotype of deletion mutants.Finally,we found that the CYP26-2 signal detected by Western Blotting corresponds to several complexes(For example PSII and LHCII)in the result of CYP26-2-HA thylakoid membrane separated by blue native gel and sucrose density gradient centrifugation,indicating that CYP26-2 protein may be involved in photosynthesis process.Next we plan to make a further study on CYP26-2 protein by CYP26-2 knockout mutant,thus two homozygous mutants with T-DNA insertion of CYP26-2 were obtained by screening T-DNA insertion line,but the T-DNA insertion sites were both located in the 3UTR region,which did not cause the complete termination of CYP26-2 gene expression.We constructed a binary vector which can target multiple loci in CYP26-2 according to the CRISPR-Cas9 system,thus the binary vector was successfully transferred into wild-type Arabidopsis thaliana,and at last,two CYP26-2 homozygous mutant without T-DNA insertion,Tagged as 66# and 19#,was obtained by screening for two or three generations.After some molecular identification,we make sure those two mutants are all knockout mutants of CYP26-2.The phenotype of two mutants was the same as wild type both under normal and high light,so we suggested the function of CYP26-2 is redundant in Arabidopsis thaliana.In the process of screening knockout mutant of CYP26-2 by CRISPR-Cas9 system,we obtained a transgenic line with obvious mutant phenotype.The identification result suggests there was no mutation in CYP26-2,so it is a new mutant resulted in CRISPRCas9 system.The mutant gene was mapped and analyzed by map-based cloning and finally the mutant gene was mapped between the two molecular markers of 4G3 and 4G8 on chromosome 4.The distance was 400 kb.The phenotypic observation of mutant plants showed that the true leaves of mutants were yellow in medium or in sugar-free medium,and they were extremely sensitive to normal light and could only grow under low light.When the concentration of sucrose in the medium increased to 3%,the cotyledons of the mutant also became yellow,which indicated that the mutant was sensitive to sucrose,therefore,We named the mutant as sugar sensitive mutant.