Exploring Physiological Roles Of CYPs On The Assembly Of Photosynthetic Multi-protein Complexes

Cyclophilin (CYP) family is located in the thylakoid lumen of Arabidopsis thaliana chloroplasto There are five CYP family members, including CYP20-2, CYP26-2, CYP28, CYP37 and CYP38. It is found that CYP38 is the key factor in the process of assembling and repairing of PSII. CYP38 deficient mutant plants could not form stable PSII and show slow growth, low photosynthetic efficiency and light hypersensitivity. Ther are four protein complexes (PSII, the cytochrome b6f complex, PSI and ATP synthase complex) of the photosynthetic electron transport chain located on the thylakoid membrane, and the electronic carrier, plastocyanin, is located in the thylakoid lumen. We believe that other members of the CYP family also functionally related to the photosynthetic macromolecular complexes. Therefore, this research aimed to find out the interacting proteins of the five CYP proteins in Arabidopsis thylakoid lumen. We try to use this method to test whether the four other proteins of CYPs is also involved in photosynthesis protein complex assembly and repair.Firstly, we have down loaded full-length CDS sequence of the five CYPs from the TAIR database and designed primers. We isolated mRNA from wild-type Arabidopsis, and obtained CDS DNA fragements of the five CYP gene via reverse transcription method., Then six histidine tag was added to the five CYP gene CDS DNA fragments respectively. The His-tagged DNA fragments were further introduced into a binary vector, pRIl101-AN to form five CYPs(His)-pRI101-AN constructs. Then, the five constructs were transferred into Agrobacterium GV3101 and transgenic plants were obtained by infecting wild type Arabidopsis. Resistance screening was conducted to TO generation of transgenic seeds and obtained T1 generation transgenic plants. The DNA and protein level of T2 transgenic plants were identified and the successfully identified plants were grow to T3 generations. I extracted each Cyclophilin protein containing His tag from T3 transgenic plants and conducted Affinity Chromatography assay with Ni-beads. as After I got CYP20-2, CYP26-2,CYP37 and CYP38 proteins and their interaction protein, mass spectrometry analysis was done after the concentration of CYP20-2, CYP26-2 and CYP37. Results from mass spectrometry analysis showed a total of 42 proteins co-purified with the CYP20-2 protein,There are 17 proteins located in the chloroplast,including 7 thylakoid lumen proteins and 10 stroma proteins. Most of the others interacting proteins located on the nucleus, there are a few located in the cytoplasm and nucleus.It is highly likely that they are interacting proteins of CYP20-2, and the further confirming tests are on the way. The mass spectrometry analysis on CYP37 interacting proteins found 2 proteins from thylakoid lumen might interact with the CYP37 protein.The endogenous wild type CYP proteins in A the transgenic plants may affect the accuracy of experiment results, we have infected homozygous mutants lacking individual CYP protein to eliminate the effect. CYP38 homozygous mutant seeds, CYP38-2, was identified in our lab previously, and four other homozygous mutant seeds of CYP protein gene were bought form ABRC(Arabidopsis Biological Resource Center) for obtaining their TO generation transgenic seeds after infection. But due to the time shortage of my graduate study, the protein was only extracted from transgenic plants of CYP38 mutants and conducted the Affinity Chromatography assay.