The Function Of Chloroplast Electron Transfer Proteins In Programmed Cell Death

In the intrinsic pathway of apoptosis,mitochondria are at the center of the cell.Mitochondrial cytochrome C?mtCytC?functioning in electon transfer would induce cell apoptosis once it is released from mitochondria into the cytoplasm.In the chloroplast of plants,several proteins play a similar role as mtCytC in electron transfer,including cytochrome C6?CytC6?and two plastocyanins?PC1 and PC2?.We hypothesized that PCs and Cytc6 may be important factors affecting plant programmed cell death in plant cells.In this study,we built both constitutive and inducible expression vectors for these three genes and transferred them to wild-type Arabidopsis?Columbia?.The function of PCs and Cytc6 in plant programmed cell death was observed and compared.The main results are as follows:?1?Construction of 12 kinds of constitutive expression vectors.Full-length sequences coding for PC1,PC2 and Cytc6 were downloaded in the Tair database.The primers were designed and the gene fragments were constructed on the plasmid vector pB003 downstream of 35S promoter.There are 3 kinds of recombinant vectors for each target gene to constitutivly express full length polypeptide?FLPP?,FLPP-GFP fusion protein,matured polypeptide?MAPP?absent of signal peptide,and MAPP-GFP,respectively.GFP fusion protein was constructed to facilitate the subsequent confirmation of protein localization.12 kinds vectors were successfully obtained and confirmed by sequencing.?2?Construction of 7 kinds of inducible expression vectors.The coding sequencse for MAPP of three target genes were ligated into plasmid vector pTA7002 downstream of inducible promoter GAL4?Pind?,generating 6 binaray vectors to express MAPP and MAPP-GFP respectively.These constructs were transferred into Columbia?Col?,and expressed after transgenic plants were sprayed by inducer DEX.The expressed target proteins were localized in the cytoplasm since the signal peptides were absent in their sequence.In addition,the empty vector pTA7002 EV was used as control.7 kinds of inducible expression vectors were successfully obtained and confirmed by sequencing.?3?Obtaining T2 generation transgenic plants.All the recombinant plasmids were transformed into Agrobacterium GV3101 and then transferred into Col.T1 plants were screened against corresponding resistance.Positive transgenic T1 lines were confirmed at DNA and protein level,and advanced to T2 generation for the study.?4?Induction of pTA7002 related transgenic plants by dexamethasone?DEX?.DEX is a kind of glucocorticoid,which can be used to induce pTA7002 series transgenic plants by DEX.The expression of GVG gene is expressed in specific tissues by using the characteristics of pTA7002 system.The concentration of DEX used in this experiment was 15 ?M.?5?The validation of salicylic acid?SA?on singlet oxygen in Col and mutant petel and pete2.SA can increase the content of singlet oxygen in plant leaves,resulting in programmed cell death.5mM SA was used to observe the phenotype of Arabidopsis leaves.The degree of programmed death of the three plants was Co1>pete2>petel,and the mortality rate of the plants was counted for three times.The results indicated that the increase of reactive oxygen species in the three plants under SA stress might be related to the content of PC.?6?The phenotype of the transgenic plants were observed and their death status was recorded.Overexpressing MAPP and FLPP proteins both leaded heavier death syndrome than wild type col after SA treatment,suggesting that Cytc6,PC1 and PC2 highly expressed in chloroplast or cytoplasm may increase the frequency of death.But there are many factors that cause cell death,due to the time,we can not exclude the various environmental factors and the impact of the expression of the system itself,the need for more detailed study.?7?Identification of T-DNA insertion mutant of Cytc6.From the Arabidopsis mutant resource center?NASC?,we bought three lines of Cytc6 mutants with T-DNA insertion,and they are Salk₀60652,Salk₀60643 and Salk₀11266c.After the plant was examined at the DNA and RNA levels,we got the homozygous plants from two insertion lines,including Salk₀60652 and Salk₀11266c with a T-DNA insertion at the exon and intron,resepectively.?8?Preparation of Cytc6 protein antibody.CDS of Cytc6 mature peptide was fused downstream of the GST ORF in the inducible expression vector pGEX4T.3 by genetic engineering technology.GST-Cytc6 fusion protein was isolated by affinity purification method and enough amout protein was obtained for immuing rabbit later.In conclusion,we found that overexpressing MAPP and FLPP proteins both leaded heavier death syndrome,suggesting that Cytc6,PC1 and PC2 highly expressed in chloroplast or cytoplasm may increase the frequency of death.However,it is not yet known this mechanism,further studies will be required to clarify this phenomenon.Furthermore,we identified knock-out homo-mutant of cyc6,purified the protein of Cytc6,these works provide a valuable resource for preparing antibody of Cytc6 protein,then help to researching the function and mechanism of Cytc6.