Research On The Function Mechanism Of Carboxyl-terminal Of PD1 Protein

Plants use solar energy to turn inorganic matter such as water and CO2 into organic matter and release oxygen at the same time during the process of photosynthesis,thus provide material and energy foundation for the survival and development of the entire biosphere life.Chloroplast is an important site for photosynthesis.PS II is the most basic photosynthetic system complex in chloroplast.The properly assembly and timely repair of PS II are the prerequisites for chloroplast to maintain its function.D1 protein,encoded by the chloroplast psbAgene,is the core protein of PSII reaction center.The precursor of D1 protein has a C-terminal tail,and once the C-terminal tail was cleaved by protease CtpA,it turns into mature D1.and then the PS II system can work normally.There are no C-terminal tail of D1 protein and ctpA enzyme in some procaryotes,D1 protein has become matured form after it has been synthesized.At present,the mature mechanism of D1 protein in higher plants and the function of C-terminal tail is remained unclear.In order to solve these problems,we use tobacco as the material,and builda tobacco chloroplast expression vector with chimeric tailless or doubletail D1 gene,then transformed these vectors into chloroplast by particle bombardment.Some mutants(with double tails or without D1 tail)were obtained by antibiotic pressure screening.In addition,using GFP as a reporter gene and the leader sequence of the Arabidopsis rbcS gene as chloroplast peptide sequence,the vectors of a chimeric one or double tail D1 were constructed to detect the effectiveness of chloroplast target peptide.Some Arabidopsis D1 mutant plants were obtained through floral dip transformation.We can use them for the further research on phenotype analysis.Finally,a few prokaryotic expression vector with different C terminal tail of the D1 protein were constructed,and exogenous protein was expressed in vitro for the protease enzyme digestion and analysis of CtpA in the future.Some results are made as following:In tobacco,six tobacco chloroplast transformation vectors were generated according to the expected design by using the DNA recombination technique,using aadA gene as selection marker,GFP as report gene.The six plasmid vectors were made into micro-bombs.The tobacco chloroplasts were transformed via particle bombardment.Tobacco resistant seedlings were obtained on screening MS medium containing 500 mg/L spectinomycin.The corresponding primers were designed and identified,and the target genes were verified to be integrated into the chloroplast genome by PCR analysis.Binary expression vector containing GFP was generated and transiently expressed in tobacco.GFP fluorescence was observed under laser confocal microscopy,which proved that this chloroplast target sequence was effective.Some Arabidopsis mutants with modified D1 protein were obtained via Agrobacterium mediated transformation method.The positive transgenic plants were verified through the screening of glufosinate and PCR analysis.The transgenic plants of Arabidopsis thaliana mutants were analyzed by RT-PCR and Western blot.In the experimental analysis of the CtpA enzyme cleavage efficiency,it was found that the D1 protein with single tail was more likely to be cleaved by the C-terminal tail than the double-tail D1 protein at the same pH condition.These two kinds of D1 proteins were cut with the highest efficiency by CtpA at pH 9,but the digestion efficiency of CtpA was significantly higher for D1 with single tail than for D1 with double tail.