Prokaryotic Expression, Purification And Bioinformatics Analysis Of P42A Gene In Alternaria Spp.

The p42A gene fragment which was screened out from cDNA library was used in this experiment. The prokaryotic expression vectors containing p42A gene were constructed, induced, and then the expression protein were purified. At the same time, some bioinformatics analysis of p42A gene and its coding protein were carried out. This experiment had provided an important evidence that the activator protein and its function mechanism at molecular biological level.1. The ITS region was cloned by PCR amplification from one Fungi. After sequencing and blast with other sequences in GeneBank, the result showed that it was completely identical with the ITS region in Alternaria spp..2. The p42A gene fragment containing enzyme digestion sites was obtained by PCR amplification, then separately cloned into fusion expression vector pGEX-KG and unfusion expression vector pBV221 in correct ORE The results showed that expression vectors was successfully constructed. They expressed two proteins with size of 44KD and 18KD detected by SDS-PAGE, respectively. Moreover, the yield of fusion protein was markedly higher than that of unfusion protein.3. The best fusion expression conditions were obtained by establishing bacterial growing curve and by optimizing inducing conditions such as thalli density, the concentration of IPTG, growing time and temperature. The results indicated when induced at 4 hours after culture and after the induction of 1.0mmol/L IPTG at 37℃for 4 hours, the yield of fusion protein reached tiptop-over 56% of total bacterial proteins. Expression fusion protein was soluble detected by SDS-PAGE after breaking up with ultrasonic and centrifugation.4. The expression fusion protein was purified by two steps: Firstly, the P42A-GST fusion protein was purified by affinity chromatographic column, then the GST tag was cut off by thrombin. Finally, the P42A protein could be gained by affinity chromatographic column again. Western Blot indicated that the specific band was the P42A-GST fusion protein.5. With the seeds dipping in P42A protein solution for 8 hours, the activities of antioxidant enzymes and MDA content were determined in early wheat growth period. The results were as follows: The activity of SOD and POD increased after being treated. The MDA content descended after being treated. All these results indicated that as the assistant of activator protein-P42A protein could enhance the ability of antioxidant and resistance to wheat.6. Bioinformatics analysis were carried out by some different methods. Forecast content include: The P42A coding protein had a 61.0% degree similar to TCTP family. As to secondary structure, random coil content was abundant. Motif search showed it was single domain protein. Its stereostructure was obtained by homologous modeling.