Study On Anti-tobacco Mosaic Virus(TMV) Protein Gene Of Y3 Cloning And Prokaryotic, Eukaryotic Expression

Plant virus disease is an important class of plant diseases which have a serious impact on agricultural production, it is not yet effective anti-viral agents, especially natural anti-viral agents to combat viral diseases of plants. The study found edible mushrooms contain many unique features of the biological activity of the protein, such as ribosome inactivating protein(RIP), anti-animal and plant virus protein, lectin and so on. They have a strong biologically active of anti-tumor, anti-animal and plant viruses, antifungal, immunomodulatory, it provides a new way for the prevention and treatment of virus diseases in plants growing.Y3 is a protein has a certain inhibitory on Tobacco mosaic virus(TMV) which discovered from Coprinus comatus, this protein is alkaline, high in vitro stability, conducive to development, with potential applications. The test of their gene sequences and prokaryotic, eukaryotic expression characteristics were studied, and the results are as follows:(1) By cloning got a full-length DNA sequences of 1000 bp and 530 bp cDNA sequence of y3 gene. Homogeneous detection by the NCBI BLAST, DNA sequence was found only with the anti-TMV protein partial DNA sequence Y3 has the greatest homology, GenBank accession number is HM204931.1, maximum identity is 94%. cDNA sequences include a maximum length of 393 bp of the ORF, the largest ORF may encodes a 130 amino acids of the polypeptide chain, which is compared to partial cDNA sequence of the y3 protein on NCBI, the consistency reached 95.69%. Meanwhile the DNA and cDNA sequences of y3 gene sequences found both identity of 89.96%, almost perfectly matched, indicating that the two sequences obtained in this test has a high homology, further illustrated that the present DNA and cDNA sequences were obtained is target sequence.(2) Designed with a restriction site Bamh1 and Xho1 primers, cDNA sequences obtained 530 bp as a template for PCR, then the PCR product with carrier PET28a(+) connection prokaryotic expression vector PET28a(+)-y3, antiviral activity was measured after induction of expression of the protein was purified using semi-leaf method, means that when y3 protein concentration is 12.5μg/ml, inhibition of TMV infection rate is 70.22% +1.25%, illustrated that the obtained y3 protein by prokaryotic expression still has a relatively good resistance activity to TMV, but lower than the anti-TMV activity of y3 protein directly extracted from the fruiting body comatus, indicating there are some shortcomings and deficiencies of prokaryotic expression, but also need to further explore better protein expression systems.(3) In sequence with restriction sites SnaBⅠ and NotⅠ as primers, cDNA sequences obtained 530 bp as a template for PCR,the fragment was ligated with Tcarriers, conversion, and then extracted with eukaryotic expression vector pPic9 k connection transformed into yeast competent cells, after induced by methanol to obtain anti-TMV protein Y3. Through the exploration of inoculation ratio, culture conditions the methanol concentration, pH value, rotation speed, induce time affect on the Y3 protein expression, determined the optimum fermentation conditions: the Mut+ transformants were plated on BMGY growth medium and incubated to OD600 =5.0, after centrifugation to 1.5:1 ratio transfer to inoculation containing 1.0% methanol, pH value of 6.0 BMMY induction medium. 300 rmp, 30 ℃, induced for 5 days, every 24 h add to 1.0% of methanol. Results showed that expression optimization Y3 about 40.7mg/L, higher than before the 24.5mg/L increased nearly 1 times. Protein by SDS-PAGE electrophoresis, about the size of 14.4kD, proved protein is Y3. semi-leaf method was determined by protein antiviral activity, show Mut+ transformants expression product of TMV average inhibition rate 83.7%, while the control group was only 25.74 percent. The results showed that, Mut+ transformants expression products have significant anti-viral activity.