Isolation Two Copper Binding Proteins Related With Plant Resistance To Pathogens From Algae, Its Properties And The Characteristics Of Gene

Striking with plant diseases is one of the most important matters in human living, while seeking for the natural products with the activity against plant diseases from biology and applying to the practice of plant diseases management is one of the key tasks many scientists focus their interests on. The proteins isolated from algae and the genes encoded such proteins must have the particularity for the special characteristic of algae, therefore, it is very significant to study the proteins and the genes of algae related with the plant resistance to pathogens.1. The anti-virus and anti-fungi activities of protein digestion from 16 algae were determined. The result showed proteins which could inhibit the infection of Tobacco mosaic virus ( TMV ) and Potato virus Y, strain N (PV YN ) contained in algae widely, and the proteins had the activity of inhibiting the fungi growth. Some protein resolution digested from algae also had the property of inhibiting the conidia germinating of Gloeosporium musarum.The procedure of salting out with (NH^SC^ and followed with ion-exchange column chromatography and gel-filtration chromatography was carried out to isolate the proteins from Sargassum fusiforme -. Hypnea cervicorrnis and Sargassum hemiphyllum. The examination result of activity showed some proteins partially purified could inhibit the infection of TMV, the mythium growth and conidia germinating of fungi.2. According to the result of scanning the proteins with activity from algae, Ulva pertusa Kjellm was selected for further study to purify the protein with anti-virus activity. A protein named UPG75 was purified to homogeneity through DEAE Sepharose Fast Flow ion-exchange chromatography and Sephadex G-75 gel-filtration chromatography from the 40%~80% saturation of ammonium sulfate salting out proteins, which could inhibit the infection of PVYN. The MW of the protein is 10,000Da determined with SDS-PAGE, no carbohydrate could be founded in the protein. 20 amino acid residues were determined and the result of blast showed the homology to the amino sequences of plastocyanin from 3 algae was high, which was above 85%. Therefore, UPG75 was considered as onemember of plastocyanin family. Plastocyanin is a copper binding protein, which play an important role in electron transmission. The discovery of anti-virus property of UPG75 is very intresting.Another protein was also be purified to homogeneity through CM Sepharose ion-exchange chromatography from the 40%~80% saturation of ammonium sulfate salting out protein of Ulva pertusa . The protein, which was named as UPCM40 could inhibit the infection of TMV. The MW of UPCM40 is 36.000Da determined with SDS-PAGE, and no carbohydrate could be founded in the protein.3. Polyphenol oxidase(PPO) is one of the metalloprotein containing copper ion and also an enzyme of organisms related with the resistance mechanism to the pressure. The results showed the activity of PPO existed widely in algae. The activity of PPO from Ulva pertusa, Ishige okamurai and Sargassum fusiforme was tested. The result showed the PPO activity in the reaction system needed an appropriate proportion of PPO and catechol, the high content of catechol could inhibit the activity. PPO from 3 algae all presented a wide suitable reaction temperature range and had certain heat stability treated with high temperature. Some inhibitors could inhibit the activity of these PPO. The most interesting fact was that the opium pH of the PPO from Ishige okamurai and Sargassum fusiforme was 9.1 and 12 respectively, while little or no activity could be found when pH was 3.0, 4.0 or 5.0, even 6.0. It was different from many other enzymes for the point.4. Touch-Down PCR and RACE were carried out to clone the gene of UPG75. The complete cDNA sequence containing 787 base pares were obtained, which including 5'UTR(40bp) and ORF(417bp) and 3'UTR(333bp). The gene encoded the UPG75 peptide precursor containing signal peptide with 41amino acid residues and mature peptide with 98 amino acid residues.The analysis of homology show...