| Trichogramma spp. is widely used as natural enemies of pests. Through horizontal thansfering Wolbachia artificially, the reproduction of part of the sexual reproduction of Trichogramma was induced into parthenoensis. As result, female increased significantly and application cost droped largely. Insect prophenoloxidase activating system (proPO-AS) is the most important insect humoral immune system, which take important part in resisting the invasion and in other important physiological responses.The success of the technology that thansfer Wolbachia artificially among Trichogramma was related to its own immune of proPO. To explore the correlation, and for further define the characteristics of PO,to analysis the different immunity to Wolbachia of Trichogramma, this study carried out the following contents with Trichogramma dendrolimi, T.Ostriniae, T. chilonis:1.Set different buffer pH, different temperature and use catechol, L-dopa, pyrogallol as substrate to determine the most suitable value of PO. The results showed that the optimum pH value of three kinds of Trichogramma was 4.5-5.0,low pH and alkaline environment strongly affectted enzyme activity;The most appropriate temperature of T.dendrolimi and T.ostriniae was 40℃, accordingly, T. chilonis was 37℃, high temperature also strongly inhibitted the PO activity of Trichogra- mma; Km values were small when Trichogramma react with catechol and L-dopa,which have no significant each other; Km values was great with pyrogallol,so catechol and L-dopa had higher compatibility with three kinds of Trichogramma.2. PO activities of three Trichogramma species homogenate induced by several chemical factors (Ca2+,laminarin,dextran, LPS and PMSF) was studied,determined PO value respec tively. The results indicated that Ca2+ was required for PO activation,and the maximal activation was observed at the Ca2+ concentration of 0.03 mol/L;Laminarin and LPS could also effectively activated proPO in the four glucan,while cellulose and dextran were proved to be significant inhibitors to PO.We also found that Ca2+ and PMSF in a different order would affect the PO activity. Ca2+, after adding PMSF, significantly inhibited the PO activity (P <0.05),while in turn had no inhibition performance. Different types of glucan had completely different activation on proPO-AS.It could make selective reaction on different glucan. Specific binding proteins of Trichogramma homogenate could identify specific glucan and activate proPO-AS.3. The immune activity value on Wolbachia was determined, through set the same host,different host,different instars of the same host and different reproductive of Trichogramma, Find a specific state insect with lower immune,so that provide theoritical basis of transfering Wolbachia artificially among Trichogramma. The results indicated that when all the host were Corcyra cephalonia, T. dendrolimi had the weakest immunity to Wolbachia,while T. ostriniae strongest;Whether the host is Corcyra cephalonia or Antheraea pernyi, there had no significant difference on immunity; PO activity of pupa Trichogramma was significantly lower than adult (P< 0.05), particularly T. dendrolimi;Affter transfering Wobachia artificially into T. dendrolimi,the immunity droped,and this specific mechanism remains unclear.4. LPS is on the surface of Wolbachia as sensitive elements,which can be identified by specific protein molecule of Trichogramma. LPS binding protein of three Trichogramma were seprated with laminarin precipitation method.Detectd by SDS-PAGE and measured with the PO activity of homogenate after precipitated. The results showed that the main band was unclear, and the electrophoresis background was heavy, while the molecular was about 40 kD or so. The homogenate precipitated by laminarin still could be activated by Ca2+,that was why LPS can not precipitated completely through centrifugation. Improve the protein extraction method is necessery for clear band. |
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