| Metarhizium anisopliae, a kind of entomopathogenic fungus widely applied at home and abroad, plays an important role in biological control of locusta migratoria. Compared with chemical insecticides, mycoinsecticides are not infective or toxic to human and have no environmental pollution, but the slow insecticidal speed has been recognized as a potential drawback to prevent the utilization of these fungi against insects. Researches on the interactions between pathogenic fungi and locusta migratoria, exploring the mechanisms of fungal pathogenesis and host defense may suggest strategies for the development of more efficient anti-insect mycoinsecticides.During the period from the pathogenic fungi getting into the haemocoel of insects to the moment the insects die, there happen complicated interactions between pathogens and their hosts. On the one hand, fungi suppress the defensive responses of the host cells by relasing destruxins, utilize nutrition from insects by secreting hydrolases, such as g?lucosidase, trehalase, acid phosphatase and so on. On the other hand, the insects defend themselves by cellular and humoral immune responses actively.Glycoprotein and phosphorylated protein, as two kinds of post-translational modified proteins, exist widely in various tissues and play great roles in various physiological activities. In recent years, their roles related to immune responses and diseases are paid much attention. Researches on glycoproteins and phosphorylated proteins can provid theoretic basis for understanding some diseases and provide curing targets for these diseases by genic engineering method.In this research, migratory locust, Locusta migratora manilensis and Metarhizium anisopliae var. acridum strain CQMa102 were used. For the first time, 13 differentially-expressed glycoproteins were screened from locust haemolymph glycoproteins mycosed with Metarhizium anisopliae by high throughput and high resolution 2-DE (two-dimensional electrophoresis) technology. Among these differentially-expressed glycoproteins, three glycoproteins were identified by de novo sequencing technology. For the first time, 2 targetting phospoproteins for the PTPase (Protein tyrisine phosphosase) purified from Metarhizium anisopliae were found by 2-DE and following tandem mass spectrometry analysis. The possible roles of the 3 glycoproteins and 2 phosphoproteins were deduced from the previous researches. This research provids some clues for understanding the pathogenic mechanism of Metarhizium anisopliae to Locusta migratora manilensis and supplies some basis for further research on their actual roles in this course. The main results were as the following:①From the 5th day to the 13 day after the final ecdysis, the content of free amino acids in the haemolymph of healthy male adult locusts were costant at about 125μg/ml, while the content in the haemolymph of the pathogen infected locusts increased with the days prolonging and it increased dramatically to 454.5μg/ml at the telophase of the infection.②From the 5th day to the 13 day after the final ecdysis, the content of proteins in the haemolymph of healthy male adult locusts were costant at about 15.25mg/ml, while the content of proteins in the haemolymph of the pathogen infected locusts decreased gradually with the days prolonging. On the 8th day after infection, the content of proteins in the hemolymph of the infected locusts was only 11.85mg/ml.③For the first 4 days after infection, the expression pattern of the total hemolymph proteins extracted from the pathogen infected locusts analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was similar to that of the healthy locusts. But on the 6th day after infection, the expression pattern of the total hemolymph proteins extracted from the pathogen infected locusts changed greatly compared with that of the healthy locusts. There appeared 3 new bands on the gel for the pathogen infected locusts and one band became weaker significantly than that on the healthy locusts.④After being separated from the haemolymph of locusts, the glycoproteins from the healthy locusts and pathogen infected ones were analyzed by 2-DE. Thirteen differentially-expressed glycoproteins were found from the 2-DE pictures for the infected locusts compared with those of the healthy ones. Among these proteins, 9 glycoproteins were up-regulated and 4 glycoproteins were down-regulated.⑤Ten clear PMF maps of the differentially-expressed glycoproteins were acquired by MALDI-TOF-MS. But none signicantly results acquired by searching in the NCBI database through MASCOT searching engine with these mass data due to the limited genome or protein sequence information available.⑥By peptide de novo sequencing, 13 internal peptides sequences of 4 differentially-expressed glycoproteins were analyzed, and 3 signifacantly results were searched by the peptide sequences. The three glycoproteins were identified to be transferrin,Apolipophorin precursor protein, hexamerin storage protein with their corresponding internal peptide sequence SGLDDVLSLDGGEALTAVR, EIVAEGLLIDDNNEK,YDAYGNEMTLDEAR, respectively. Hexamerin storage protein and transferrin were reported to be related to the immune responses of insect . The other 10 differentially-expressed glycoproteins are still unknown.⑦We separated the phosphoproteins from the haemolymph of locusts by affinity chromatography and treated the phosphoproteins with purified PTPase from Metarhizium anisopliae. Then, the phosphoprotein sample was analyzed by 2-DE and two targetting phosphoproteins were found. In the two targetting proteins, one was found to be highly homology to toll-like receptor and the other was found to be highly homology to golgi autoantigen, golgin subfamily A member 4 (Trans-Golgi p230) by tandem mass analysis and following searching in related database. Toll-like receptor is involved in mediating the information transduction of immune response for insects, and this indicates that the PTPase probably plays some roles in information transduction by the dephosphorylation of toll-like protein. Golgi autoantigen,golgin subfamily A member 4 (Trans-Golgi p230) is related to the animal auto immune diseases and the transportation of some glycoproteins from golgi to other places, this indicates that PTPase possibly disturbs the transportation of some glycoproteins, especially some important glycoproteins related to insect immune functions to decline the defensive ability to the fungal pathogen by dephosphorylation of golgi autoantigen.
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