| Monochamus alternatus is a serious forest pest, which attacks pine trees and tunnels in the host tree stems and branches, and is a major vector of pine wood nematode, Bursaphelenchus xylophelus. Entomologenous fungi are important biological control agents, and are widely applied to control insect pest. It is for some years to control M. alternatus by entomogenous fungi. But it is limited to control pest because only spore can be used. Screening toxic proteins to M alternatus from entomogenous fungi is the base of control pest. It will reveal the mechanism how the fungi infect their host from the view of biochemistry and molecular biology, and base for culture trees of anti-longhorn beetle.Six strains of entomogenous fungi are selected in the experiment from the collections in our laboratory. Six extracellar total protein extracts are obtained by dialysis after ammonium sulfate precipitation. Six intracellar total proteins are extracted by grinding hyphae. Forty eight proteins, including 26 intracellar and 22 extracellar ones, are purified from 12 total protein extracts by preparative polyacrylamid gel electrophresis and eluticn gel strip.The result of primary bioassy for 48 proteins shows that 25 proteins are no toxic to the larvae of M alternatus, 19 are low toxic , four are high toxic. Low toxic proteins can be grouped into two groups, i.e., group I and groupII. Group I has 10 ,which are Bb36W-E, Bb36N-B, Bb36N-D, Bb36N-E, Bb03W-A, Bb03N-C, Bb03N-D, Bb03N-E, Ma09W-B, Pf01W-A. GroupII has nine, which are Bb36N-A, Bb36N-F, Bb06W-D, Bb06W-E, Bb29W-B,Ma09W-C,Ma09N-C,Pf01N-A,Pf01N-B. High toxic proteins are Bb36W-C, Bb36W-D, Bb06N-C, Ma09N-A. The result of second bioassy for four high toxic proteins shows that the toxicity of protein Bb36W-D is the highest among all proteins. According to result of second bioassy, the sequence of toxicity, which is from high to low, is Bb36W-D, Bb36W-C, Bb06N-C and Ma09N-A.At the beginning, the host larvae injected with toxic protein refuse to feed, then act less, obviously reduce the activity of tunneling in the artificial diet, and are lying in most time. Most of them laier became dead con.ing out from the diet. The complexion of the host larvae keep normal for a short time after death. The host body will change to soft, and to be brown, and finally black in color with in 1-2 days tifter death. Little difference of symptom was observed after the host larvae were injected with toxic proteins. Part of them treated were alive, but they can't pupate in normal way, the pupa were abnormal, pupa couldn't moult for emergence, the adult was abnormal.The highest toxic protein Bb36W-D is composed of two subunits, which are big subunit and small subunit. Molecular weight of them is calculated to be 24 kD and 17 kD respectively by SDS-PAGE. Isoelectric point is 9.59 and 9.40 respectively by 1EF. N-terminal ammonia acid sequence of Bb36\V-D is Glu→Pro→Ser→Ile→Val.The protein Bb36W-D is composed of 17 ammonia acids. The contents of alkaline ammonia acid is higher than acidic ammonia acid. Concentration of arginine, histidine and alamine is 0.13, 0.12, 0.11mg/mL respectively.There are significant difference in zymog:ams of csterasc isozyrnes between the host larvae injected with Bb36W-D and the control. The zymograms of the treated increase band E3, and decrease band E5. Band E6 first become short, then become wide rapidly. There was obvious physiological change in the host larvae injected with Bb36W-D.According to the above, Bb36W-D from B. bassiana shows very strong toxicity to the larvae of M. alternatus. The primer should be designed by N-terminal ammonia acid sequence, then the gene will be cloned by PCR. Trees of anti-longhorn beetle will be cultured after the gene transferred into trees.
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