The Analyses Of Antimicrobial Activity And Tricine-SDS-PAGE About Antimicrobial Peptides From Immune Tenebrio Molitor Induced By Different Microbial Treatments

In this thesis, hungry Tenebrio molitor larvaes induced to generate immune response via feeding with four strains of bacteria, and with ten strains of fungi and a strain of actinomycete after pricking. The antimicrobial peptides extracts were obtained by grinding, heating and refrigerated centrifugation. Its antimicrobial activity against 24 plant pathogenic fungi and two animal pathogenic fungi were measured by inhibition zone diameters, and evaluated by trisection to four types and inhibitory spectrum. The frequency of the first three antimicrobial activity treatments were calculated. Differences of antimicrobial peptides from immune T. molitor induced by different microbial treatments were isolated and purified by Tricine-SDS-PAGE, and analysed by Gel-Pro analyzer software.The molecular weight range of the main active ingredient were determined. The results are followed below:1. It is remarkably different that antimicrobial activity of the antimicrobial peptides from immune T. molitor induced by different microbial treatments.The maximum antimicrobial activity diameter(Dmax) of the antimicrobial peptides against various pathogens were Botrytis cinerea (from tomato), Botrytis cinerea (from cucumber), Alternaria brassicae, Fusarium solani, etc, with a maximal of 6.23 mm in Botrytis cinerea (from tomato) useing antimicrobial peptides induced by Beauveria bassiana (from Bombyx mori) and a minimal of 0.94 mm in Bipolaris sorokiniana useing antimicrobial peptides induced by Streptomyces griseolus. To a pathogen, the highest sum of high activity and middle activity treatments were 32 in both Botrytis cinerea (from tomato) and Rhizopus stolonifer, each accounting for 53% of their total treatments(60),the lowest sum was 2 in Ceratocystis fimbriata, accounting for 3%. The most number of treatments without activity was 44 in Colletetrichum orbiculare (from melon), accounting for 73% of their total treatments(60), the least number was 4 in Staphylococcus aureus, accounting for 7%.2. It is remarkably different that the inhibitory spectrum of induction treatments of antimicrobial peptides from T. molitor.Compared with the control groups, the number of inhibitory spectrum of treatments induced by Bacillus sublitis, Bacillus thuringiensis, Staphylococcus aureus and Esch- erichia coli present an increasing number of inhibitory spectrum at high activity, medium activity and low activity level and a decreasing number of inhibitory spectrum at inactive level. Bacillus sublitis showed the highest antimicrobial activity. Inducing with ento- mogenous fungi and biocontrol-used Streptomyces albogriseolus increased inhibitory spectrum of high activity remarkably, and medium activity and decreased inactivity remarkably. It should be noted that the number of inhibitory spectrum at the 1^st, 2^nd and 3^rd d was different, as well as species of inhibitory spectrum, while common fungus Saccharomyces cerevisiae basically maintained at the level of the control group.3. It is remarkably different that the frequency of the first three antimicrobial activity treatments that induced T. molitor to the antimicrobial peptides.The frequency order are biocontrol bacteria, entomogenous fungi, insecticidal actino- bacteria and animal pathogenic bacteria etc, with a maximal of 11.5 induced by Bacillus sublitis and a minimal of 2.6 induced by Beauveria bassiana (from Bombyx mori), Bacillus thuringiensis and CK-water. The maximal frequency is about 4.4 times to the minimal frequency. Bio-control microbes can fully induce insects to express antimicrobial peptides.4. It is different to some degrees that the component of antimicrobial peptides from immune T. molitor induced by different microbial treatments.There is some difference in antimicrobial peptides component and amount of immune T. molitor induced by different microbial treatments based on extraction time, as well antimicrobial peptides induced by the same microbial treatments. A series of polypeptides bands (electrophoretically pure) were obtained, for example 4.2kD, 5.2kD, 6.6kD, 6.9kD, 11.0kD, 12.6kD, 13.2kD, 14.7kD, 17.8kD, 18.7kD, 23.6kD, 24.4kD, 25.1kD, 25.3kD,25.8kD, 26.0kD, 27.6kD, 28.5kD(About 4.5kD was repored),etc. The antimicrobial peptides induced by Beauveria bassiana (from Bombyx mori) against Botrytis cinerea (from tomato) have 5.2kD, 6.6kD, 11.0kD, 18.7kD, etc.5. Choosing the appropriate way of induction and time of extraction is very important to explore antimicrobial peptides resource.The research found out that the species of inducer and extraction time have great influence on types, quantity and activity of antimicrobial peptides. Different ways and extracting time differently can induce antimicrobial peptides in number and variety. Types of antimicrobial peptides are the main reason for its antimicrobial activity and inhibitory spectrum difference. Inducible expression of antimicrobial peptides has a complex regulation mechanism. The inducible antimicrobial peptides play a major role in the antimicrobial activity.