Study On The Pathogenicity Modification Of Beauveria Bassiana And Its Application In Biological Control And Production Of Bombyx Batryticatus

Beauveria bassiana belongs to filamentous fungi,which has been widely studied.As an important entomopathogenic fungus,Beauveria bassiana is mainly used in biological control,but the large-scale application of related insecticides is limited to a certain extent due to its slow insecticidal speed.For silkworm,Beauveria bassiana has another major application,which is Bombyx batryticatus,a kind of Chinese traditional medicine.Bombyx batryticatus is the dried dead larvae of Bombyx mori L.（silkworm of4～5 instars）infected by Beauveria bassiana.With anticonvulsant,antioxidant and other pharmacological activities,it is widely used in clinic.However,the current market of Chinese herbal medicine is dominated by unified supply,which makes Bombyx batryticatus have some disadvantages such as small size,light weight and so on.Silkworm is an important economic insect and a lepidopteran model insect.Ecdysone（20E）plays a very important role in silkworm.Molting is regulated by ecdysone.The spinning and cocooning of the last instar silkworm are also related to ecdysone.Previous studies have shown that ecdysteroid uridine diphosphate glucosyltransferase（ecdysteroid UDP-glucosyltransferase,EGT）can inactivate ecdysone.In addition,overexpressing EGT in the last instar silkworm can reduce ecdysone titer.In this study,egt was used as the target gene to construct the recombinant fungal expression vector.Transgenic Beauveria bassiana was obtained by transforming the recombinant fungal expression vector into Beauveria bassiana,and then inoculated into the early instars silkworm and the last instar silkworm respectively,so as to evaluate the effectiveness of transgenic Beauveria bassiana in biological control and Bombyx batryticatus.The main results are presented as follows:1.The isolation and purification of Beauveria bassiana and screening of antibioticsFirstly,Beauveria bassiana strains were successfully isolated from Bombyx batryticatus by PDA solid medium.And then purified Beauveria bassiana strains were screened after 20 generations of single spore culture.Because the acquisition of transgenic Beauveria bassiana required the use of antibiotics to screen positive strains,so we carried out the screening of antibiotics.After consulting massive related references,hygromycin,zeocin and G418 sulfate solution were selected for the test.It was found that G418 sulfate solution at the concentration of500μg/m L could completely inhibit the growth of Beauveria bassiana for 4 days,so it could be used as a screening marker for genetic transformation and the selection pressure of transformed progeny.2.The construction of transgenic fungal expression vectorAccording to the codon preference of Beauveria bassiana provided by NCBI database,the target gene containing EGT was optimized and connected to the fungal expression vector p G418 containing G418 resistance gene.The transgenic fungal expression vector was successfully constructed.3.The transformation and identification of Beauveria bassianaIn this study,the method of fungal transformation was protoplast transformation.The protoplasts of Beauveria bassiana were obtained by preparing conidia suspension,exploring spore germination conditions and enzymolysis.The morphology of the protoplasts was translucent and round under the microscope.Then,the recombinant fungal expression vector was transferred into the protoplasts of Beauveria bassiana.And the transformants were inoculated into PDA solid medium for further culture.After that,the identification of transgenic Beauveria bassiana was carried out at the genome and RNA levels.The results indicated that the transformant 6 could detect the target gene and sequenced correctly.It showed that the transgenic Beauveria bassiana was successfully obtained.4.The effectiveness evaluation of transgenic Beauveria bassiana in biological controlIn this part,silkworm larvae on the first and second days of the second instar and the first day of the third instar were selected for the experiment.Infecting silkworm larvae on the first and second days of the second instar,the half lethal time(LT₅₀)of transgenic Beauveria bassiana was 24 hours shorter than that of the wild-type.On the first day of inoculation,the number of young silkworms killed by the transgenic strain before second instar molting was three times that of the wild-type;On the second day of inoculation,the number of young silkworms killed by the transgenic strain before second instar molting was 20%,while that of the wild-type was 0%.Infecting with silkworm larvae on the first day of the third instar,the LT₅₀of the transgenic strain was12 hours shorter than that of the wild-type.There was no significant difference in the number of silkworms died after moulting,but silkworms infected with the transgenic strain became more rigid within the same period of time.The above results showed that the pathogenic time of transgenic Beauveria bassiana was shortened,and the effect on the second instar silkworm was more obvious.The genome and RNA of Beauveria bassiana was extracted from silkworm carcasses and the target gene was detected.The results demonstrated that the target gene were successfully detected in the experimental group,but not in the control group.It was indicated that EGT played a role in shortening the pathogenic time of transgenic Beauveria bassiana.5.The influence analysis of transgenic Beauveria bassiana on the formation time of Bombyx batryticatusTransgenic and wild-type Beauveria bassiana were used to infect silkworm larvae in different days of the fifth instar,and the effectiveness of transgenic Beauveria bassiana in Bombyx batryticatus was evaluated.When inoculating silkworm larvae on the second day of the fifth instar,the weight of Bombyx batryticatus obtained after treatment with transgenic Beauveria bassiana was less than that of the wild-type;When inoculating on the third and fourth days of the fifth instar,the weight of Bombyx batryticatus obtained after treatment with transgenic Beauveria bassiana increased,and the effect was more obvious when inoculating at a lower concentration on the third day of the fifth instar.Furthermore,the 20E titer in silkworm blood decreased at 96 hours after inoculation with transgenic Beauveria bassiana;When inoculating on the fifth day of the fifth instar,all silkworms entered prepupal or pupal stage after treatment with transgenic and the wild-type Beauveria bassiana.Compared with the wild-type,transgenic Beauveria bassiana with EGT can shorten the death time of growth silkworm,thus affecting the formation time of Bombyx batryticatus.Therefore,the inoculation time of Beauveria bassiana can be postponed to a certain extent.Through this part of the research,it was confirmed that the transgenic Beauveria bassiana obtained at present inoculated on the third day of the fifth instar was the most suitable:it can significantly increase the weight of Bombyx batryticatus obtained and reduce the proportion of Bombyx batryticatus in the cocoon compared with the control.