Cloning And Expression Of The Virulence Genes From Isaria Fumosorosea And Generation Of Transgenic Beauveria Bassiana Strains With These Genes

Isaria fumosorosea is a kind of entomogenous fungi belongs to Ascomycota and have a worldwide distribution.It was recorded to be able to infect many species insects belonging to 25 families including some important agricultural insects,plutella xylostella, diuraphis noxia and bemisia tabaci,etc.In order to invade the hosts,the fungus mostly excretes several varieties of ectoenzyme hydrolases for degradating the body wall of the insect.Those hydrolases mainly contain protease,chitinase,lipase,and many others.The biocontrol fungus Beauveria bassiana plays an important role in biological control against insect pests,and it also has been studied and applied widely recently.In China,it has been mass-produced in metric tons and applied in large scale against Masson's pine caterpillar, Dendrolimus punctatus(Walker),making this one of the largest,successful microbial pest control programs in the world.However,the fungal insecticide has such shortcomings for example,slow kill,susceptible to environment,unstable kill effect,and relative narrow insecticidal range,resulting in the limit for being used widely.Thus in ordor to gain the commercial fungal insecticide,it is necessary to apply genetic and cell engineering technologies to improve strain virulence.This thesis conducts the following several researches on Isaria fumosorosea and Beauvewria bassiana:1.The screening of high yielding protease strain and the optimization of conditions for chitinase producing by the corresponding strain.By analyzing a couple of tens of Isaria fumosorosea preserved in this lab,we firstly selected 16 strains of Isaria fumosorosea with different hosts and different habitats,and then screened out the strain RCEF3304 which has a relatively high protease activity by protease activity assay.The further study on the optimization enzymatic kinetics study of yielding chitinase from this strain demonstrates that the appropriate condition are:2%(m/v) of colloid chitin,3%(m/v) of NaNO₃,0.05%(m/v) of Mg²⁺,pH6.0 as the initial value of the culture medium is,inoculum concentration is 12%,temperature is 25℃,time is 36 h.2.The clone and analysis of Isaria fumosorosea protease gene,chitinase gene and upstream regulatory elementsWith SMART RACE and DNA walking technology,we successfully cloned two important new genes from Isaria fumosorosea RCEF3304,protease gene and chitinase gene,which are associated with its virulence.And we deposited the two genes to the GenBank database coded as FJ423001 and FJ377733 respectively.The subsequent studies was conducted as follows:1) The full-length cDNA of lfuprl gene is 1704bp,with 1287bp,233bp and198 bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region (3'-UTR),respectively.The open reading frame(ORF) encodes 428 amino acids.The theoretical molecule quantity of mature protein is 46.3kDa with a calculated pI of 7.37.In the 428 amino acids,there are 50 with strong alkali(K,R),52 with strong acid(D,E),146 hydrophobic amino acids(A,I,L,F,W,V),and 105 polar amino acids(N,C,Q,S,T,Y), taking up 11.8%,12.2%,34.3%and 24.7%respectively.Using of the biological sofeware DNAman,Alignments with the deduced amino acid of mature proteins in 4 species of fungi showed 88%,71%,71%and 70%,respectively,identical with those of Isaria farinosa(AAY87460),Hypocrea lixii(ABI84117),Trichoderma atroviride(ABG57252), and Metarhizium anisopliae var.anisopliae(CAD13274),respectively.Meanwhile,the active site residues for serine proteases could be readily identified in the mature protease.2) The full-length cDNA of IfuchilI gene is 1704bp,with 1542bp,33 bp and 234bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region (3'-UTR),respectively.The open reading frame(ORF) encodes 425 amino acids.The theoretical molecule quantity of mature protein is 47.6 kDa with a calculated pI of 4.89.In the 425 amino acids,there are 39 with strong alkali(K,R),54 with strong acid(D,E),139 hydrophobic amino acids(A,I,L,F,W,V),and 118 polar amino acids(N,C,Q,S,T,Y), taking up 9.2%,12.7%,32.6%and 27.8%respectively.Alignments with the deduced amino acid of mature proteins in 4 species of fungi showed 90%,71%,71%and 70%, respectively,identical with those of Lecanicillium lecanii(AAX56960),Hypocrea lixii (KAA05855),Hypocrea virens(AAL78814),and Isaria javanicus(AAZ83728), respectively.It is prognosticated that,on the guarding area by Blast,these chitinases all belong to the family of Glycosyl hydrolase 18,and contain many conserved regions, including 2 highly- conserved regions SIGG and DGIDVDWE,which are the centred point of active hydrolysis of 18 family.3) It is shown that,by analysis to the structural gene of Ifuprl and the upstream promoter region,there is no intron existing in the structural gene,and this sequence not only contains the central stucture order CAAT frame of the promoter,but also covers some differential response components and the binding sites of transcription factors(including GATA-1,GATA-2,CdxA,etc.).The CAAT frame is a component of controlling transcription speed,but other binding sites of transcription factors(including GATA-1, GATA-2,CdxA,etc.) may take on distinct controlling function.The team work of several GATA binding sites of transcription factors can notably enhance the report of genetic transcription activity,however,there is also research illustrating that GATA is able to restrain gene.As a part of the promoter,the CdxA component usually lies at the end of 5', and it can increase the genetic transcription activity within the carrier in the system of delivering CdxA protein report.4) It is shown that,by gaining the structural gene of Ifuchil and the upstream promoter regions,there is 2 introns existing in the structural gene,and the uptream promoter has no obvious TATA frame and CAAT frame,but includes many binding sites of transcription factors,eg.GATA-1,etc..The uptream region also has the conjoint sequence between pyrimidine-rich and purine-rich,and it may be the binding site of the Glucose restraining component Mig1 and Mig2,which are activated by Glucose directly or indirectly so as to restrain the expression of Ifuchila.The promoter region lfuprlb and Ifuchilb both have the potential pressure-response componet STREs(CCCCT).The reason might be the genetic transcription or expression of Isaria fumosorosea's parasitic life is induced by insect nutrient to gain virulence gene.3.Generation of transgenic Beauveria bassiana strains with Protease gene and chitinase gene from Isaria fumosorosea and its increased virulence against Dendrolimus punctatus.In order to obtain Bb13-Ifupr1 and Bb13-Ifuchi1,the blastospore preparation methods of genetic transformation was resorted to transformate strain Bb13 produced by Beauveria bassiana—The enzymatic activity measurement assay,result shows that activity in transgenic compared with wild-type strains has been improved significantly.Wild-type strains were 2.65 times and 1.73 times of activity.In the biometrics study on Dendrolimus,the average LT₅₀ of recombinant strain Bb13-Ifuprl was reduced by 33.7%and the average LT₅₀ of recombinant strain Bb13-Ifupr1 was reduced by 29%by comparing with wide-type strain while the spore concentration is Bb13 is 1×10⁷ /ml,In addition,the average LT₅₀ of recombinant strain Bb13-Ifupr1 was reduced 4.2 times of spore usage,and Bb13-Ifuchi1 was reduced 1.7 times of spore usage comparing with wide-type.The cost of biological control,therefore,has been decreased dramatically.