Cloning And Expression Of The Chitinase Ⅱ Genes From Isaria Fumosorosea And Inductive Transcription Of Two Chitinase Genes

Entomogenous fungi, an important lethal factor of insect in ecosystem, have significantpandemic potentiality, and play an important role in maintaining ecological balance. Someentomogenous fungi have been developed into fungal pesticides to widely use in thecontrol of pests. The pan-global Isaria fumosorosea infects host species in numerousorders of insects, such as Lepidoptera, Homoptera, Diptera, Hymenoptera, Coleoptera andso on. Isaria fumosorosea have been used in greenhouses in North America and Europe.These mycoinsecticides are registered for use in greenhouses to control whiteflies, aphids,thrips and scales.Multiple chitinases were found in entomogenous fungi in the previous study, butwhich chitinase plays a majority role in the infection process has not been elucidated. Achitinase (Ifuchit1) of Isaria fumosorosea was previously cloned using RT-PCR and DNAwalking technology, and another novel chitinase gene (Ifuchit2) has been cloned fromIsaria fumosorosea in the study. The Ifuchit2gene sequence was deposited to the GenBankdatabase coded as HQ267382. The full length of the cDNA is1584bp containing a ORF of1272bp that encoded423amino acids with a stretch of22amino acid residues displayingcharateristics of signal peotide. The theoretical molecule weight of the mature protein is46.57kDa with a calculated pI of5.169. Among the423amino acids, there are37withstrong alkali amino acids (K, R),48with strong acid amino acids (D, E),150hydrophobicamino acids (A, I, L, F, W, V), and119polar amino acids (N, C, Q, S, T, Y). It waspredicted that, according to the conserved region by Blast, these chitinases all belonged tothe family of glycosyl hydrolase18and contained many conserved regions, includingactive site (SIGG) and binding domain (DGIDVDWE). With genome walking technology,the structural gene of Ifuchit2and the upstream promoter region were cloned. The length ofthe structural gene is1425bp, containing3introns with size of54bp,53bp and51bplocated at the sites of121st~176th base,277th~331th base and380th~431st base,respectively. In the current study, the upstream sequence of chitinases (Ifuchit2) fromIsaria fumosorosea was cloned by genome walking based on its structural gene. It contains1794bp, of which209bp are overlapped compared with its cDNA sequences. The sequenceanalysis indicated that the upstream sequence has a typical eukaryotic gene TATA box andseveral binding sites of potential highly conserved transcription factor, such as Oct1,GATA-1, GATA-2, CdxA. Additionally, it contains some specific response elements, suchas the heat responsive element (HSE), stress response elements (STRE).The cloned gene Ifuchit2of Isaria fumosorosea was ligated with secreting expression vector pPIC9K and then transformed into Pichia pastoris GS115using electroporation.The activity of Ifuchit2was37.3U/ml after the induction of methyl hydrate for144h. Thecrude enzyme solution was precipitated by80%ammonium sulfate and the centrifuged.The precipitate was dissolved in Tris-HCl buffer solution (50mmol/L, pH8.0) and dialyzedin the same buffer for24h. After centrifugation, the supernatant was purified byDEAE-Sepharose column and detected the activity of Ifuchit2. The collected solution withactivity of Ifuchit2was pooled together and concentrated by PEG20000. The molecularweight of enzyme was determined to be46kDa by SDS-PAGE, which was close to thepredictive molecular weight (46.57kDa).The total extracted RNA of Isaria fumosorosea RCEF3304strain was extracted afterinduction of chitin for12h,36h and60h, respectively. Meanwhile, the expressions ofIfuchit1and Ifuchit2were quantified to compare the expression difference between the twogenes and the differences of two gene transcriptions before and after induction byFluorescent Quantification PCR System. The result indicated that the expressions of thetwo genes increased after induction, and the expressions of Ifuchit1both were higher thanthat of Ifuchit2whether induced by chitin or not. We therefore speculate that Ifuchit1playsa major role in the infection of host, and it may be a major gene of chitinase in Isariafumosorosea RCEF3304.