Disruption Of PksCT Gene In Monascus Aurantiacus And Construction Of The Long Homologous Arms Replacement Vector

In this paper, PCR analysis was used to research the conservatism of citrininbiosynthesis gene—pksCT in different Monascus species. Fourteen producingcitrinin Monascus spp. coming from seven Monascus species were used inexperiment. A pksCT-replacement vector pHD 116 was constructed to disrupt pksCTgene by the genetic engineering technique, and was transformed into M. aurantiacusAS 3.4384 by protoplast PEG transformation and electroporation transformation.Transformants were screened with hygromycin B resistance and PCR analysis, thenanalysis of the expression of the red pigments and citrinin were used to gain thepksCT-disrupted strain. A method to construct the replacement vector by RecETrecombination system was found, and it provided a fast way to construct thereplacement vector of gene-knockout in Monascus.The major findings were expounded as follows:1. Two DNA fragments from the transcriptional start region and the stop codonregion of pksCT, which are 669bp and 591bp, were gained by genomic PCR fromfourteen Monascus spp effectively. Results of NCBI BLAST showed that PCRproducts exhibited high identity with each other, and were in accordance with thepartial sequence of pksCT genein M. purpureus which was announced in Genbank. Itis suggested that pksCT gene is highly conserved in the fourteen citrinin-producingMonascus spp.2. PksCT was the targeting gene which is responsible for citrinin biosynthesis inMonascus. Homologous arm sequence fragments A and B from the transcriptionalstart region (65-733) and the stop codon region (8349-8940) of pksCT were gainedby genomic PCR from M.aurantiacus AS3.4384, which is a citrinin high-producingstrain. A pksCT-replacement vector (pHD116) was constructed by the DNArecombinant technique such as restriction enzyme cutting and ligase ligation andtransformation. A hygromycin resistance gene in the middle of homologous armsequence fragments A and B was used for the selection marker. The vector couldaim at the targeted gene pksCT on chromosome of Monascus accurately with Homologous arm sequence fragments. A hygromycin resistance gene was used toscreen target gene disruptants.3. The conditions of protoplast-PEG transformation and protoplast-electroporationwere optimized. The pksCT-replacement vector pHD116 was transformed intoprotoplast of M.aurantiacus AS3.4384 to disrupt pksCT. Then the methods ofhygromycin resistance and genomic-PCR were used to screen disruptants. ApksCT-disrupted strain PHDS26 was gained, whose capacity of producing citrininwas decreased by about 97.2％compared with the original strain. At same time, itscapacities of producing red pigments and yellow pigments were increased by 49.4％and 28.8％4. Two short homologous sequences (55bp), which were homologous to hph geneand pUC18 vector respectively, were designed on the 5'end of primers. PCRfragments E and F from exterior sequences of pksCT, were transformed intoEscherichia coli YZ2005 by electroporation in turn. When homologousrecombinations happened twice with the catalysis of homologous recombinationenzyme RecE and RecT, fragments E (2437bp) and F (2694bp) were replaced thehomologous fragments A(668bp ) and B (591bp) in pHD116. A newpksCT-replacement vector pHF08 with longer homologous sequences E and F wasreconstructed.