Cloning And Characterization Of The Mrpks10 Gene From Monascus Ruber

The polyketide synthease (PKS) is able to catalyze the vast amount of products of polyketides during the metabolism of Monascus spp., including monascus pigment, monacolin K and citrinin. The gene clusters of monacolin K and citrinin in Monascus spp has already been submitted to NCBI. However, the polyketide synthease gene which can control the monascus pigment, have not been reported. Cloning the potential polyketide synthease gene from Monascus ruber will lay the foundation for the further study of the polyketide biosynthesis.In this study, a pks gene called MrpkslO was cloned by degenerate PCR using Monascus ruber M-7 as the starting strain. The technique of gene knockout was used to study the function of this gene, and the main research contents are as follows:1. Cloning and analysis the MrpkslO geneAccording to the conserved sequence of the amino acid and nucleic acid sequence of the KS domain of pks gene from the species which have the closer genetic relationships, a pair of degenerate primer was designed and a fragment of MrpkslO about 700 bp was cloned. Using the routine PCR and SON-PCR, both sides of this sequence were extended, and the total sequence was 11787bp eventually. The bioinformatics analysis showed that this sequence includes two genes, and the longer one is the target gene with 7304 bp of ORF, which contains 10 exons and 9 introns and coding 2038 amino acids. The DNA sequence of this gene has high homologue with the pksl gene of Monascus purpureus and the similarity is 91%. Removed the introns and then made BlastX match, the gene was predicted to control the synthesis of monascus pigment; and the result of Blastp indicated that MrpkslO has four domains such as Ketoacyl synthase, Acyl transferase, Acyl carrier protein and Thioesterase, which belongs to the non-reducing PKS.2. Knockout of MrpkslO from M.ruber M-7The MrpkslO replacement vector was constructed using a hygromycin B resistance gene (hph) as selective marker and transformed into M. ruber M-7 by Agrobacterium tumefaciems mediated transformation, and a MrpkslO deleted mutant (AMrpks10) was obtained after the identification by PCR and southern blotting.3. Primary studies on the function of MrpkslO geneIn order to identify the function of this gene in the the process of growth in M. ruber M-7, the basic biological characteristics of△Mrpks10, including the colony color and the colony morphology, the structure of conidium、cleistothecium and mycelium, and the quantity of citrinin,intracellular and extracellular pigments, The results showed that, there was no significant difference in the basic biological characteristics between AMrpksl0 and M-7.It seems that gene silencing may have happened in Mrpks10.RT-PCR should be used to identify the level of mRNA of MrpkslO in the next experiment, and prove that the gene can not be expressed at the level of mRNA.