Study On Silencing Of The Carra Gene By RNA Interfetrnce In Filamentous Fungus Blakeslea Trispora

The filamentous fungus Blakeslea trispora is the best strain used for the production of carotenoids.However,the study on gene function and metabolic regulation of Blakeslea trispora is limited.The traditional approach,homologous recombination,is both time-consuming and labor-intensive compared with RNA interference(RNAi) in filamentous fungi.In this paper,RNAi method was tried to silence lycopene cyclase gene carRA in Blakeslea trispora.The relevant techniques suitable for filamentous fungi and the effect of shRNA-mediated RNAi in Blakeslea trispora were investigated.The results obtained in this paper will make us understand the metabolic pathway more thoroughly and provide a new powerful genetic tool for strain improvement and genome-wide analysis of this biotechnically important filamentous fungus.In our investigation,four shRNA-expressing plasmids(shRNA-carRA1, shRNA-carRA2,shRNA-carRA3,shRNA-control) were constructed using the mU6 pro vector.Firstly,the oligonucleotide sequences of three different carRA shRNAs and a control shRNA were designed and synthesized.These oligonucleotide sequences encoding shRNAs for targeted gene carRA coding region were taken from GenBank,and they were evaluated for sequence specificity by a BLAST search and did not show homology to other known genes.The shRNA expression cassette driven by U6 promoter was engineered into the XbaI and BbsI sites of plasmid mU6 pro vector,resulting in the following plasmids:mU6 pro shRNA-carRA1,mU6 pro shRNA-carRA2,mU6 pro shRNA-carRA3 and mU6 pro shRNA-control.The mU6 pro shRNA-control was designed as an unspecific shRNA which has no homology to carRA or other known genes.To generate plasmids for cloning shRNAs,mU6 pro vector was linearized with XbaI and BbsI restriction endonuclease and the annealed oligonucleotides were ligated with the linearized mU6 pro vector at XbaI-BbsI sites using T4 DNA ligase.All the constructed plasmids were confirmed by restriction endonuclease digestion and DNA sequencing.The effects of key parameters on the preparation and regeneration of protoplast from theβ-carotene-producing fungus Blakeslea trispora were discussed in our study,including combination of various enzymes, mycelial age,digesting time and temperature,pH value,osmotic stabilizers,pretreatment,culture medium and culture method.Under the condition of mixed enzymes in osmotic stabilizer(0.6 mol/L NaCl) combined with 2%lysozyme,3%cellulase and 3%snailase,the highest protoplast yield as high as 7.48×10~6 protoplasts/mL was obtained when mycelial age was 60 h at pH 6.0 with digesting for 14h at 28℃.After purification of the obtained protoplasts,they were regenerated in PDA regenerative medium using bilayer plate culture method.To investigate the activity of protoplasts and usability of the mU6 pro vector,we assayed genetic transformation of these protoplasts with the mU6 pro vector.It is clear that the transformants with mU6 pro-GFP showed strong GFP expression,which can prove that the protoplasts were active and applicable in further gene manipulation experiments,the method of transformation is befitting and mU6 pro vector was suitable for Blakeslea trispora.Monitoring of downregulation of carRA gene expression at mRNA level was conducted.Isolation and purification of biologically active RNA from filamentous fungi is difficult because of the complex cell wall and the high level of polysaccharides which bind to or co-precipitate with RNA.Using benzyl chloride and guanidine thiocyanate,RNA was successfully isolated from Blakeslea trispora in which other RNA extraction methods and commercially available kits failed to deliver suitable results.The integrity of the RNA was further substantiated by RT-PCR and Northern hybridization,respectively.This procedure should be useful for isolating RNA from other filamentous fungi and therefore, will serve as an important tool for the molecular analysis of these organisms.Then Real-time quantitative RT-PCR was carried out for mRNA expression level assay.Two plasmids(mU6 pro shRNA-carRA1 and mU6 pro shRNA-control) were respectively transfected into protoplast of Blakeslea trispora and their effects on carRA mRNA levels were determined by comparison with the untreated cells by Real-time quantitative RT-PCR analysis.The results showed that carRA mRNA levels were decreased by 56.8%in the mU6 pro shRNA-carRA1 transfected group at 84h after transfection,compared with nontransfected (untreated-control) group.The results obtained in this paper indicated carRA mRNA expression was effectively suppressed by mU6 pro shRNA-carRA1.Monitoring of downregulation of carRA gene expression at protein level was conducted.For production of specific anti-carRA antibodies,a polypeptide(KC-15)antigene was designed and synthesized.KC-15 is a kind of hapten that must conjugate to carrier protein KLH with MBS methods.Highly titer and specifc antiserum is prepared.At the same time, the system of protein extraction suitable for Blakeslea trispora was established.Then to determine whether the decreased mRNA expression could be translated into decreased carRA protein levels,Western blot hybridization analysis was employed in the carRA.The results showed carRA protein expression was significantly reduced by mU6 pro shRNA-carRA1,compared with nontransfected(untreated-control) cells, whereas mU6 pro shRNA- control transfection could not affect the carRA protein expression.These decreases in carRA protein levels correlate well with the reduction in its mRNA levels.Monitoring of downregulation of carRA gene expression at performance level was conducted.To further evaluate whether silencing carRA gene in Blakeslea trispora may inhibit cell growth and proliferation,the morphology of protoplasts and mycelia nontransfected and transfected with mU6 pro shRNA-carRA1 and mU6 pro shRNA-control were detected by microscope.It was found that shRNA-mediated carRA gene silencing had no distinct effect on cell growth and viability. Furthermore,the carotenoid production of Blakeslea trispora nontransfected and transfected with mU6 pro shRNA-carRA1 and mU6 pro shRNA-control was analyzed by HPLC.Our results revealed that the production of lycopene andβ-carotene of Blakeslea trispora in fermentation was reduced remarkably,after treatment with mU6 pro shRNA-carRA1,compared with nontransfected(untreated-control) group, whereas mU6 pro shRNA-control transfection could not affect their production.The results were well consistent with recent report that carRA gene encodes lycopene cyclase and phytoene synthase.Because phytoene is the first carotenoid in the biosynthesis pathway of Blakeslea trispora, inhibition of phytoene synthase results decrease of production of both lycopene andβ-carotene.Our results demonstrated that carRA RNAi could effectively down-regulate carRA expression with great specificity. The shRNA-expressing plasmids could successfully suppress the expression of carRA at different levels.