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20081217 06李利 紅麴菌G蛋白Alpha亞基Mga1介導的信號轉導 (Pp Tminimizer)
 

20081217 06李利 紅麴菌G蛋白Alpha亞基Mga1介導的信號轉導 (Pp Tminimizer)

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20081217_06李利_紅麴菌G蛋白alpha亞基Mga1介導的信號轉導

20081217_06李利_紅麴菌G蛋白alpha亞基Mga1介導的信號轉導

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    20081217 06李利 紅麴菌G蛋白Alpha亞基Mga1介導的信號轉導 (Pp Tminimizer) 20081217 06李利 紅麴菌G蛋白Alpha亞基Mga1介導的信號轉導 (Pp Tminimizer) Presentation Transcript

    • Li Li, Fusheng Chen DEC. 2008 College of Food Science and Technology Huazhong Agricultural University Signal Transduction by Mga1 , a Group I G Protein Alpha Subunit of Monascus ruber
    • Contents
      • Ⅰ . Introduction
      • Ⅱ . Cloning of G α subunit gene Mga1
      • Ⅲ . Deletion of G α subunit gene Mga1
      • Ⅳ . Effects of Mga1 -mediated signaling on growth and development
      • Ⅴ . Effects of Mga1 -mediated signaling on secondary metabolite production
      • Ⅵ . Conclusion
      • All cells have the capacities to sense and respond to various external signals
      • Nutrients
      • Hormones
      • Physical stimuli
      • Chemical stimuli
      • Environmental stress
      Ⅰ . Introduction
      • In this signal transduction, the G protein system plays a pivotal role
      Nobel Prize for Physiology / Medicine Alfred G. Gilman and Martin Rodbell "for their discovery of G-proteins and the role of these proteins in signal transduction in cells" 1994
      • In fungi, G-protein signaling pathways have been shown to regulate various aspects
      • growth
      • morphogenesis
      • mating
      • virulence
      • secondary metabolite production
      • Aspergillus nidulans
      • the model filamentous fungus
      • genome
      • expressed sequence tags (ESTs)
      • Many of the components of the G protein / cAMP signaling pathways have been identi fi ed
    • GPCRs (16) gprA-K gprM-P NopA α-subunit (3) fadA ganA ganB β-subunit (1) sfaD γ-subunit (1) gpgA PhLPS(3) phnA phnB phnC RGS(4) flbA rgsA rgsB rgsC Adenylate cyclase (1) cyaA PKA(3) pkaA pkaB pkaR Fig.1. Basic components of G protein signaling in A. nidulans (Adapted and modified from Yu, 2006 and Lafon et al.,2006 )
    • Fig.2. G protein mediated regulation of development and ST production in A. nidulans ( Yu, 2006)
    • Fig. 3. The roles of G-protein α subunit FadA in controlling growth, development and ST production in A. nidulans (Yu, 2006)
    • Tab.1. G-protein α subunit subunits cloning from ascomycetous fungi Yu et al., 2008 Gibberella zeae 2008 Garcia-Rico et al., 2007 Penicillium chrysogenum 2007 Yamagishi et al., 2006 Alternaria alternata 2006 Reithner et al., 2005 Trichoderma atroviride 2005 Mukherjee et al., 2004 Trichoderma virens 2004 Jain et al., 2002 Fusarium oxysporum 2002 Cronover et al., 2001 Botrytis cinerea 2001 Chen et al., 2001 Arthrobotrys dactyloides 2001 Loubradou et al., 1999 Podospora anserina 1999 Horwitz et al., 1999 Cochliobolus heterostrophus 1999 Liu and Dean, 1997 Magnaporthe grisea 1997 Yu et al., 1996 Aspergillus nidulans 1996 Smulian et al., 1995 Pneumocystis carinii 1995 Choi et al., 1995 Cryphonectria parasitica 1995 Turner and Borkovich, 1993 Neurospora crassa 1993 Reference Ascomycetous fungi Year
    • NCBI taxonomy database Eukaryota (真核生物域) Fungi (真菌界) Dikarya (双核亚界) Ascomycota (子囊菌门) Pezizomycotina (子囊菌亚门) Eurotiomycetes (散囊菌纲) Eurotiomycetidae (散囊菌亚纲) Eurotiales (散囊菌目) Elaphomycetaceae( 大团囊科 ) Monascus ( 红曲菌属)
    • Ⅱ . Cloning of G α subunit gene Mga1 Fig.4. Analysis of Degenerate Primers PCR and SON-PCR Products by Agarose Gel Electrophoresis . (a) PCR products obtained with degenerate primers: M, DNA marker DL2000; Lane 1, negative control; Lane 2, product with degenerate primers. (b,c) SON-PCR products amplifying 5’ and 3’flanking sequence respectively: M, 250bp DNA marker; Lane 1, products of the primary reaction; Lane 2, products of the secondary reaction. The arrows indicate amplicons that were cloned and sequenced .
    • Fig. 5. FGENESH 2.6 predicted potential genes in sequence Fragment: 3832bp (650+1242+1940) ORF: 1242bp (651~1892) mRNA: 1062 bp (4 exons) Protein: 353 aa
    • Fig. 6. Putative conserved domains in the predicted protein GTP/Mg 2+ binding site Adenylyl cyclase interacteration site Putative receptor binding site Beta-gama complex interaction site GoLoco bibding site Switch I region Switch II region G1~G5 box
    • Fig.7. Phylogenetic tree of heterotrimeric G protein alpha subunits from different ascomycetous fungi.
    • Tab. 2 Similarity Analysis of the Deduced Protein 96 93 0.0 692 353aa CGA1 [ Cochliobolus heterostrophus ] 95 92 0.0 685 353aa gna1 [ Neurospora crassa ] 96 93 0.0 693 353aa AGA1 [ Alternaria alternata ] 98 95 0.0 707 353aa gasA [ Penicillium marneffei ] 98 96 0.0 711 353aa pag1 [ Penicillium chrysogenum ] 98 96 0.0 716 353aa FadA [ Aspergillus. nidulans ] Positives (%) Identites (%) E Value Score (bit) Over- lapping Homologs
    • Ⅲ . Deletion of G α subunit gene Mga1 Fig.8. Deletion of Mga1. Black bar, PtrpC-hygB-TtrpC; open bar, Mga1 ORF; checked bar, 5’ flanking sequences of Mga1 ORF; diagonal bar, 3’ flanking sequences of Mga1 ORF. Restriction enzyme sites: S, Sty I; X, Xba I; H, Xho I.
    • Fig.9. Identification of pCMGA1 by PCR
    • Fig.10. Identification of pCMGA1. The plasmids were digested by Xbal I and XhoI. M, DNA marker DL2000 plus; Lane 1, digested products of pCMGA1; Lane 2, digested products of pCAMBIA3300 . pCMGA1
    • Condition for ATMT of Monascus ruber ( Shao et al., 2006 ) Correct gene replacement rates: 7.03%(9 /128) : 3d Co-cultivated time : 30℃ Co-cultivated temperature : OD 600 = 0.5 EHA105 concentration : 200μmol/L AS concentration : 10 6 Conidia /mL Conidia concentration : 15d Cocultivation time : PDA Cocultivation medium
    •  
    • Fig.11. Analysis of Mga1 deletion mutans F3 R3 F1 R1 F2 R2
    • Ⅳ . Effects of Mga1 -mediated signaling on growth and development
    • Fig.12. Colony morphology. Strains were grown on PDA, and photographed 7 days(A) or 10days(B) after inoculation. Front of Petri plates are shown. A B 25℃ 28℃ 31℃ 37 ℃ M-7 M-7 GKmga1 GKmga1
    • Fig.13. Colony morphology. Strains were grown on PDA, and photographed 7 days(A) or 10days(B) after inoculation . Back of Petri plates are shown . A B 25℃ 28℃ 31℃ 37 ℃ M-7 M-7 GKmga1 GKmga1
    • Fig.14. Radial growth on PDA plates. Strains were grown on PDA at 25 ℃, 28℃, 31℃ and 37 ℃. The colony diameter were measured every after cultivating for 2days.
    • Fig.15. Radial and aerial hyphal growth. Strains were grown on PDA at 31℃. The colony diameter were measured 2days after inoculation and photographs were taken 10days after inoculation .
    • Fig.16. Radial growth on PDA plates. Strains were grown on PDA at 25 ℃, 28℃, 31℃ and 37 ℃. The colony diameter were measured 7days after inoculation.
    • Fig.17. Colony morphology . Strains were grown at 28℃ on PDA added with 0, 1, 2, 5mM cAMP and photographed 7 days(A) or 10days(B) after inoculation . Front of Petri plates are shown. A B 0mM 1mM 2mM 5mM M-7 M-7 GKmga1 GKmga1
    • Fig.18. Colony morphology. Strains were grown at 28℃ on PDA added with 0, 1, 2, 5mM cAMP and photographed 7 days(A) or 10days(B) after inoculation . Back of the Petri plates are shown . A B M-7 M-7 GKmga1 GKmga1 0mM 1mM 2mM 5mM
    • Fig.19. Radial growth on PDA plates with cAMP. Strains were grown at 28℃ on PDA added with 0, 1, 2, 5mM cAMP. The colony diameter were measured every after cultivating for 2 days.
    • Fig.20. Radial growth on PDA plates with cAMP. Strains were grown at 28 ℃ on PDA added with 0, 1, 2, 5mM cAMP. The colony diameter were measured 7days after inoculation.
    • Fig.21. Aerial hyphal growth on PDA plates with cAMP . Strains were grown at 28℃ on PDA added with 0 or 1mM cAMP. The photographs were taken 10days after inoculation.
    • Fig.22. Biomass production . Colonies were grown on cellophane – overlaid PDA at 28℃. Biomass accumulation was determined by stripping the mycelia off the cellophane and weighing directly.
    • 4 × 4 × 4 × 4 × 40 × 40 × Fig. 23. Microscopic morphology. Single-spore colonies were grown at 28℃ on cellophane–overlaid PDA and photographed after 24h and 48h. M-7 GKmga1 24h 48h 48h
    • 10 × 10 × 40 × 40 × Fig. 24. Microscopic morphology. Colonies were grown at 28℃ on cellophane–overlaid PDA and photographed after 48h. M-7 GKmga1
    • Fig. 25. Microscopic morphology. Strains were grown in YES liquid cultrue at 28℃ for 6days. M-7 GKmga1 40 × 40 × 40 × 40 ×
    • Fig.26. Microscopic morphology. Colonies were grown at 28℃ on PDA and photographed after 5d. 4× 4× 10× 10× M-7 GKmga1
    • Fig.27. Microscopic morphology. Colonies were grown at 28℃ on PDA and photographed after 5d. M-7 GKmga1 40× 40× 40× 40× 40× 40×
    • Fig.28. Microscopic morphology. Colonies were grown at 28℃ on PDA and photographed after 6d. 4× 4× 10× 10× M-7 GKmga1
    • Fig.29. Microscopic morphology. Colonies were grown at 28℃ on PDA and photographed after 8d. 4× 4× 10× 10× M-7 GKmga1
    • 40 × 40 × 40 × 40 × Fig. 30. Microscopic morphology . Strains were grown on PDA at 28℃for 10days. Aerial hyphae of the colony were stamped and photographed. M-7 GKmga1 GKmga1 GKmga1
    • Ⅴ . Effects of Mga1 -mediated signaling on secondary metabolite production
    • Fig.31. Fermention process without agitation in YES medium 8d 12d 16d 10d 14d 19d GKmga1 M-7 GKmga1 M-7
    • Fig. 32. Pigment production in YES medium 0d 4d 6d 8d 10d 12d 14d 16d 19d GKmga1 M-7 GKmga1 M-7 4d 6d 8d 10d 12d 14d 16d 19d
    • Fig.33. Optical density of pigments. All the filtrate of fermented broth were diluted 42 times by distilled water and absorbance measurements were performed with a scanning spectrophotometer.
    • Fig.34. Production of yellow, orange, and red pigments. All the filtrate of fermented broth were diluted 42 times by distilled water and absorbancemeasurements were performed with a scanning spectrophotometer .
    • Fig.35. Production of citrinin. Citrinin was extracted from the filtrate of fermented broth using TEF, and citrinin was monitored by comparison with an authentic sample by using TLC on silica gel G, Toluene -Ethyl actate – Formic acid, 7:3:1. The toxin was detected by fluorescence under a 365-nm UV light.
    • Ⅵ . Conclu s ion
      • Mga1 is required for normal growth
      • Colony diameter:
      • 79% (25℃) , 83% (28℃), 76% (37℃)
      • Fresh weight:
      • 52%(2d), 49%(3d), 60%(5d), 71% (6d)
      • Aerial hyphae
      • Aspergillus nidulans, Penicillium marneffei, Neurospora
      • crassa, …
      • Hyphae growth
      • ☆ highly branched vs straight, less branched
      • ☆ radially expanding hyphae vs long hyphal axis
      • Fusarium oxysporum, Trichoderma virens, …
      • · Thermosensitivity? 31℃
      • 91%(4d), 99%(5d), 100%(6),100%(7d), 99%(8d), 97%(9d), 96%(10d)
      • · PKA is the downstream target? exogenous cAMP
      • partially restored the aerial hyphal growth
      • In A. nidulans, FadA-mediated vegetative growth signaling is
      • transduced in part by PkaA
      • Mga1 is required for sexal reproduction
      • ☆ failed to producing cleistothecium
      • female sterile ?
      • Cochliobolus heterostrophus, Neurospora crassa,
      • Gibberella zeae, …
      • asexual sporulation?
      • ☆ reduced conidiation on plate
      • Fusarium oxysporum, Cryphonectria parasitica, …
      • ☆ hyper-active asexual sporulation in submerged culture
      • A. nidulans, Penicillium marneffei , …
      • Mga1 negatively regulates toxin production
      • ☆ enhanced CIT production
      • Aspergillus nidulans ST
      • Gibberella zeae DON and ZEA
    • The roles of G - protein α subunit FadA in controlling growth, development and ST production in A. nidulans (Yu, 2006) Other mutants? constitutively activated mutant, dominant interfering mutants Nutrients, Hormones, Physical stimuli , Chemical stimuli ,Environmental stress External signals? Other components? We have seccessfully cloned genes orthologous to sfaD/ gpgA/ pkaA/flbA /cyaA
    • ﹍﹍ ﹍﹍ The End ﹍﹍ ﹍﹍ Thank you for your attention!