20081217_05邵彥春_與紅麴菌菌絲發育相關基因的克隆及序列分析

1. Isolation and analysis of mrf A involved in aerial hyphae development of Monascus ruber Yanchun Shao College of Food Science and Technology Huazhong Agricultural University

2. Contents Isolation and analysis of mrf A involved in aerial hyphae development Introduction: Experiment:

3. 1 The hypha is the basic unit of a filamentous fungus, which is the main mode of vegetative growth, and is collectively called a mycelium. Introduction

4. Hypha grows at their tips. During tip growth, cell walls are extended by the external assembly and polymerization of cell wall components, and the internal production of new cell membrane. 2 Hyphae growth

5. In most fungi, hyphae are divided into cells by internal cross-walls called septa (singular septum). Septa are usually perforated by pores large enough for ribosomes, mitochondria and sometimes nuclei to flow among cells.

6. 3 Types of hyphae 3.1 Classification based on cell division Septate (with septa) Aseptate or coenocytic (without septa). 3.2 Classification based on cell wall and overall form Generative Skeletal hyphae

7. Understanding the mechanisms of hyphal growth is still a major challenge in fungal cell biology. 4 Genes Involved in hyphae growth and development

8. A Cellulose synthase-Like Protein involved in hyphal tip growth and morphological differentiation in Streptomyces ( Xu et al, A Cellulose Synthase-Like protein involved in hyphal tip growth and morhological differentiation in Streptomyces , Journal of Bacteriology, 2008,7, 4971–4978) A.gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. (G. Bauer et al, A Ras-like GTPase is involved in hyphal growth guidance in the filamentous Fungus Ashbya gossypii, Molecular Biology of the Cell, 2004,10( 15), 4622–4632) The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC , which are involved in septum formation. (M.Masayuki et al, Class I and Class II Chitin synthases are involved in septum formation in the fungus Aspergillus nidulans, Eukaryotic Cell, 2005, 6,1125–1136) .

9. Deletion of Wd STUA in W.dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich yeast maintenance agar medium yeast extract-peptonedextrose agar (YPDA) at 37 ℃ . Additionally, deletion of Wd STUA repressed aerial hyphal growth, conidiation, and invasive hyphal growth on the nitrogen-poor, hypha-inducing agar medium potato dextrose agar (PDA) at 25 ℃ . Ectopic overexpression of Wd STUA repressed the convoluted colony surface growth on YPDA at 37 ℃ , and also strongly repressed hyphal growth on PDA at 25 ℃ and 37 ℃ . Qin Wang and Paul J. Szaniszlo , 2007

10. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae,premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. ( F.Douglas Ivey et al.Shared and independent roles for a G-alpha protein and adenylyl Cyclase in Regulating Development and Stress Responses in Neurospora crassa .Eukaryotic Cell,2002,(4):634-642. Strains containing rho-4 loss-of-function mutations in the filamentous fungus Neurospora crassa lead to a loss of septation. （ Carolyn G. Rasmussen and N. Louise Glass. A Rho-Type GTPase, rho-4 , is requied for septation in Neurospora crassa Eukaryotic Cell, 2005, 11,191–1925) SO , a protein involved in hyphal fusion in Neurospora crassa , localizes to septal plugs (Andre´ Fleiner and N. Louise Glass, Eukaryotic Cell, 2007, 1, 84–94)

14. Experiment- mrfA , involved in aerial mycelium development of Monascus ruber

15. 28℃, PDA 10d Fig.1 original strain and mutants with abnormal hyphea M-7 805

16. Fig 2-1 PCR amplification Fig 2-2 Southern analysis

17. Fig.3 The locusic schematic of specific primers in T-DNA for TAIL-PCR amplification

18. Fig.4 the result of TAIL-PCR amplification

19. Fig.5-1 Similarity analysis of DNA sequences amplification by TAIL- PCR With blast tool

20. Fig.5-2 Similarity analysis of DNA sequences amplification by TAIL- PCR With blast tool

21. Fig .6 Degenerate primers designation

22. Fig. 7 The results of PCR and nested-PCR of 5’ of DNA sequence

23. Fig .8 Amplification of full length DNA by SON-PCR

24. Fig.9 FGENESH 2.6 Prediction of potential genes in Aspergillus / Pezizomycotina genomic DNA

25. Fig.10 Blast Prediction of conseved domain of derived protein

26. Fig .11 The distance tree of derived protein with Blast tool

27. Fig.12 the Diagram of DNA conding for mrf A

28. Hin d Ⅲ Kpn I Hin d Ⅲ 和 Kpn Ⅰ 双酶切后，连接 pC3300 T-Border(left) CaMV35s polyA kan(R) SacI KpnI SmaI XbaI HindIII T-Border(right) EcoRI phosphinothricin(R) XhoI SalI KpnI SacII XhoI BstXI pBR322bom pBR322ori SacII CaMV35S promoter lacZ

29. Fig .13 the Construction procedure of knock-out vector pC805S Sac I+ Sma I 双酶切后，连接

30. Fig.14 PCR amplification for long DNA fragment 1: Negative control ： PCR result with steriled ddH 2 O as template ； 2~5: PCR of long DNA fragment with primers P1 and P2 and genomic DNA of M. ruber as the template.

31. Fig.15 Identification of recombinant vector pC805 digested by Hin d Ⅲ and Kpn Ⅰand PCR amplification 1: Recombinant vector pC805 ； 2: Recombinant vector pC805 digested by Hin d Ⅲ and Kpn I; 3~4: the PCR result with recombinant vector pC805 as template ； 5: the PCR result with genomic DNA of M. ruber as template

32. Fig.16 Identification of recombinant vector of pC805S digested by Sac I and Sma I 1: Recombinant vector of pC805S digested by Sac I and Sma I

33. Fig.17 Identification of EHA105 containing recombinant vector pC805S by PCR 1~7 represented the PCR result with steriled H2O, pSKH, pCAMBIA3300, M.ruber , pC805, pC805S and pC805S in A tumefaciens as template

34. Fig.18 Pictures of putative transformants with autolytic aerial hyphae

36. Thank you for your attention!