Rezumatul tezei de doctorat - USAMV Cluj-Napoca

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developed on PDA medium, then was placed on a histological slide, minced with a scalpel, was added a droplet of Lugol solution for coloring, homogenized, and in the end the sample was covered with a histological slide. The observations were performed with the phase contrast microsope and the images were analyzed with a adapted digital camera. I.3.5. Fungal DNA extraction and detection DNA extraction was made pursuant to the protocol of Colao Maria Chiara (1999). DNA was extracted from fungal mycellium developed in pure culture on PDB (Potato dextrose broth) liquid medium. We used Qiagen extraction kit Dneasy Plant Minikit (pursuant to http://www.qiagen.com) and DNA rapid extraction using PBS (phosphate saline buffer) solution. The DNA extraction was applied to 69 samples, obtained on Potato Dextrose Agar (PDA) solid medium-Potato Dextrose Broth (PDB) liquid medium combination. Each sample from each fishpond was divided in two equal parts, being realized a total number of 138 samples, performing a comparative extraction for the two methods used. I.3.5.1. Fungal DNA extraction using extraction kits (QIAGEN) The DNeasy Plant minikit is a spin column procedure that incorporates sample lysis, removal of RNA, removal of proteins and polysaccharides, DNA precipitation, and binding to the spin column membrane. Multiple washes are performed to remove contaminants, and DNA is then eluted from the membrane. Equipment, materials and reactives used: - Stationary phase fungal colonies (approximate 1 x 10 9 ), 69 samples, on fishponds provenance; Qiagen kit. I.3.5.2. Fungal DNA extraction using solutions 52
The DNA rapid extraction using PBS solution, applied on 69 samples, performed in the following way: there was weighted 120 mg of fungal tissue in an Eppendorf tube and added 200 µl PBS solution. The samples were centrifuged at 3000 rpm, for 20 minutes, then the supernatant was removed. This operation repeated five times, until the fungal DNA pellet was clean. Then, the samples were introduced in a marine bath, at 95°C for 15 minutes, to disrupt the cell wall. The samples were dried for 30 minutes, and then was addes TE (Tris-EDTA) solution for DNA rehydrating. For the DNA rapid extraction method with NE solution are used some chemical substances, the solutions being made in a laboratory. The materials and expendables were the following: Eppendorf pipettes, with adjustable volumes; Eppendorf tubes; pipette tips of different dimensions (large, medium and small); sterile water. I.3.6. DNA quantification using direct method of DNA purity and concentration with Nanodrop ND-1000 spectrophotometer For the establishing of DNA purity and concentration, there was used Nanodrop ND- 1000 spectrophotometer. To be able to find the DNA concentration extracted from fungal samples, we used the direct method, which involves the optical density measurement of DNA sample, at 260/280 nm ripple length on a spectrophotometer, in UV/VIS domain. I.3.7. ITS region from fungi and primers used At the fungi and other eukaryotes, there are two locations for rDNA: the nuclear and the mitochondrial genome. The last one contains two genes which are coding the small and large mitochondrial genes of rDNA. Fungal nuclear rDNA is generally structured in a tandem repeated nuclear unit. A rDNA unit, illustrated in fig.2, includes 3 rRNA genes: small nuclear (18S) rRNA, 5,8S rRNA and large nuclear (28S) rRNA gene. In a unit, the 53
Page 1 and 2: UNIVERSITATEA DE ŞTIINłE AGRICOLE
Page 3 and 4: II.6. REZULTATELE RESTRICłIEI ENZI
Page 5 and 6: diverse specii piscicole. De aici s
Page 7 and 8: Fig.1. LocaŃiile în care s-au efe
Page 9 and 10: A fost constituit un număr de 207
Page 11 and 12: Sabouraud Dextrose Broth (SDB-bulio
Page 13 and 14: Echipament, materiale şi reactivi
Page 15 and 16: Primerii folosiŃi de către noi î
Page 17 and 18: CAPITOLUL II REZULTATELE CERCETĂRI
Page 19 and 20: Mediile diametrului coloniilor de S
Page 21 and 22: laborator specifice metodologiei de
Page 23 and 24: Deoarece am utilizat în experiment
Page 25 and 26: fragmente: primul la aproximativ 45
Page 27 and 28: II.7. REZULTATE PRIVIND STUDIUL ÎN
Page 29 and 30: mai mare genetică, indivizii despr
Page 31 and 32: MenŃionăm faptul că în toate ba
Page 33 and 34: CAPITOLUL III CONCLUZII ŞI RECOMAN
Page 35 and 36: isexualis (761 pb, o identitate de
Page 37 and 38: BIBLIOGRAFIE SELECTIVĂ 1. Colao Ma
Page 39 and 40: 19. Oroian, T.E., 2006, SelecŃia a
Page 41 and 42: UNIVERSITY OF AGRICULTURAL SCIENCES
Page 43 and 44: SPECIES FROM SAPROLEGNIACEAE FAMILY
Page 45 and 46: The specific literature presents up
Page 47 and 48: Fig.1. The locations where the rese
Page 49 and 50: estimation of the spawns infestatio
Page 51: pellets were filtred, washed with d
Page 55 and 56: We used a comparatively mixture of
Page 57 and 58: II.1. THE RESULTS OF SAPROLEGNIA CU
Page 59 and 60: Reading time 24 48 72 n Culture med
Page 61 and 62: asexual one. We can precisely concl
Page 63 and 64: 1 2 3 4 5 6 7 8 9 700pb- Fig.3. Ele
Page 65 and 66: pairs length. The sequencing result
Page 67 and 68: II.7. RESULTS REGARDING THE GENETIC
Page 69 and 70: higher genetic distance, the indivi
Page 71 and 72: We mention the fact that in al the
Page 73 and 74: extraction have values between 1,10
Page 75 and 76: 13. In researches on the aquatic fu
Page 77 and 78: 40. Frisvad, J.C., Bridge, P.D., Ar
Page 79: 61. *** http://www.sigmaaldrich.com

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