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Sequence-characterized Amplified Region (SCAR) Marker Linked to Acid Resistance-related Gene in Oenococcus Oeni

Author: LiKai
Tutor: LiuShuWen
School: Northwest University of Science and Technology
Course: Grapes and wine ,
Keywords: Oenococcus oeni acid-resistant gene RAPD marker SCAR marker
CLC: Q933
Type: Master's thesis
Year: 2013
Downloads: 12
Quote: 0
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Abstract


Malolactic fermentation (MLF) is an important process for red wine of high quality.MLF can not only increase the stability of the wine, also can modify the wine flavor andimprove the sensory quality. However, for the lactic acid bacteria (LAB) conductiong MLF,wine is a harsh living environment, including low pH, high alcohol, high SO2concentrationand lack of nutrients etc. Therefore, application of LAB of high stress resistance would bepromising for carrying out MLF successfully. Oenococcus oeni is considered to be dominantLAB during the process of MLF. Therefore, it is very necessary to study the stress resistanceof Oenococcus oeni, so as to abtain strains with excellent fermentation properties.However, the good traits-related genes in wine Oenococcus oeni are rarely reported. Thisstudy aims to investigate the differences between the acid-resistant strains and acid-sensitivestrains, screening acid-resistance RAPD markers in Oenococcus oeni and developing a morestable and reliable SCAR markers, so as to provide a theoretical basis for the rapid screeningof well-preformed MLF strain and for the further research of related gene function. Theexperiment conclusion as follows.1.30Oenococcus oeni strains were supplied as experimental material by the winemicrobiology laboratory to screen acid-resistant and acid-sensitive strains, as a result,7acid-resistant strains and11acid-sensitive strains were obtained.2. Random amplified polymorphic DNA (RAPD) and bulked segregate analysis (BSA)techniques were employed in this study. Through a selection from55random primers,8primers reflected the differences between acid-resistant and acid-sensitive gene pool to acertain extent, then confirmed the8primers in a single Oenococcus oeni strain, eventuallyfound primer S200can stably amplify a1000bp band in acid-resistant strains, namedS200-1000RAPD marker related to acid resistance in Oenococcus oeni.3. The specific fragments were recovered and ligated into pGEM-T Easy vector, and thensequenced, and we have obtained the sequence information of specific fragment, provideing atheoretical basis for the further research of related gene function.4. According to the sequence information, the specific primers(SA1/SA2) were designed, and SA1/SA2could achieve stable amplification of a1000bp specific band in7acid-resistantstrains, named SA-1000acid resistance-related marker. This study proved that there was asignificant difference between Oenococcus oeni strains of acid-resistance and Oenococcusoeni strains of acid-sensitive in their genome.

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