食品级重组产朊假丝酵母菌的构建及其遗传稳定性测定
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摘要
旨在构建食品级酵母工程菌株,并对其遗传稳定性进行评价。采用重叠延伸PCR(gene splicing by overlap extension PCR,SOE-PCR)方法,将实验室已构建完成的原核表达载体PGZM18中的原核基因序列删除,设计2组引物,以PGZM18原核载体序列为模板进行引物设计,将片段序列分为2个片段P1-1和P2-1,通过重叠延伸PCR技术删除来自原核的DNA序列(包括抗药性标记Amp在内的细菌质粒序列DNA片段及原核复制区),将最终获得的PCR产物GZM18转化至产朊假丝酵母中,通过菌落计数和PCR方法测定外源基因遗传稳定性。结果表明,食品级产朊假丝酵母工程菌株构建成功,外源基因具有较高的遗传稳定性。研究结果为产朊假丝酵母工程菌在微生物饲料开发方面的应用提供了科学依据。
The aim of this study was to construct a food-grade yeast engineering strain and to evaluate its genetic stability. The prokaryotic gene sequences in the PGZM18, a previously constructed prokaryotic expression vector, was deleted by SOE-PCR;two sets of primers were designed based on the prokaryotic vector sequence of PGZM18, and the fragment sequence was divided into two fragments, P1-1 and P2-1; the prokaryotic DNA sequences(bacterial plasmid sequence, including antibiotic resistance marker Amp, and prokaryotic replication region) were deleted by SOE-PCR; the finally obtained PCR product GZM18 was transformed into Candida utilis, and the genetic stability of the exogenous gene was determined by colony counting and PCR. The results showed that a food-grade genetically engineered strains of Candida utilis was successfully constructed, and the exogenous gene had high genetic stability. Our results provide a scientific basis for application of the engineering strain of Candida utilis in development of microbial feed.
The aim of this study was to construct a food-grade yeast engineering strain and to evaluate its genetic stability. The prokaryotic gene sequences in the PGZM18, a previously constructed prokaryotic expression vector, was deleted by SOE-PCR;two sets of primers were designed based on the prokaryotic vector sequence of PGZM18, and the fragment sequence was divided into two fragments, P1-1 and P2-1; the prokaryotic DNA sequences(bacterial plasmid sequence, including antibiotic resistance marker Amp, and prokaryotic replication region) were deleted by SOE-PCR; the finally obtained PCR product GZM18 was transformed into Candida utilis, and the genetic stability of the exogenous gene was determined by colony counting and PCR. The results showed that a food-grade genetically engineered strains of Candida utilis was successfully constructed, and the exogenous gene had high genetic stability. Our results provide a scientific basis for application of the engineering strain of Candida utilis in development of microbial feed.
引文
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[14]赵佳琪,王婉莹,范业宁,等.基于SOE-PCR的核酸疫苗pIRES2-MLAA34-HSP70的构建及免疫效果研究[J].重庆医学,2017,46(19):2612-2614.
[15]徐芳,姚泉洪,熊爱生,等.重叠延伸PCR技术及其在基因工程上的应用[J].分子植物育种,2006,4(5):747-750.
[16]宋柬,马会勤,陈尚武.SOE法对大豆苯丙氨酸解氨酶(PAL)基因拼接的应用研究[J].大豆科学,2006,25(3):228-233.
[17]陈燕宇,何丹,陈兆华,等.SOE-PCR重构SLA-Ⅰ复合体研究[J].动物医学进展,2010,31(9):58-62.
[18]范贺超.新型木糖利用酵母的评价及其遗传表达系统构建[D].无锡:江南大学,2015.
[19]杨森.Bacillus subtilis基因调控表达系统构建及其在发酵工程中的应用[D].无锡:江南大学,2017.
[20]叶小飞.重组毕赤酵母遗传稳定性的研究及其表达产物猪α干扰素的分离与纯化[D].南京:南京农业大学,2010.
[2] HANSEN E B. Commercial bacterial starter cultures for fermented foods of the future[J]. International Journal of Food Microbiology,2002,78(1/2):119-131.
[3] CHEN J,JIN Z,GAI Y,et al. A food-grade expression system for D-psicose 3-epimerase production in Bacillus sub tilils using an alanine racemase-encoding selection marker[J].Bioresources and Bioprocessing,2017,4(1):9.
[4] LABRIE S,BART C, VADEBONCOEUR C,et al. Use of an alpha-galactosidase gene as a food-grade selection marker for Streptococcus thermophilus[J].Journal of Dairy Science, 2005,88(7):2341-2347.
[5] LANDETE J M. A review of food-grade vectors in lactic acid bacteria:from the laboratory to their application[J].Critical Reviews in Biotechnology,2017,37(3):296-308.
[6] WATANASRISIN W,IWATANI S,OURA T,et al.Identification and characterization of Candida utilis multi drug efflux transporter CuCdrlp[J].FEMS Yeast Research,2016,16(4):42.
[7] PEDERSEN M,IVERSEN S,SORENSEN K,et al. Thelong and winding road from the research laboratory to industrial applications of lactic acid bacteria[J].FEMS Microbiology Reviews, 2005,29(3):611-624.
[8]王琛.以食品级乳酸菌为载体的抗幽门螺杆菌疫苗的构建及免疫效果[D].郑州:郑州大学,2018.
[9] MARTIN M C,ALONSO J C,SUAREZ J E,et al.Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination[J].Applied and Environmental Microbiology, 2000,66(6):2599-2604.
[10]郭钦.食品级酿酒酵母高效分泌/展示表达系统构建[D].杭州:浙江大学,2009.
[11] HORTON R M,CAI Z L,HO S N,et al. Gene splicing by overlap extension:tailor-made genes using the polymerase chain reaction[J].Biotechniques, 1990,8(5):528-535.
[12] MANIVASAKAM R,WEBER S,MCEIVER J,et al.Micro-homology mediated PCR targeting in Saccharomyces cerevisiae[J].Nucleic Acids Research,1995,23(14):2799-2800.
[13]钟志成,黎诚耀,朱利,等.一种可用于多DNA片段连接的新SOE-PCR方法[J].热带医学杂志,2010,10(3):253-257.
[14]赵佳琪,王婉莹,范业宁,等.基于SOE-PCR的核酸疫苗pIRES2-MLAA34-HSP70的构建及免疫效果研究[J].重庆医学,2017,46(19):2612-2614.
[15]徐芳,姚泉洪,熊爱生,等.重叠延伸PCR技术及其在基因工程上的应用[J].分子植物育种,2006,4(5):747-750.
[16]宋柬,马会勤,陈尚武.SOE法对大豆苯丙氨酸解氨酶(PAL)基因拼接的应用研究[J].大豆科学,2006,25(3):228-233.
[17]陈燕宇,何丹,陈兆华,等.SOE-PCR重构SLA-Ⅰ复合体研究[J].动物医学进展,2010,31(9):58-62.
[18]范贺超.新型木糖利用酵母的评价及其遗传表达系统构建[D].无锡:江南大学,2015.
[19]杨森.Bacillus subtilis基因调控表达系统构建及其在发酵工程中的应用[D].无锡:江南大学,2017.
[20]叶小飞.重组毕赤酵母遗传稳定性的研究及其表达产物猪α干扰素的分离与纯化[D].南京:南京农业大学,2010.