l32基因的敲除和过表达菌株的构建及其生长特性研究
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摘要
核糖体蛋白L32(ribosome protein L32)是一种在动物、植物和酵母等不同生物类群中普遍存在的蛋白,其进化上相对保守。大多数真核生物的核糖体蛋白L32仅由一个基因编码,而粟酒裂殖酵母核糖体蛋白L32由两个基因编码,表达的蛋白质分别是L32-1和L32-2。本实验室前期已对132-2基因进行了克隆表达,并对其功能进行了研究,表明其可能是一种潜在的转录调控因子。由于核糖体蛋白L32-1和L32-2的氨基酸序列相似度极高,为96.85%,为了更深入地研究和确定粟酒裂殖酵母核糖体蛋白L32-1和L32-2的功能,本文首先构建了132基因的敲除和过表达酵母菌株,并对这些菌株的生长特性进行了研究。
本文首先提取了野生型粟酒裂殖酵母Sohizosacchharomyces pombe的基因组DNA,通过PCR反应扩增得到编码核糖体蛋白L32-1的基因132-1,将其连接到pMD18-T载体上,用双酶切连接的方法将其连入酵母表达载体pESP-3,构建质粒pESP-3/ι32-1,醋酸锂法转化SP-QO1菌株,构建过表达菌株SP-QO1(pESP-3/ι32-1).另外,本实验室前期研究中已成功构建过表达菌株SP-Q01(pESP-3/ι32-2).
本文还成功构建了敲除132基因的酵母菌株。通过设计三对引物,两轮over-lapPCR反应获得含有132基因上下游同源臂的融合片段,将其导入SP-Q01菌株,通过同源置换原理,G418抗性筛选,最终成功构建了敲除菌株SP-Q01(Δι32-1)和SP-Q0l(△132-2)的突变株。同时,在敲除菌的基础上,我们回补了相关基因,得到了基因回补菌株SP-Q01(Δι32-1,pESP-3/ι32-1).SP-Q01(Δι32-1,pESP-3/ι32-2).SP-Q01 (△ι32-2,pESP-3/ι32-1).SP-Q01(△ι32-2,pESP-3/ι32-2)。
最后,本文对132基因的敲除或过表达菌株的生长特性进行了研究。测定其生长曲线发现,132-1基因的过表达菌株的生长速度最慢；形态观察发现,敲除132基因的酵母菌株SP-Q01(△ι32-1)和SP-Q01(△ι32-2)出现凝絮现象,过表达132基因的菌株SP-Q01(pESP-3/ι32-1)和SP-Q01(pESP-3/ι32-2)出现了个体形态变长的细胞。具体的机制有待继续研究探讨。
The evolutionarily conservative ribosomal protein L32 exists widely in different biological groups such as animals, plants and yeast and so on. Most of the eukaryotic ribosomal protein L32 is only encoded by one gene, but the Schizosaccharomyces pombe ribosomal protein L32 is encoded by two genes which respectively express ribosomal protein L32-1 and L32-2. In our laboratory,we have not only cloned and expressed 132-2 gene,but also done researches in its functions before to demonstrate that maybe it is a transcriptional regulation factor. Considering their high amino acid similarity of L32-1 and L32-2,that is 96.85%,we try to discover and conform their functions. Therefore, this thesis does researches in gene cloning, gene knockout,and growth features of overexpression or knockout of 132 gene.
In this thesis, wild Schizosaccharomyces pombe genome DNA was extracted, and then,132-1 gene was synthesized with specific primers by PCR.132-1 was ligated into pMD18-T vector,creating plasmid pMD18-T/ι32-1. After that,ι32-1 gene was digested from pMD18-T/ι32-1 by restriction endonuclease enzyme,then, ligated into pESP-3 vector to creat plasmid pESP-3/ι32-1. The recombinant plasmid was transformed into SP-Q01 cell to construct expression strain SP-Q01 (pESP-3/ι32-1).Besides, expression strain SP-Q01 (pESP-3/ι32-2) has already been constructed in our labs before.
The 132 gene knockout strains SP-Q01(Δι32-1) and SP-Q01(Δι32-2) were also costructed in this thesis. Through designing three pairs specific primers and two turns over-lap PCR reactions,we successfully required fusion gene cosist of the upstream and downstream gene of 132.After that,we transformed the gene into SP-Q01 cell, by the theory of homology exchange, G418 screening, we successfully constructed yeast strains of SP-Q01(Δι32-1) and SP-Q01(Δι32-2).Besides,based on the konckout strains, we successfully complemented related genes into them to constrct SP-Q01 (Δι32-1, pESP-3/ι32-1), SP-Q01 (Δι32-1,pESP-3/ι32-2), SP-Q01 (Δι32-2,pESP-3/ι32-1), SP-Q01 (Δι32-2,pESP-3/ι32-2) yeast strains.
Finally, growth features of over-expression or knockout of 132 gene were studied.The differences of the growth curve showed thatι32-1 overexpression strain SP-Q01 (pESP-3/ι32-1) grows the slowest; Through the observation of their modalities, phenomenon of flocculation appeared in the 132 gene knockout strains SP-Q01 (Δι32-1) and SP-Q01 (Δι32-2); Besides,large cells appeared in the overexpression strains SP-Q01 (pESP-3/ι32-1) and SP-Q01 (pESP-3/ι32-2).Intensive studies were needed to explain all of the above.
本文首先提取了野生型粟酒裂殖酵母Sohizosacchharomyces pombe的基因组DNA,通过PCR反应扩增得到编码核糖体蛋白L32-1的基因132-1,将其连接到pMD18-T载体上,用双酶切连接的方法将其连入酵母表达载体pESP-3,构建质粒pESP-3/ι32-1,醋酸锂法转化SP-QO1菌株,构建过表达菌株SP-QO1(pESP-3/ι32-1).另外,本实验室前期研究中已成功构建过表达菌株SP-Q01(pESP-3/ι32-2).
本文还成功构建了敲除132基因的酵母菌株。通过设计三对引物,两轮over-lapPCR反应获得含有132基因上下游同源臂的融合片段,将其导入SP-Q01菌株,通过同源置换原理,G418抗性筛选,最终成功构建了敲除菌株SP-Q01(Δι32-1)和SP-Q0l(△132-2)的突变株。同时,在敲除菌的基础上,我们回补了相关基因,得到了基因回补菌株SP-Q01(Δι32-1,pESP-3/ι32-1).SP-Q01(Δι32-1,pESP-3/ι32-2).SP-Q01 (△ι32-2,pESP-3/ι32-1).SP-Q01(△ι32-2,pESP-3/ι32-2)。
最后,本文对132基因的敲除或过表达菌株的生长特性进行了研究。测定其生长曲线发现,132-1基因的过表达菌株的生长速度最慢；形态观察发现,敲除132基因的酵母菌株SP-Q01(△ι32-1)和SP-Q01(△ι32-2)出现凝絮现象,过表达132基因的菌株SP-Q01(pESP-3/ι32-1)和SP-Q01(pESP-3/ι32-2)出现了个体形态变长的细胞。具体的机制有待继续研究探讨。
The evolutionarily conservative ribosomal protein L32 exists widely in different biological groups such as animals, plants and yeast and so on. Most of the eukaryotic ribosomal protein L32 is only encoded by one gene, but the Schizosaccharomyces pombe ribosomal protein L32 is encoded by two genes which respectively express ribosomal protein L32-1 and L32-2. In our laboratory,we have not only cloned and expressed 132-2 gene,but also done researches in its functions before to demonstrate that maybe it is a transcriptional regulation factor. Considering their high amino acid similarity of L32-1 and L32-2,that is 96.85%,we try to discover and conform their functions. Therefore, this thesis does researches in gene cloning, gene knockout,and growth features of overexpression or knockout of 132 gene.
In this thesis, wild Schizosaccharomyces pombe genome DNA was extracted, and then,132-1 gene was synthesized with specific primers by PCR.132-1 was ligated into pMD18-T vector,creating plasmid pMD18-T/ι32-1. After that,ι32-1 gene was digested from pMD18-T/ι32-1 by restriction endonuclease enzyme,then, ligated into pESP-3 vector to creat plasmid pESP-3/ι32-1. The recombinant plasmid was transformed into SP-Q01 cell to construct expression strain SP-Q01 (pESP-3/ι32-1).Besides, expression strain SP-Q01 (pESP-3/ι32-2) has already been constructed in our labs before.
The 132 gene knockout strains SP-Q01(Δι32-1) and SP-Q01(Δι32-2) were also costructed in this thesis. Through designing three pairs specific primers and two turns over-lap PCR reactions,we successfully required fusion gene cosist of the upstream and downstream gene of 132.After that,we transformed the gene into SP-Q01 cell, by the theory of homology exchange, G418 screening, we successfully constructed yeast strains of SP-Q01(Δι32-1) and SP-Q01(Δι32-2).Besides,based on the konckout strains, we successfully complemented related genes into them to constrct SP-Q01 (Δι32-1, pESP-3/ι32-1), SP-Q01 (Δι32-1,pESP-3/ι32-2), SP-Q01 (Δι32-2,pESP-3/ι32-1), SP-Q01 (Δι32-2,pESP-3/ι32-2) yeast strains.
Finally, growth features of over-expression or knockout of 132 gene were studied.The differences of the growth curve showed thatι32-1 overexpression strain SP-Q01 (pESP-3/ι32-1) grows the slowest; Through the observation of their modalities, phenomenon of flocculation appeared in the 132 gene knockout strains SP-Q01 (Δι32-1) and SP-Q01 (Δι32-2); Besides,large cells appeared in the overexpression strains SP-Q01 (pESP-3/ι32-1) and SP-Q01 (pESP-3/ι32-2).Intensive studies were needed to explain all of the above.
引文
[1]Wool IG. Extraribosomal function of eukaryotic ribosomes.Biochem.1979,48:719～754.
[2]Uechi T,Tanaka T and Kenmochi N. A complete map of the human ribosomal protein genes:assigment of 80 genes to the cytogenetic map and implication for human disord-ers[J].Gemomics.2001,72(3):223～230.
[3]Chavez-Rios R and Arias-Romero LE. L10 ribosomal protein from Entam oeba histolytica share structuraland functional homologies with QM/Jif-1:proteins with extraribosomal functions. Mol.B iochem. Parasitol.2003,127(2):151～160.
[4]Chen FW, Ioannou YA. Ribosomal proteins in cell proliferation and apoptosis. Int.Rev.Immunol.1999,18(5-6):429～448.
[5]Wool IG. Extra-ribosomal function of ribosomes proteins.Trends.Biochem. Sci.1996,21:164～165.
[6]Lee HS, Mun JH and Kim SG. Characterization of cDNA encoding cytoplasmic ribosomal protein L15 and L27a in petunia.(Petunia hybrida):Primary structures and coordinate expression.Gene.1999,226(2):155～163.
[7]Wang Q,Yang C and Zhou J,et al. Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer.Gene.2001,263(1-2):205～209.
[8]Morita T, Sato T and Nyunoya H,et al.Isolation of a cDNA encoding DNA-binding protein(TAXREB107) that binds specifically to domain C of the tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I,AIDS Res. Hum.Retroviruses.1993,9:115～121.
[9]Neumann F and Krawinkel U. Constitutive expression of human ribosomal protein L7 arrests the cell cycle in G1 and induces apoptosis in JurkatT-Lymphoma cells.Experim ental Cell Research.1997,230:252～261.
[10]Liu ZH and YangQ.Cloning and characterization analysis of 60S ribosomal protein L1Oa gene from Chaetomium globosum.Microbiology.2006,33(1):24
[11]Andreas KEK,Baier G and Wittmann-Liebold B. An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family.1989,FEB,247:167～172.
[12]Park S and Jeong DG. Ribosomal protein L10 interacts with the SH domain and regulates GDNF-induced neurite growth in SH-SY-5y cells.Cell Biochem. 2006,99(2):624～634.
[13]Nguyen YH, Mills AA and Stanbridge EJ.Assembly of the QM protein onto the 60S ribosomal subunit occurs in the cytoplasm.Cell Biochem.1998,68(2):281～285.
[14]Chan YL,Diaz J and Denoroy L, et al.The primary structure of rat ribosomal protein L10:Relationship to jun-binding protein and to a putative wilms tumor suppressor. Biochemical And Biophysical Research Communications.1996,225:952～956.
[15]卜友泉,杨正梅和宋方洲.新基因功能研究策略与方法.生命科学研究.2006,10(2)：95-99.
[16]杜玉梅和左正宏.基因功能的研究方法新进展.生命科学.2008,20(4)：590-592.
[17]王敏,郑飞云和李崎等.敲除啤酒酵母fksl基因对啤酒酵母自溶性能的影响.2010,40(1)：32-39.
[18]宋浩雷,郭晓贤和王艳尊等.敲除sfal基因提高酿酒酵母乙醇合成能力的研究[J].微生物学通报.2007(03)：67-72.
[19]于廷曦,朱应葆和童坦君HR24L基因过表达抑制细胞凋亡.北京大学学报.2002,34(1)：28-32.
[20]高国赋,陈信波和刘爱玲等.水稻OsWAX2-1基因过表达及RNAi研究.湖南农业科学.2008,(3)：1-3.
[21]黄湘,谭家余和王穗海等.过表达HSPC238对Be17402细胞周期的影响.中国免疫学杂志.2011,27：120-125.
[22]Su-Chiung Fang and Donna E Fernandez. Effect of regulated over-expression of the MADS domain factor AGL15 on flower senescence and fruit maturation[J].Plant physiol,2002,130:78～79.
[23]许亮,卢向阳和田云.后基因组时代基因功能分析的策略.中国生物工程杂志.2003,23(8)：29-34.
[24]Guidotti JE,Mignon A and Haase G, et al.Adenoviral gene therapy of the Tay-Sachs disease in hexosaminidase A-defilent knock-out mice[J].Hum Mol Genet,1999,8 (5):831～838.
[25]Dutton R,Yamada T and Turnley A,et al.Sonic hedgehog promote neuronal differentiation of murine spinal cord precursors and collaborates with neutrophin 3 to induce islet-1 [J]. Neurosci,1999,19:2601～2608.
[26]许杨和涂追.丝状真菌基因敲除技术研究进展.食品与生物技术学报.2007,26(01)：126-131.
[27]邓春梅,葛玉强和刘丽等.外源基因表达系统的研究进展.现代生物医学进 展.2010,10(19)：3744-3746.
[28]岳强和周惠.粟酒裂殖酵母——一种良好的真核模式生物.韶关学院学报(自然科学版).2002,23(12)：120-125.
[29]Michelle D and Wayne PW.High-efficiency Gene Targeting in Schizosaccharomyces pombe Using a Modular,PCR-based Approach with Long Tracts of Flanking Homology. Yeast15,1999,1419-1427.
[30]Bahler J, Wu JQ and Longtime MS,et al. Heterologous Modules for Efficient and Ver-satile PCR-based Gene Targering in Schizosaccharomyces pombe. Yeast14 1998,943～951.
[31]Angela Atwood-Moore, Kenechi Ejebe and Henry L. Levin. Specific Recognition and Cleavage of the Plus-Strand Primer by Reverse Transcriptase. Journal of Virology.2005.79 (23):14863～14875.
[32]吕彩霞,杨捷和叶江等.林可霉素生物合成基因lmbU的敲除及其遗传回补.华东理工大学学报(自然科学版).2008,34(1)：60-65.
[33]Strauss CJ, Botes PJ and Pohl CH. Oxylipin Associated Co-Flocculation in Yeasts. J.Inst. Brew.2006,112(1):66～71.
[34]Chan YL,Olvera J and Gluck A,et al. A leucine zipper-liker motif and a basic region-leucinezipper-likerelementinratribosomalproteinL13a.J.Biol.Chem.1994,269,5589-5594.
[35]Tabuchi N,Tanaka S and Iwahara, et al. The Schizosaccharomyces pombe gmsll gene encodes an UDP-galactose transporter homologue required for protein galactosylation. Biochem. Biophys. Res.Commun.1997,232:121～125.
[36]Bony M, Barre P and Blondin B. Localization and cell surface anchoring of the Sac-charomyces cerevisiae flocculation protein Flolp. J. Bacteriol.1997,179:4929～4936.
[37]Naotaka Tanaka, Atsuro Awai and M. Shah Alam Bhuiyan, et al. Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe.1999,181(4):1356～1359.
[38]Peng X,Sun J and Iserentant D,et al. Flocculation and coflocculation of bacteria by yeasts. Appl Microbiol Biotechnol.2001,55:777～781.
[39]Anna M and Anna G. Ethanol-induced Cell Aggregation and Its Physiological Background in Schizosaccharomyces pombe. ActaBiological Hungarica2001,52(2-3):231～ 239.
[40]Kim KH, Cho YM and Kang WH,et al. Negative Regulation of Filamentous Growth and Flocculation by Lkhl, a Fission Yeast LAMMER Kinase Homolog. Biochemical and Biophysical Research Communications.2001,289:1237～1242.
[41]Brian LA, Miki NHP and Allan PJ, et al.Possible Mechanism for Flocculation Interactions Governed by Gene FLOI in Saccharomyces cerevisiae. Journal of Bacteriology,1982,150(2):878～889.
[42]Anna G and Anna M.Cell wall ultrastructure of flocculent and non-flocculent Schizo-saccharomyces pombe strains.Effect of cell wall hydrolysing enzymes on flocculation and cell wall ultastructure.Acta Microbiologica et Immunologica Hungarica.2007,54(1):35～ 46.
[43]Mohammed EB, Gerald G and Joel C, et al. Evidence for a lectin in Kluyveromyces sp. that is involved in co-(?)occulation with Schizosaccharomyces pombe.FEMS Microbiology Letters.2000,184:41～46.
[44]汪洋,王哲和沈妍等.石蒜碱对裂殖酵母生长的抑制作用.复旦学报(医学版).2010,37(6)：642-646.
[45]Kim DU, Hayles J and Kim D,et al. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. nature biotechnology.2010,28:617～623.
[46]Strauss CJ, Kock JLF and Van Wyk,et al.Bioactive oxylipins in Saccharomyces cerevisiae. J. Inst. Brew.,2005,111,:304～308.
[47]Tsitsigiannis D, Kowieski TM and Keller NP.Three putative oxylipins biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans. Microbiology, 2005,151:1809～1821.
[48]Strauss CJ, Kock JLF and Viljoen BC. Lipid turnover during inverse flocculation patterns in Saccharomyces cerevisiae UOFS Y-2330. J. Inst. Brew.,2004,110:207～212.
[49]Sebolai OM, Kock JLF and Pohl CH. Report on the discovery of a novel 3-hydroxy oxylipins cascade in the yeast Saccharomycopsis synnaedendra. Prostag. Oth. Lipid M., 2004,74:139～146.
[50]Rania S,William PD and Susan LF.Choosing and using Schizosacharomyces pombe plasmids.ELSEVIER,2004,33:189198.
[51]Verstrepen KJ, Derdelinckx G and Veractert H. Yeast flocculation, what brewers should know. Appl. Microbiol. Biotechnol.,2003,61:197～206.
[52]Strauss CJ, Kock JLF and Van WYK, et al. Inverse flocculation patterns in Saccharo-myces cerevisiae Uofs Y-2330. J. Inst. Brew.2003,109:3～7.
[53]Tang Z, Kuo T and Shen J,et al. Biochemical and genetic conservation of fission yeast Dskl and human SR protein-specific kinase 1. Mol. Cell. Biol.2000,20:816～824.
[54]Costerton JW, Stewart PS and Greenberg EP.Bacterial biofilms:a common cause of persistent infections. Science 1999,284:1318～1322.
[55]Kobayashi O, Hayashi N and Kuroki R, et al.Region of Flo 1 proteins responsible for sugar recognition. J Bacteriol.1998,180:6503～6510.
[56]Tanaka N, Ohuchi N and Mukai Y, et al. Isolation andcharacterization of an invertase and its repressor genes from Schizosaccharomyces pombe.Biochem.Biophys.Res. Commun. 1998,245:246-253.
[57]Machiko M, Makoto M and Tadashi I. Induction of sexual coflocculation of heterothallic fission yeast(Schizosaccharomyces pombe) cells by mating pheromones. J. Gen. Appl.MicrobioL.1997,43:169～174.
[58]Vilardell J and Warner JR. Regulation of splicing at an intermediate step in the formation of the spliceosome. Genes Dev.1994 Jan:8(2):211～220.
[59]Presutti C,Caifre S and Bozzoni I. The ribosomal protein L2 in S. cererisiae controls the level of accumulation of its own mRNA.EMBO J.1991,10:2215～2221.
[60]Lorraine S and Symington. Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae.the EMBO Journal.1991,10(4):987～996.
[61]Matyas S, Wolf-Dietrich H and Jurg K. Preparation and Regeneration of Protoplasts and Spheroplasts for Fusion and Transformation of Schizosaccharomyces pombe.1985,12:169～174.
[62]Calleja GB. Flocculation in Schizosaccharomyces pombe. Journal of General Micro-biology.1970,64:247～250.
[63]Teunissen AWRH, Van den Berg JA and Steensma HY.Transcriptional regulation of flocculation genes in Saccharomyces cerevisiae. Yeast 11:435～446.
[64]韩聪,张惟材和游松等.大肠杆菌ptsG基因敲除及其缺陷株生长特性研究.生物工程学报.2004,20(1)：16-20.
[2]Uechi T,Tanaka T and Kenmochi N. A complete map of the human ribosomal protein genes:assigment of 80 genes to the cytogenetic map and implication for human disord-ers[J].Gemomics.2001,72(3):223～230.
[3]Chavez-Rios R and Arias-Romero LE. L10 ribosomal protein from Entam oeba histolytica share structuraland functional homologies with QM/Jif-1:proteins with extraribosomal functions. Mol.B iochem. Parasitol.2003,127(2):151～160.
[4]Chen FW, Ioannou YA. Ribosomal proteins in cell proliferation and apoptosis. Int.Rev.Immunol.1999,18(5-6):429～448.
[5]Wool IG. Extra-ribosomal function of ribosomes proteins.Trends.Biochem. Sci.1996,21:164～165.
[6]Lee HS, Mun JH and Kim SG. Characterization of cDNA encoding cytoplasmic ribosomal protein L15 and L27a in petunia.(Petunia hybrida):Primary structures and coordinate expression.Gene.1999,226(2):155～163.
[7]Wang Q,Yang C and Zhou J,et al. Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer.Gene.2001,263(1-2):205～209.
[8]Morita T, Sato T and Nyunoya H,et al.Isolation of a cDNA encoding DNA-binding protein(TAXREB107) that binds specifically to domain C of the tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I,AIDS Res. Hum.Retroviruses.1993,9:115～121.
[9]Neumann F and Krawinkel U. Constitutive expression of human ribosomal protein L7 arrests the cell cycle in G1 and induces apoptosis in JurkatT-Lymphoma cells.Experim ental Cell Research.1997,230:252～261.
[10]Liu ZH and YangQ.Cloning and characterization analysis of 60S ribosomal protein L1Oa gene from Chaetomium globosum.Microbiology.2006,33(1):24
[11]Andreas KEK,Baier G and Wittmann-Liebold B. An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family.1989,FEB,247:167～172.
[12]Park S and Jeong DG. Ribosomal protein L10 interacts with the SH domain and regulates GDNF-induced neurite growth in SH-SY-5y cells.Cell Biochem. 2006,99(2):624～634.
[13]Nguyen YH, Mills AA and Stanbridge EJ.Assembly of the QM protein onto the 60S ribosomal subunit occurs in the cytoplasm.Cell Biochem.1998,68(2):281～285.
[14]Chan YL,Diaz J and Denoroy L, et al.The primary structure of rat ribosomal protein L10:Relationship to jun-binding protein and to a putative wilms tumor suppressor. Biochemical And Biophysical Research Communications.1996,225:952～956.
[15]卜友泉,杨正梅和宋方洲.新基因功能研究策略与方法.生命科学研究.2006,10(2)：95-99.
[16]杜玉梅和左正宏.基因功能的研究方法新进展.生命科学.2008,20(4)：590-592.
[17]王敏,郑飞云和李崎等.敲除啤酒酵母fksl基因对啤酒酵母自溶性能的影响.2010,40(1)：32-39.
[18]宋浩雷,郭晓贤和王艳尊等.敲除sfal基因提高酿酒酵母乙醇合成能力的研究[J].微生物学通报.2007(03)：67-72.
[19]于廷曦,朱应葆和童坦君HR24L基因过表达抑制细胞凋亡.北京大学学报.2002,34(1)：28-32.
[20]高国赋,陈信波和刘爱玲等.水稻OsWAX2-1基因过表达及RNAi研究.湖南农业科学.2008,(3)：1-3.
[21]黄湘,谭家余和王穗海等.过表达HSPC238对Be17402细胞周期的影响.中国免疫学杂志.2011,27：120-125.
[22]Su-Chiung Fang and Donna E Fernandez. Effect of regulated over-expression of the MADS domain factor AGL15 on flower senescence and fruit maturation[J].Plant physiol,2002,130:78～79.
[23]许亮,卢向阳和田云.后基因组时代基因功能分析的策略.中国生物工程杂志.2003,23(8)：29-34.
[24]Guidotti JE,Mignon A and Haase G, et al.Adenoviral gene therapy of the Tay-Sachs disease in hexosaminidase A-defilent knock-out mice[J].Hum Mol Genet,1999,8 (5):831～838.
[25]Dutton R,Yamada T and Turnley A,et al.Sonic hedgehog promote neuronal differentiation of murine spinal cord precursors and collaborates with neutrophin 3 to induce islet-1 [J]. Neurosci,1999,19:2601～2608.
[26]许杨和涂追.丝状真菌基因敲除技术研究进展.食品与生物技术学报.2007,26(01)：126-131.
[27]邓春梅,葛玉强和刘丽等.外源基因表达系统的研究进展.现代生物医学进 展.2010,10(19)：3744-3746.
[28]岳强和周惠.粟酒裂殖酵母——一种良好的真核模式生物.韶关学院学报(自然科学版).2002,23(12)：120-125.
[29]Michelle D and Wayne PW.High-efficiency Gene Targeting in Schizosaccharomyces pombe Using a Modular,PCR-based Approach with Long Tracts of Flanking Homology. Yeast15,1999,1419-1427.
[30]Bahler J, Wu JQ and Longtime MS,et al. Heterologous Modules for Efficient and Ver-satile PCR-based Gene Targering in Schizosaccharomyces pombe. Yeast14 1998,943～951.
[31]Angela Atwood-Moore, Kenechi Ejebe and Henry L. Levin. Specific Recognition and Cleavage of the Plus-Strand Primer by Reverse Transcriptase. Journal of Virology.2005.79 (23):14863～14875.
[32]吕彩霞,杨捷和叶江等.林可霉素生物合成基因lmbU的敲除及其遗传回补.华东理工大学学报(自然科学版).2008,34(1)：60-65.
[33]Strauss CJ, Botes PJ and Pohl CH. Oxylipin Associated Co-Flocculation in Yeasts. J.Inst. Brew.2006,112(1):66～71.
[34]Chan YL,Olvera J and Gluck A,et al. A leucine zipper-liker motif and a basic region-leucinezipper-likerelementinratribosomalproteinL13a.J.Biol.Chem.1994,269,5589-5594.
[35]Tabuchi N,Tanaka S and Iwahara, et al. The Schizosaccharomyces pombe gmsll gene encodes an UDP-galactose transporter homologue required for protein galactosylation. Biochem. Biophys. Res.Commun.1997,232:121～125.
[36]Bony M, Barre P and Blondin B. Localization and cell surface anchoring of the Sac-charomyces cerevisiae flocculation protein Flolp. J. Bacteriol.1997,179:4929～4936.
[37]Naotaka Tanaka, Atsuro Awai and M. Shah Alam Bhuiyan, et al. Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe.1999,181(4):1356～1359.
[38]Peng X,Sun J and Iserentant D,et al. Flocculation and coflocculation of bacteria by yeasts. Appl Microbiol Biotechnol.2001,55:777～781.
[39]Anna M and Anna G. Ethanol-induced Cell Aggregation and Its Physiological Background in Schizosaccharomyces pombe. ActaBiological Hungarica2001,52(2-3):231～ 239.
[40]Kim KH, Cho YM and Kang WH,et al. Negative Regulation of Filamentous Growth and Flocculation by Lkhl, a Fission Yeast LAMMER Kinase Homolog. Biochemical and Biophysical Research Communications.2001,289:1237～1242.
[41]Brian LA, Miki NHP and Allan PJ, et al.Possible Mechanism for Flocculation Interactions Governed by Gene FLOI in Saccharomyces cerevisiae. Journal of Bacteriology,1982,150(2):878～889.
[42]Anna G and Anna M.Cell wall ultrastructure of flocculent and non-flocculent Schizo-saccharomyces pombe strains.Effect of cell wall hydrolysing enzymes on flocculation and cell wall ultastructure.Acta Microbiologica et Immunologica Hungarica.2007,54(1):35～ 46.
[43]Mohammed EB, Gerald G and Joel C, et al. Evidence for a lectin in Kluyveromyces sp. that is involved in co-(?)occulation with Schizosaccharomyces pombe.FEMS Microbiology Letters.2000,184:41～46.
[44]汪洋,王哲和沈妍等.石蒜碱对裂殖酵母生长的抑制作用.复旦学报(医学版).2010,37(6)：642-646.
[45]Kim DU, Hayles J and Kim D,et al. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. nature biotechnology.2010,28:617～623.
[46]Strauss CJ, Kock JLF and Van Wyk,et al.Bioactive oxylipins in Saccharomyces cerevisiae. J. Inst. Brew.,2005,111,:304～308.
[47]Tsitsigiannis D, Kowieski TM and Keller NP.Three putative oxylipins biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans. Microbiology, 2005,151:1809～1821.
[48]Strauss CJ, Kock JLF and Viljoen BC. Lipid turnover during inverse flocculation patterns in Saccharomyces cerevisiae UOFS Y-2330. J. Inst. Brew.,2004,110:207～212.
[49]Sebolai OM, Kock JLF and Pohl CH. Report on the discovery of a novel 3-hydroxy oxylipins cascade in the yeast Saccharomycopsis synnaedendra. Prostag. Oth. Lipid M., 2004,74:139～146.
[50]Rania S,William PD and Susan LF.Choosing and using Schizosacharomyces pombe plasmids.ELSEVIER,2004,33:189198.
[51]Verstrepen KJ, Derdelinckx G and Veractert H. Yeast flocculation, what brewers should know. Appl. Microbiol. Biotechnol.,2003,61:197～206.
[52]Strauss CJ, Kock JLF and Van WYK, et al. Inverse flocculation patterns in Saccharo-myces cerevisiae Uofs Y-2330. J. Inst. Brew.2003,109:3～7.
[53]Tang Z, Kuo T and Shen J,et al. Biochemical and genetic conservation of fission yeast Dskl and human SR protein-specific kinase 1. Mol. Cell. Biol.2000,20:816～824.
[54]Costerton JW, Stewart PS and Greenberg EP.Bacterial biofilms:a common cause of persistent infections. Science 1999,284:1318～1322.
[55]Kobayashi O, Hayashi N and Kuroki R, et al.Region of Flo 1 proteins responsible for sugar recognition. J Bacteriol.1998,180:6503～6510.
[56]Tanaka N, Ohuchi N and Mukai Y, et al. Isolation andcharacterization of an invertase and its repressor genes from Schizosaccharomyces pombe.Biochem.Biophys.Res. Commun. 1998,245:246-253.
[57]Machiko M, Makoto M and Tadashi I. Induction of sexual coflocculation of heterothallic fission yeast(Schizosaccharomyces pombe) cells by mating pheromones. J. Gen. Appl.MicrobioL.1997,43:169～174.
[58]Vilardell J and Warner JR. Regulation of splicing at an intermediate step in the formation of the spliceosome. Genes Dev.1994 Jan:8(2):211～220.
[59]Presutti C,Caifre S and Bozzoni I. The ribosomal protein L2 in S. cererisiae controls the level of accumulation of its own mRNA.EMBO J.1991,10:2215～2221.
[60]Lorraine S and Symington. Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae.the EMBO Journal.1991,10(4):987～996.
[61]Matyas S, Wolf-Dietrich H and Jurg K. Preparation and Regeneration of Protoplasts and Spheroplasts for Fusion and Transformation of Schizosaccharomyces pombe.1985,12:169～174.
[62]Calleja GB. Flocculation in Schizosaccharomyces pombe. Journal of General Micro-biology.1970,64:247～250.
[63]Teunissen AWRH, Van den Berg JA and Steensma HY.Transcriptional regulation of flocculation genes in Saccharomyces cerevisiae. Yeast 11:435～446.
[64]韩聪,张惟材和游松等.大肠杆菌ptsG基因敲除及其缺陷株生长特性研究.生物工程学报.2004,20(1)：16-20.