摘要
芽孢杆菌产生许多由多功能复合酶系非核糖体合成的生物活性脂肽。其中以表面活性素(surfactin)在基因水平上研究的较为深入。Surfactin是由SrfA-C三个NRPSs合成,硫酯酶/酰基转移酶SrfD刺激该过程的起始。Surfactin是一个非常强大的生物表面活性剂,可以有效降低水的表面张力。其在生物膜上具有类似生物洗涤剂的作用,并具有特殊的乳化、发泡、抗病毒和抗支原体活性。其在植物病害的生物防治和生物医学中具有潜在的应用价值。此外,脂肽可广泛应用于食品、化妆品等行业,同时可以用于提高原油采收率和生物除污。但是,通常的芽孢杆菌菌株脂肽产量很低,迄今为止,抗菌脂肽产生菌的产量一般低于1.0g/L,少的甚至在0.1g/L以下,因此提高抗菌脂肽产量显得尤为重要。目前已经有许多学者尝试采用不同的方法来增加抗菌肽的产量,但几乎所有的人都集中在菌株传统诱变、发酵优化、分离提纯或利用基因工程方法调节脂肽的合成等方面。
本实验室刊、立军从药用植物黄芩中分离出一株抗菌脂肽产生菌——淀粉液化芽孢杆菌(Bacillus amyloliquefaciens) ES-2,其对植物性病原菌、食源性病原菌和腐败菌以及真菌都有较强的抑制作用。方传记通过低能N+注入技术对该菌进行诱变,获得抗菌脂肽产量提高15.2%的高产突变菌株ES-2-4。在本研究中,以此作为出发菌株进行全基因组重组方法来迅速改善抗菌脂肽产量。同时利用RT-PCR和蛋白质组学的方法来分析出发菌株和改组高产菌株之间的差异,具体的研究描述如下：
1.通过紫外线、亚硝基胍及低能N+离子注入诱变建立具有遗传多样性的亲本库.以实验室筛选的抗菌脂肽高产菌株淀粉液化芽孢杆菌ES-2-4为出发菌株,分别采用紫外线照射(UV),亚硝基胍(NTG),或低能N+离子注入诱变,然后稀释涂布于PDA平板上。通过致死曲线的测定,确定UV诱变的条件为：紫外线功率20W,垂直距离15cm,照射时间120s；NTG诱变的条件为：NTG浓度0.4mg/mL,37℃保温30min；N+离子束诱变条件为：注入能量10kev,时间10s。Bacillus amyloliquefaciens ES-2-4分别采用以上三种方法进行诱变,经琼脂柱初筛、摇瓶发酵复筛后获得六株脂肽产量从40.5-45.8mg/L有所改善的突变株UV89、UV101、N114、 N32、Ni49、Ni5。它们经10代摇瓶传代实验证明,其产surfactiin性能稳定。以此作为基因组改组的亲本菌株,进行递推式原生质体融合。
2.对Bacillus amyloliquefaciensES-2-4的原生质体制备、再生、灭活及融合条件进行研究。对淀粉液化芽孢杆菌原生质体制备及再生条件进行了研究。实验结果表明原生质制备的最佳条件为：菌体培养时间为4h,采用0.2mg/mL溶菌酶酶解液,在37℃的条件下,酶解处理15min。采用PDA为基础再生培养基,0.6mol/L的NaCl做为高渗稳定剂,能够使原生质体的再生率达到24.7%。采用亲本原生质体灭活的方法检出融合子,并分别对紫外线灭活和热灭活的条件进行了优化。实验结果表明原生质体紫外灭活和热灭活的最佳条件分别为：20W,15cm,60min与100℃,30min。灭活后的原生质体在pH9.0,37℃条件下,用40%PEG6000融合12min,融合率为1.04×10-5。
3.利用基因组改纽技术选育surfactin高产菌株.以紫外线照射、亚硝基胍、和离子束诱变产生的六株高产突变株UV89、UV101、N114、N32、Ni49及Ni5进行递归原生质体融合。通过致死的紫外线照射灭活和热处理灭活来创建原生质体融合株库。经过两轮基因组改组,获得遗传性能稳定的高产突变株F2-38,其在1L摇瓶和19L发酵罐中surfactin产量分别是出发菌株的3.5和10.3倍。
4.利用荧光定量PCI检测突变菌株中surfactin成酶基因表达量.选取surfactin合成酶基因srfA作为靶基因,16S tDN(?)作为内参基因,利用2-△△ct相对定量法检测出发菌株Bacillus amyloliquefaciens ES-2-4及脂肽高产菌FMB38(F2-38)中(?)rfA基因在转录阶段的表达差异。2-△△CT目对定量分析表明,表面活性素合成酶基因(srfA)在FMB38中转录水平的表达量是ES-2-4的15.7倍,与抗菌脂肽产量的增加相一致。
5.建立本实验室双向电泳体系.对2-DE方法进行改进,分别对淀粉液化芽孢杆菌蛋白质样品的制备、上样量、染色方法、胶条pH以及2-DE参数的选择等关键步骤进行优化。其优化后条件如下：染色方法选择银染；胶条pH值为4-7；24cm胶条上样量为200μg。实验发现,采用含7M尿素和2M硫脲的水化液,结合低电压脱盐的等电聚焦可以获得高分辨率、重复性好的蛋白质2-DE图谱。银染后经软件分析,在单张2-DE图谱上可以检测1000以上蛋白点。为本实验室双向电泳实验的开展奠定了基础。
6.利用比较蛋白质组学的方法分析出发菌株与突变菌株的蛋白质表达差异.以上述实验为基础,研究了基因组改组高产菌株FMB38的可溶性全蛋白2-DE图谱的变化,并对差异表达的蛋白点进行质谱分析。2-DE图谱分析结果表明：在FMB38中,凝胶银染后经软件匹配,对其中丰度变化在2倍以上的51个差异蛋白点进行MALDI-TOF-MS分析,有46个蛋白点得到有效地鉴定。其中有3种转录调节蛋白与脂肽合成相关。其中ComA和DegU丰度变化与其它研究报道一致,CodY丰度变化与先前的研究相悖。对所鉴定的蛋白质通过COG直系同源簇网站以及相关的文献报道对其功能进行了分类,发现这些蛋白质的功能涉及代谢、能量产生和转化、细胞分裂和染色体分配、翻译,核糖体结构和生物合成、DNA复制,重组和修复、细胞运动和分泌、翻译后修饰、一般功能预测等方面。其中明确定位的蛋白质有84.4%位于细胞质中。
Bacillus strains produce many kinds of bioactive lipopeptides synthesized nonribosomally by a large multifunctional enzyme complex. Of these, the lipopeptide surfactin is well characterized at the genetic level. Surfactin is biosynthesized by three NRPSs, SrfA-C; the thioesterase/acyltransferase enzyme SrfD stimulates the initiation of this process. Surfactin is an extraordinarily powerful biosurfactant that is known to decrease the surface tension of water; it exerts a detergent-like action on biological membranes, and is distinguished by its exceptional emulsifying, foaming, antiviral and anti-mycoplasma activities. Surfactin has a great number of potential applications in plant disease biocontrol and biomedicine. Moreover, lipopeptide can be widely used in the food, cosmetic industries and for enhanced oil recovery and for the bioremediation of oil-contaminated sites. However, the production of antimicrobial peptides in Bacillus is generally lower than1.0g/L, some even below0.1g/L, thereby it is particularly important to improve the antimicrobial peptide production. There have been many attempts to increase lipopeptide production, but almost all of them have focused on traditional mutagenesis, fermentation optimization, isolation and purification, or on the regulation of lipopeptide synthesis using genetic engineering methods. Although rational methods and global techniques have been successfully applied to strain improvement, the need to engineer more complex phenotypes requires a more combinatorial approach.
Bacillus amyloliquefaciens ES-2was an endophytic bacterium isolated from the Chinese medicinal plant Scutellaria baicalensis Georgi by Sun Lijun in our laboratory, which strongly inhibited plant pathogens, foodborne pathogens, spoilage bacteria and fungi. B. amyloliquefaciens ES-2-4was a high-yield mutant strain obtained with N+ion beam implantation. The concentration of the lipopeptides in fermentation broth increased by 15.2%compared to ES-2. In this study, the strain ES-2-4was determined for the initial strain. The technology of genome shuffling has been used as a novel whole-genome engineering approach for the rapid improvement of antimicrobial lipopeptide yield. Comparative RT-PCR and proteomic analysis were conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. The detailed works were described as following:
1. Make the diversity of phenotype in parental library by UV, NTG and N+ion implantation mutagenesis. To start the genome shuffling process, parent strain library should be constructed in the first place. The initial strain was firstly engineered to generate more genotypes, and then the strains of interest were collected to form the parental library for the next step of recursive protoplast fusion. In order to make the diversity of phenotype in parental library, ES-2-4cells were mutagenized with either nitrosoguanidine, ultraviolet irradiation, or ion implantation (implantation sources were produced by an ion-beam bioengineering instrument devised by Chinese Academy of Sciences, Institute of plasma physics) and then spread on PDA agar plates. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. The mutation conditions were as follows, UV treatment:UV for20W,15cm,60s; NTG treatment:0.4mg/ml NTG,37℃for30min; N+ion implantation treatment:10kev of energy,10s of time. The six mutant strains with high yield, namely UV89、UV101、N114、N32、Ni49、Ni5, were obtained by UV、NTG and N+ion implantation mutagenesis, respectively. These six mutants showed small increases, from40.5to45.8mg/L in the production of surfactin. In addition, their high producing capacity was stable and was maintained after at least10transfers in shake-flasks. Consequently, these six mutants were used as the starting population for genome shuffling.
2. The conditions of Bacillus amyloliquefaciens ES-2-4protoplast preparation, regeneration, inactivation and fusion were studied. The optimized conditions for protoplast preparation and regeneration were as follows:cell culture for4h, lysozyme concentration of0.2mg/mL, incubation at37℃for15min to allow cell wall lysis; The PDA as a basis for the regeneration medium,0.6mol/L NaCl as medium osmotic stabilizer, and the regeneration rate of up to22.7%. An equal number of protoplasts from different populations were mixed and then divided equally into two fractions. One fraction was inactivated with UV for60min, and the other was heat treated at100℃for30min. Both inactivated protoplasts were fused by PEG6000at concentration of40%, pH9.0and37℃for12min. The fusion rate was up to1.04×10"5.
3. A surfactin high-yield recombinant was obtained by genome shuffling. This study was undertaken to enhance the yield of surfactin produced by Bacillus amyloliquefaciens ES-2-4using genome shuffling. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant F2-38strain that exhibited3.5-and10.3-fold increases in surfactin production in1L shake flask and19L fermenter respectively, was obtained.
4. The surfactin synthetase gene srfA expression in the recombinant was quantitated by FQ RT-PCR. We focused on the relative quantification of target gene transcript in comparision to a reference gene transcript.16S rDNA was selected as the housekeeping gene, the surfactin synthetase gene srfA gene is the gene of interest. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. The normalized expression was calculated by averaging three CT values for the reference gene (16S rDNA) and for the gene of interest (srfA). Delta CT (threshold cycle) relative quantitation analysis revealed that surfactin synthetase gene (srfA) expression at the transcriptional level in the F2-38strain was15.7-fold greater than in the ES-2-4. It is consistent with the increase in production of antibacterial lipopetide.
5. The2-DE protocol was optimized.2-DE is one of the key techniques used in proteomic studies and, production of a high quality2-DE image is crucial for analysis of the whole-cell proteins. In this experiment, an optimized protocol was developed paying special attention to the whole-cell protein analyses of Bacillus amyloliquefaciens based on2-DE. The optimization included sample preparation, sample loading, gel staining, IPG strips of pH scope, electrophoresis conditions, protein spots resolution and reproducibility. The optimized conditions are as follows:stained by silver staining; strips pH value of4-7;200μg sample loading of24cm strip. The results indicated that, use of rehydration solution containing7M urea and2M thiourea, in combination with low-voltage desalination in isoelectric focus (IEF) gave rise to satisfactory2-DE images. The maximal detectable protein spots number were estimated to be more than1000per gel under silver staining. This protocol may lay the foundation for2-DE studies in our laboratory.
6. The differentially expressed proteins in the recombinant were studied. The proteome was separated by2-DE and analysed by MS. In the shuffled strain FMB38,51differentially expressed protein spots with higher than2-fold spot density were detected in gel image comparison.46protein spots were detectable by silver staining and sampled for further MS analysis. The results showed that46proteins were successfully identified by MALDI-TOF-MS, among which3proteins were transcription regulation proteins concerned with lipopeptide synthesis previously reported, the abundance change of ComA and DegU in our results are consistent with others work, but that of CodY is in contrast. According to COG function category and related references, these proteins may approximately be classified into several functional categories such as, proteins involved in metabolism, energy production and conversion, cell division and chromosome partitioning, translation, ribosomal structure and biogenesis, DNA replication, cell motility and secretion, posttranslational modification, general function prediction, etc. The analysis also elucidated that84.8%of proteins detected are located in cytoplasm.
引文
[1]Arima K, Kakinuma A, Tamura G. Surfactin acrystalline peptidelipid surfactant produced by bacillus subtilis isolation, characterization and its inhibition of fibrin clot formation [J]. Biochemical and Biophysical Research Communication,1968,31(3):488-496
[2]Kowall M, Vater J, Kluge B, et al. Separation and Characterization of Surfactin Isoforms produced by Bacillus subtilis OKB 105 [J]. Journal of Colloid Interface Science,1998,204:1-8
[3]Peypoux F, Bonmatin J M, Labbe H, et al. Surfactin, a novel isoform from Bacillus subtilis studied by mass and NMR spectroscopies [J]. Europe Journal of Biochemistry,1994,224:89-96
[4]Vanittanakom N, Loeffler W. Fengycin-a novel antifugal lipopeptide antibiotics produced by Bacillus subtilis F-29-3 [J]. The Journal of Antibiotics,1986,39:888-901
[5]Kim P I, Bai H, Bai D, et al. Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26 [J]. Journal of Applied Microbiology,2004,97:942-949
[6]Ongena M, Jacques P, Toure Y, et al. Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis [J]. Applied Microbiology and Biotechnology,2005,69: 29-38
[7]Schneider J, Taraz K, Budzikiewicz H, et al. The structure of two fengycins from Bacillus subtilis S499 [J]. Z. Naturforsch,1999,54 (11):859-65.
[8]Vater J, Kablitz B, Wilde C, et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-l isolated from petroleum sludge [J]. Applied and Environmental Microbiology,2002,68:6210-6219
[9]Maget-Dana R, Peypoux F. Iturins, a special class of pore-forming lipopeptides:biological and physicochemical properties [J]. Toxicology,1994,87:151-174
[10]Sandrin C, Peypoux F, Michel G. Co-production of surfactin and iturin A, lipopeptides with surfactant and antifungal properties by Bacillus subtilis [J]. Applied Biochemistry and Biotechnology,1990,12(4):370-376
[11]Souto G I, Correa O S, Montecchia M S, et al. Genetic and functional characterization of a Bacillus sp. Strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds [J]. Journal of Applied Microbiology,2004,97:1247-1256
[12]Roongawang N, Thaniyavarn J, Thaniyavarn S, et al. Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides:bacillomycin L, plipastatin, and surfactin [J]. Extremophiles,2002,6:499-506
[13]Moyne A L, Shelby R, Cleveland T E, et al. Bacillomycin D:an iturin with antifungal activity against Aspergillus flavus [J]. Journal of Applied Microbiology,2001,90:622-629
[14]Peypoux F, Pommier M T, Das B C, et al. Structures of bacillomycin D and bacillomycin L peptidolipide antibiotics from Bacillus subtilis [J]. The Journal of Antibiotics,1984,37:1600-1604
[15]Landy M, Warren G H, Rosenman S B, et al. Bacillomycin, an antibiotic from bacillus subtilis active against pathogenic fungi [J]. Proceedings of the Society for Experimental Biology and Medicine,1948,67:539-541
[16]Mhammedi A, Peypoux F, Besson F, et al. Bacillomycin F, a new antibiotic of iturin group: isolation and characterization [J]. Journal of Antibiotics,1982,35:306-311
[17]Peypoux F, Marion D, Maget-Dana R, et al. Structure of bacillomycin F, a new pepdolipid antibiotic of the iturin group [J]. European Journal Biochemistry,1985,153:335-340
[18]Duitman E H, Hamoen L W, Rembold M, et al. The mycosubtilin synthetase of Bacillus subtilis ATCC6633:A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase [J]. Proceedings of the National Academy of Science of the United States of America,1999,96:13294-13299
[19]Peypoux F, Michel G, Delcambe L. The structure of mycosubtilin, an antibiotic isolated from Bacillus subtilis [J]. European Journal of Biochemistry,1976,63:391-398
[20]Leclere V, Bechet M, Adam A, et al. Mycosubtilin Overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities [J]. Applied and Environmental Microbiology,2005,71:4577-4584
[21]Kenji T, Takahashi A, Mitsuyo H, et al. The genes degQ, pps, lpa-8 (sfp) are responsible for conversation of Bacillus subtilis 168 to plipastatin production [J]. Antimicrobial Agents and Chemotherapy,1999,9:2183-2192
[22]Kenji T, Takahashi A, Makoto S. Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus suhtilis YB8 [J]. Archive Microbiology,1996, 165:243-251
[23]Carrillo C, Teruel J A, Aranda F J, et al. Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin [J]. Biochim Biophys Acta,2003,1611:91-97
[24]Peypoux F, Bonmatin J M, Wallach J. Recent trends in the biochemistry of surfactin [J]. App1 Microbiol Biotechnol,1999,51:553-563
[25]Ongena M, Jacques P. Bacillus lipopeptides:versatile weapons for plant disease biocontrol [J]. Trends Microbiol,2008,16:115-125
[26]Kowall M, Vater J, Kluge B, et al. Separation and characterization of surfactin isoforms produced by Bacillus subtilis OKB 105 [J]. J Colloid Interface Sci,1998,204:1-8
[27]Bie X M, Lu Z X, Lu F X, et al. Screening the main factors affecting extraction of the antimicrobial substance from Bacillus sp. fmbJ using Plackett-Burman method [J]. World J Microbiol Biotechnol, 2005,21:925-928
[28]Kanlayavattanakul M, Lourith N. Lipopeptides in cosmetics [J]. Int J Cosmet Sci,2010,32:1-8
[29]Schaller K D, Fox S L, Bruhn D F, et al. Characterization of surfactin from Bacillus subtilis for application as an agent for enhanced oil recovery [J]. Appl Biochem Biotechnol,2004,115: 827-836
[30]Mulligan C N, Yong R N, Gibbs B F. Heavy metal removal from sediments by biosurfactants [J]. J Hazard Mater,2001,85:111-125
[31]Sun L J, Lu Z X, Bie X M, et al. Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi [J]. World J Microbiol Biotechnol,2006,22:1259-1266
[32]Yoshida S, Hiradate S, Tsukamoto T, et al.Antimicrobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves [J]. Phytopathology,2001,91:181-187
[33]Jacques P, Hbid C, Destain J, et al. Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by Plackett-Burman design [J]. Appl Biochem Biotechnol,1999,77:223-233
[34]Dimitrov K, Gancel F, Montastruc L, et al. Liquid membrane extraction of bioactive amphiphilic substances:recovery of surfactin [J]. Biochem Eng J,2008,42:248-253
[35]Cao G Q, Zhang X H, Zhong L, et al. A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides [J]. Biotechnol Lett,2010,33: 1047-1051
[36]Sun H G, Bie X M, Lu F X, et al. Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter [J]. Can J Microbiol,2009,55: 1003-1009
[37]Arima K, Kakinuma A, Tamura G. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis:isolation, characterization and its inhibition of fibrin clot formation[J]. Biochem Biophys Res Commun,1968,31:488-494.
[38]Kleinkauf H, Dohren H V. A nonribosomal system of peptide biosynthesis [J]. Eur J Biochem, 1996,236:335-351.
[39]Vater J. Lipopeptides, an attractive class of microbial surfactants [J]. Prog Colloid Polymer Sci, 1986,72:12-18.
[40]Ullrich C, Kluge B, Palacz Z, et al.Cell-Free biosynthdsis of surfactin, a cyclic lipopeptide produced by Bacillus subtilis [J]. Biochemistry,1991,30:6503-6508.
[41]Menkhaus M, Ullrich C, Kluge B, et al. Structural and functional organization of the surfactin synthetase multienzyme [J]. system. J Biol Chem,1993,268:7678-7684.
[42]Lambalot R H, Gehring A M, Flugel R S, et al. A new enzyme superfamily-the phosphopantetheinyl transferases [J]. Chem Biol,1996,3:923-936.
[43]Kohli R M, Walsh C T. Enzymology and acyl chain macrocyclization in natural product biosynthesis [J]. Chem Commun (Camb),2003,7:297-307.
[44]Peypoux F, Bonmatin J M, Wallach J. Recent trends in the biochemistry of surfactin [J]. Appl Microbiol Biotechnol,1999,51:553-563.
[45]Steller S, Vollenbroich D, Leenders F, et al. Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3 [J]. Chem Biol,1999,6: 31-41.
[46]Vater J, Kablitz B, Wilde C, et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge [J]. Applied and Environmental Microbiology,2002,68: 6210-6219
[47]Cosmina P, Rodriguez F, de Ferra F, et al. Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis [J]. Mol Microbiol,1993,8:821-31.
[48]Hamoen L W, Eshuis H, Jongbloed J, et al. A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis [J]. Mol Microbiol,1995,15:55-63
[49]Nakano M M, Xia L A, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis [J]. J Bacteriol,1991, 173:5487-93
[50]Stinson M, Ezraa D, Hess W M, et al. An endophytic Gliocladium sp. of Eucryphia cordifolia producing selective volatile antimicrobial compounds [J]. Plant Science,2003,165:913-922
[51]高学文,姚仕义,Huong Pham,等.Bacillus subtilis B2菌株产生的表面活性素变异体的纯化和鉴定[J].微生物学报,2003,43(5)：647-652
[52]Duitman E H, Hamoen L W, Rembold M, et al. The mycosubtilin synthetase of Bacillus subtilis ATCC 6633:a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase [J]. Proc Natl Acad Sci USA,1999,96:13294-13299
[53]Tsuge K, Ohata Y, Shoda M. Gene yerP, involved in surfactin self-resistance in Bacillus subtilis [J]. Antimicrob Agents Chemother,2001a,45:3566-3573
[54]Sheppard J D, Jumarie C, Cooper D G, et al. Ionic channels induced by surfactin in planar lipid bilayer membranes [J]. Biochim Biophys Acta,1991,1064:13-23
[55]Maget-Dana R, Ptak M. Iturin lipopeptides:interactions of mycosubtilin with lipids in planar membranes and mixed monolayers [J]. Biochim Biophys Acta,1990,1023:34
[56]Grau A, Ortiz A, De Godos A, et al. A biophysical study of the interaction of the lipopeptide antibiotic iturin A with aqueous phospholipid bilayers [J]. Arch Biochem Biophys,2000,377: 315-323
[57]Latoud C, Peypoux F, Michel G, et al. Interactions of antibiotics of the iturin group with human erythrocytes [J]. Biochim Biophys Acta,1986,856:526-535
[58]Thimon L, Peypoux F, Wallach J, et al. Effect of the lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells [J]. FEMS Microbiol Letts,1995,128:101-106
[59]Roongawang N, Thaniyavarn J, Thaniyavarn S, et al. Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides:bacillomycin L, plipastatin, and surfactin [J]. Extremophiles,2002,6:499-506
[60]Moyne A L, Shelby R, Cleveland T E, et al. Bacillomycin D:an iturin with antifungal activity against Aspergillus flavus [J]. Journal of Applied Microbiology,2001,90:622-629
[61]Mhammedi A, Peypoux F, Besson F, et al. Bacillomycin F, a new antibiotic of iturin group: isolation and characterization [J]. Journal of Antibiotics,1982,35:306-311
[62]Koumoutsi A, Chen X H, Henne A, et al. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42 [J]. Journal of Bacteriology,2004,186:1084-1096
[63]Peypoux F, Michel G, Delcambe L. The structure of mycosubtilin, an antibiotic isolated from Bacillus subtilis [J]. European Journal of Biochemistry,1976,63:391-398
[64]Kenji T, Takahashi A, Mitsuyo H, et al. The genes degQ, pps, lpa-8 (sfp) are responsible for conversation of Bacillus subtilis 168 to plipastatin production [J]. Antimicrobial Agents and Chemotherapy,1999,9:2183-2192
[65]Kenji T, Takahashi A, Makoto S. Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8 [J]. Archive Microbiology,1996, 165:243-251
[66]Stein T. Bacillus subtilis antibiotics:structures, syntheses and specific functions[J]. Mol Microbiol, 2005,56:845-857
[67]Hamoen L W, Venema G, Kuipers O P. Controlling competence in Bacillus subtilis:shared use of regulators [J]. Microbiology,2003,149:9-17
[68]Tsuge K, Ohata Y, Shoda M. Gene yerP, involved in surfactin self-resistance in Bacillus subtilis[l]. Anti-microb Agents Chemother,2001a,45:3566-3573
[69]Stein T, Heinzmann S, Diisterhus S, et al. Expression and functional analysis of subtilin immunity genes spalFEG in the subtilin-sensitive host Bacillus subtilis MO 1099[J]. J Bacteriol,2005,187: 822-828
[70]Inaoka T, Takahashi K, Ohnishi-Kameyama M, et al. Guanine nucleotides guanosine 5'-diphosphate 3'-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis[J]. J Biol Chem,2003,278:2169-2176
[71]Yazgan A, Cetin S, Ozcengiz G. The effects of insertional mutations in comQ, comP, srfA, spoOH, spoOA and abrB genes on bacilysin biosynthesis in Bacillus subtilis[J]. Biochim Biophys Acta, 2003,1626:51-56
[72]Daniel T V, Taryn B K, Nicola R S-W. DegU co-ordinates multicellular behaviour exhibited by Bacillus subtilis[J]. Molecular Microbiology,2007,65(2):554-568
[73]Alexandra K, Chen X H, Joachim V. DegU and YczE positively Regulate the Synthesis of Bacillomycin D by Bacillus amyloliquefaciens Strain FZB42[J]. Applied and Environmental Microbiology,2007,73(21):6953-6964
[74]Kong L, Siranosian K.J, Grossman A D, et al. Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus sublilis[J]. Mol Microbiol,1993.9:365-373
[75]Branda S S, Gonzalez-Pastor J E, Dervyn E, et al. Genes involved in formation of structured multicellular communities by acillus subtilis [J]. J Bacteriol,2004,186:3970-3979
[76]Hamon M A, Stanley N R, Britton R A, et al. Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis [J]. Mol Microbiol,2004,52:847-860
[77]Kearns D B, Chu F, Rudner R, et al. Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility [J]. Mol Microbiol,2004,52:357-369
[78]Hofemeister J, Conrad B, Adler B, et al. Genetic analysis of the biosynthesis of non-ribosomal peptide-and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3 [J]. Mol Genet Genomics,2004,272:363-378
[79]Bais H P, Fall R, Vivanco J M. Biocontrol of Bacillus subtilis against infection of Arabidops is roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production [J]. J Plant Physiol,2004,134:307-319
[80]Yan L, Boyd K G, Adams D R, et al. Biofilm-specific cross-species induction of antimicrobial compounds in bacilli [J]. App1 Environ Microbiol,2003,69:3719-3727
[81]Rosenberg E, Ron E Z. High and low-molecular-mass microbial surfactants [J]. App1 Microbiol Biotechnol,1999,52:154-162
[82]Fawcett P, Eichenberger P, Losick R, et al. The transcriptional profile of early to middle sporulation in Bacillus subtilis[J]. Proc Natl Acad Sci USA,2000,97:8063-8068
[83]Gonzalez-Pastor J E, Hobbs E C, Losick R. Cannibalism by sporulating bacteria[J]. Science,2003, 301:510-513
[84]Steinborn G, Hajirezaei M R, Hofemeister J. Bac genes for recombinant bacilysin and anticapsin production in Bacillus host strains[J]. Arch Microbiol,2005,183:71-79
[85]Nicholson W L. Roles of Bacillus endospores in the environment[J]. Cell Mol Life Sci,2002,59: 410-416
[86]Gluliano B, Andres H, Luigi C. Isolation and partial purification of a metabolite from a mutant strain of Bacillus sp. with antibiotic activity against plant pathogenic agents[J]. Journal Biotenology, 2002,5:1-8
[87]Yu G Y, Sinclair J B, Hartman G L, et al. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[S]. Soil Biology &Biochemistry,2002,34:955-963
[88]Stein T, Heinzmann S, Kiesau P, et al. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis[J]. Mol Microbiol,2003b,47:1627-1636
[89]Kleerebezem M. Quorum sensing control of lantibiotic production; nisin and subtilis autoregulate their own biosynthesis[J]. Peptides,2004,25:1405-1414
[90]Klein C. Dual control of subtilin biosynthesis and immunity in Bacillus subtilis[S]. Mol Microbiol, 2002a,44:403-416
[91]Diamond D J, York J, Sun J Y, et al. Development of a candidate HLA A0201 restricted peptide-based vaccine against human cytomegalovirus infection[J]. Blood,1997,90:1751-1767
[92]中国食品添加剂生产应用工业协会编著.中国食品添加剂[M].北京：化学工业出版社,2001
[93]芦国营,张朝晖,洪伟杰.新型食品防腐剂纳他霉素的研究进展[J].中国食品添加剂,2005,2：38-41
[94]Pialoux G, Gahery-Segard H, Sermet S, et al. Lipopeptides induce cell-mediated anti-HIV immune esponses in seronegative volunteers[J]. AIDS,2001,15:1239-1249
[95]Livingston B, Crimi C, Grey H, et al. The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection[J]. J Immunol,1997,159:1383-1392
[96]Martinon F, Gras-Masse H, Boutillon C, et al. Immunization of mice with lipopeptides bypasses the prerequisite for adjuvant. Immune response of BALB/c mice to human immunodeficiency virus envelope glycoprotein[J]. J Immunol,1992,149:3416-3422
[97]Vitiello A, Ishioka G, Grey HM, et al. Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection-induction of a primary cytotoxic T lymphocyte response in humans[J]. J Clin Invest,1995,95:341-349
[98]Fonseca D P, Joosten D, Snippe H, et al. Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis[J].Mol Immunol,2000,37:413-422
[99]BenMohamed L, Thomas A, Bossus M, et al. High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules[J]. Vaccine, 2000,18:2843-2855
[100]BenMohamed L, Gras-Masse H, Tartar A, et al. Lipopeptide immunization without adjuvant induces potent and long-lasting B, T helper; and cytotoxic T lymphocytes responses against a malaria liver stage antigen in mice and chimpanzees[J]. Eur J Immunol,1997,27:1242-1253
[101]Daubersies P, Thomas A, Millet P, et al. Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3[J]. Nat Med,2000,11:1258-1263
[102]吕应年,杨世忠,牟伯中.脂肽类生物表免活性剂的研究究进展[J].生物技术通报,2004,(6)：11-16
[103]Mulligan C N, Yong R N, Gibbs B F. Heavy metal removal from sediments by biosurfactants [J]. Journal of hazardous materials,2001,85(1-2):111-125
[104]杨富廷.脂肽类生物表面活性剂研究进展[J].精细化工,2006,(2)：121-125
[105]李桂平,陈仪本.一种新融合抗菌肽Hex-Mag基因的克隆、表达及其抗菌活性的研究[J].微生物学报,2007,47(01)：115-120
[106]吴正奇.高安全性生物型防腐剂的研究进展[J].中国食物与营养,2006,10：22-24
[107]胡国良,黄兴国,杨承剑,等.抗菌肽的特性及其在畜牧生产中的应用[J].饲料博览,2007,9：46-48
[108]陈晓生,刘为民,温刘发,等.抗菌肽替代抗生素对肉鸭生产性能及血清代谢激素水平的影响[J].中国家禽,2005,27(5)：7-9
[109]Zhang Y X, Perry K, Vinci V A, et al. Genome shuffling leads to rapid phenotypic improvement in bacteria [J]. Nature,2002,415:644-646
[110]Hida H, Yamada T, Yamada Y. Genome shuffling of Streplomyces sp. U121 for improved production of hydroxycitric acid [J]. Appl Microbiol Biotechnol,2007,73:1387-1393
[111]Chen Tao, Wang J Y, Zhou S Q, et al. Trait improvement of riboflavin-producing Bacillus subtilis by genome shuffling and metabolic flux analysis [J]. J Chem Ind Eng,2004,55(11):1842-1848
[112]Gong J X, Zhao X M, Xing Q R, et al. Femtosecond laser-induced cell fusion [J]. Appl Phys Lett, 2008b,92(9). doi:10.1063/1.2890070
[113]Skelley A M, Oktay K, Suh H, et al. Microfluidic control of cell pairing and fusion [J]. Nat Method, 2009,6(2):147-152
[114]Mao F L, Xing Q R, Wang K, et al. Optical trapping of red blood cells and two-photon excitation-based photodynamic study using a femtosecond laser [J]. Opt Commun,2005,256: 358-363.
[115]John R P, Gangadharan D, Nampoothiri K M. Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes [J]. Bioresour Technol,2008,99(17):8008-8015
[116]Dai M H, Ziesman S, Ratcliffe T. Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli [J]. Metab Eng,2005, 7:45-52
[117]Zhao K, Ping W X, Zhang L N, et al. Screening and breeding of high taxol producing fungi by genome shuffling [J]. Sci China Ser C Life Sci,2008,51(3):222-231
[118]Gong G L, Sun X, Liu X L, et al. Mutation and a high-throughput screening method for improving the production of epothilones of Sorangium [J]. J Ind Microbiol Biotechnol,2007,34:615-623
[119]Patnaik R, Louie S, Gavrilovic V, et al. Genome shuffling of Lactobacillus for improved acid tolerance[J]. Nat Biotechnol,2002,20:707-712
[120]Yu L, Pei X L, Lei T, et al. Genome shuffling enhanced 1-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus[J]. J Biotechnol,2008,134:154-159
[121]Dai M H, Copley S D. Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC39723[J]. Appl Environ Microbiol, 2004,70:2391-2397
[122]Zhao M, Dai C C, Guan X Y, et al. Genome shuffling amplifies the carbon source spectrum and improves arachidonic acid production in Diasporangium sp[J]. Enzyme Microb Technol,2009,45: 419-425
[123]Zhang Y, Liu J Z, Huang J S, et al. Genome shuffling of Propionibacterium shermanii for improving vitamin B12 production and comparative proteome analysis[J]. J Biotechnol,2010,148: 139-143
[124]Xu B, Wang M R, Xia Y, et al. Improvement of the output of teicoplanin by genome shuffling[J]. Chin J Antibiot,2006,31 (4):237-242
[125]Lin J, Shi B H, Shi Q Q, et al. Rapid improvement in lipase production of Penicillium expansum by genome shuffling[J]. Chin J Biotechnol,2007,23(4):672-676
[126]Liang H Y, Guo Y. Whole genome shuffling to enhance activity of fibrinolytic enzyme producing strains[J]. China Biotechnol,2007,27(10):39-43
[127]Zhu H, Jin Z H, Cen P L. Natamycin-producing strain breeding by genome shuffling[J]. Chin J Antibiot,2006,31(12):739-742
[128]Yu L, Lei T, Pei X L, et al. Application of genome shuffling in enhancing L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus[J]. Food Sci,2007,28(9):369-373
[129]Zheng Z B, Zhao X M. Astaxanthin-producing strain breeding by genome shuffling[J]. J Biotechnol,2008,136 S:S310-311
[130]Gong G L,Wang C L, Chen M H, et al. Genome shuffling to improve the ethanol production of Saccharomyces cerevisiae[J]. J Biotechnol,2008a,136S:S311-312
[131]Shi D J, Wang C L, Wang K M. Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae[J]. J Ind Microbiol Biotechnol,2009,36(1): 139-147
[132]Jin Z H, Xu B, Lin S Z, et al. Enhanced production of spinosad in Saccharopolyspora spinosa by genome shuffling[J]. Appl Biochem Biotechnol,2009, doi:10.1007/s12010-008-8500-0
[133]Wang Y H, Li Y, Pei X L, et al.Genome-shuffling improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosns[J].J biotechnol,2007,129:510-515
[134]Xu B, Jin Z H, Wang H Z, et al. Evolutionof Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling[J]. Appl Microbiol Biotechnol,2008,80(2): 261-267
[135]Wei P Y, Li Z L, He P, et al. Genome shuffling in the ethanologenic yeast Candida krusei to improve acetic acid tolerance[J]. Biotechnol Appl Biochem,2008,49:113-120
[136]Kumar M. Improving polycyclic aromatic hydrocarbons degradation by genome shuffling[J]. Asian J Microbiol Biotechnol Environ Sci,2007,9(1):145-149
[137]Jin Q, Jin Z, Xu B, et al. Genomic variability among high pristinamycin-producing recombinants of Streptomyces pristinaespiralis revealed by amplified fragment length polymorphism [J]. Biotechnol Lett,2008,30(8):1423-1429
[138]Santos C N, Stephanopoulos G. Combinatorial engineering of microbes for optimizing cellular phenotype [J]. Curr Opin Chem Biol,2008,12(2):168-176
[139]魏开华,应天翼.蛋白质组学实验技术精编[M].北京：化学工业出版社,2010
[140]特怀曼.蛋白质组学原理[M].北京：化学工业出版社,2007
[141]郭葆玉.药物蛋白质组学[M].北京：人民卫生出版社,2007
[142]汉弗莱史密斯,黑克尔.微生物蛋白质组学[M].北京：化学工业出版社,2009
[143]颜真,张英起.蛋白质研究技术[M].西安：第四军医大学出版社,2007
[144]索伦森.蛋白质与蛋白质组学[M].北京：科学出版社,2007
[145]费格斯.工业蛋白质组学——在生物技术和制药中的应用[M].北京：科学出版社,2007
[146]雷姆.蛋白质生物化学与蛋白质组学[M].北京：科学出版社,2007
[147]哈马驰.药物研究中的蛋白质组学[M].北京：科学出版社,2008
[148]陶士衍.生物信息学[M].北京：科学出版社,2007
[149]钟扬,张亮,赵琼.简明生物信息学[M].北京：高等教育出版社,2001
[1]于鹏,张兰威,等.亚硝基胍诱变选育丁二酮高产菌株[J].乳品科学与技术,2006,120(5)：218-220
[2]董会平,夏帆.紫外LiCl复合诱变选育高产糖化酶菌株[J].酿酒科技,2009,8(182)：34-37
[3]余增亮.离子束生物技术引论[M].合肥：安徽科学技术出版社,1998
[4]沈娟,别小妹,陆兆新等.N+注入诱变芽孢杆菌选育高产抗菌物质菌株[J].核农学报,2006,20(4)：296-298
[5]Sun L J, Lu Z X, Bie X M, et al. Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi [J]. World J Microbiol Biotechnol,2006,22:1259-1266
[6]Fang C J, Lu Z X, Sun L J, et al. Study on mutation breeding and fermentation of antimicrobial lipopeptides yielding bacterium with 20 keV N+ ion beam implantation[J]. J Radiat Res Radiat Process,2006,24:333-336
[7]Yu L, Pei X L, Lei T, et al. Genome shuffling enhanced 1-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus[J]. J Biotechnol,2008,134:154-159
[8]Wang Y H, Li Y, Pei X L, et al. Genome-shuffling improved acid tolerance and 1-lactic acid volumetric productivity in Lactobacillus rhamnosus[J]. Journal of Biotechnology,2007,129: 510-515
[9]孔祥清,沈怀君,等.亚硝基胍对链格孢菌LD2-13的诱变改造[J].黑龙江八一农垦大学学报,2009,21(3)：1-4
[10]Liu J, Liu M, Wang J, et al. Enhancement of the Gibberella zeae growth inhibitory lipopeptides from a Bacillus subtilis mutant by ion beam implantation [J]. Applied Microbiology and Biotechnology,2005,69:223-228
[11]Gong G H, Zheng Z, Chen H, et al. Enhanced Production of Surfactin by Bacillus subtilis E 8 Mutant Obtained by Ion Beam Implantation[J]. Food Technology and Biotechnology,2009,47: 27-31
[12]Liu Q M, Yuan H, Wang J, et al.A mutant of bacillus subtilis with high-producing surfactin by ion beam implantation[J]. Plasma Science and Technology,2006,8:491
[13]Liu Q M, Yao J M, Pan R R, et al. A mutant strain of a surfactant-producing bacterium with increased emulsification activity[J]. Plasma Science and Technology,2005,7:2889
[14]Li S C, Wu M, Yao J M, et al. Mutation-Screening in Xylanase-Producing Strains by Ion Implantation[J]. Plasma Science & Technology,2005,01:2697-2700
[15]郑凤娥.纳他霉素产生菌诱变、种内原生质体融合及发酵条件研究[D].沈阳农业大学,2008
[16]王伟,王宝刚.琼脂柱预筛选法在头孢菌素菌种选育中的应用[J].安徽农业科学,2008,36(27)：11628-11629
[1]Zhang Y X, Perry K, Vinci V A, et al. Genome shuffling leads to rapid phenotypic improvement in bacteria [J]. Nature,2002,415:644-646
[2]Hida H, Yamada T, Yamada Y. Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid [J]. Appl Microbiol Biotechnol,2007,73:1387-1393
[3]Chen Tao, Wang J Y, Zhou S Q, et al. Trait improvement of riboflavin-producing Bacillus subtilis by genome shuffling and metabolic flux analysis [J]. J Chem Ind Eng,2004,55(11):1842-1848.
[4]Skelley A M, Oktay K, Suh H, et al. Microfluidic control of cell pairing and fusion [J]. Nat Method, 2009,6(2):147-152.
[5]John R P, Gangadharan D, Nampoothiri K M. Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes [J]. Bioresour Technol,2008,99(17):8008-8015
[6]Gong G L, Sun X, Liu X L, et al. Mutation and a high-throughput screening method for improving the production of epothilones of Sorangium [J]. J Ind Microbiol Biotechnol,2007,34:615-623.
[7]Patnaik R, Louie S, Gavrilovic V, et al. Genome shuffling of Lactobacillus for improved acid tolerance[J]. Nat Biotechnol,2002,20:707-712
[8]Yu L, Pei X L, Lei T, et al. Genome shuffling enhanced l-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus[J]. J Biotechnol,2008,134:154-159
[9]Dai M H, Copley S D. Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC39723[J]. Appl Environ Microbiol, 2004,70:2391-2397
[10]Zhao M, Dai C C, Guan X Y, et al. Genome shuffling amplifies the carbon source spectrum and improves arachidonic acid production in Diasporangium sp[J]. Enzyme Microb Technol,2009,45: 419-425
[11]Lin J, Shi B H, Shi Q Q, et al. Rapid improvement in lipase production of Penicillium expansum by genome shuffling[J]. Chin J Biotechnol,2007,23(4):672-676
[12]Zheng Z B, Zhao X M. Astaxanthin-producing strain breeding by genome shuffling[J]. J Biotechnol, 2008,136S:S310-311
[13]Gong G L,Wang C L, Chen M H, et al. Genome shuffling to improve the ethanol production of Saccharomyces cerevisiae[J]. J Biotechnol,2008a,136S:S311-312
[14]Chen Y, Lin Z X, Zou Z Y, et al. High yield antibiotic producing mutants of Streptomyces erythreus induced by low energy ion implantation[J].Nucl Instrum Methods Phys Res B,1998,140:341-348
[15]Shi D J, Wang C L, Wang K M. Genome shuffling to improve thermotoleranee, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae[J]. J Ind Microbiol Biotechnol,2009,36(1): 139-147
[16]Jin Z H, Xu B, Lin S Z, et al. Enhanced production of spinosad in Saccharopolyspora spinosa by genome shuffling[J]. Appl Biochem Biotechnol,2009, doi:10.1007/s12010-008-8500-0
[17]Wang Y H, Li Y, Pei X L, et al.Genome-shuffling improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus[Y].J biotechnol,2007,129:510-515
[18]Kumar M. Improving polycyclic aromatic hydrocarbons degradation by genome shuffling[J]. Asian J Microbiol Biotechnol Environ Sci,2007,9(1):145-149
[19]Sun H G, Bie X M, Lu F X, et al. Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter[J]. Can J Microbiol,2009,55: 1003-1009
[20]Cao G Q, Zhang X H, Zhong L, et al. A modified electro-transformation method for Bacillus subtilis and its application inthe production of antimicrobial lipopeptides[J]. Biotechnol Lett,2010,33: 1047-1051
[21]Gong J X, Zheng H J, Wu Z J, et al. Genome shuffling:progress and applications for phenotype improvement[J].Biotechnol Adv,2009,45:996-1005
[22]Zhang Y, Liu J Z, Huang J S, et al. Genome shuffling of Propionibacterium shermanii for improving vitamin B12 production and comparative proteome analysis[J]. J Biotechnol,2010,148: 139-143
[23]Gibson U E M, Heid C A, Williams P M.A novel method for real time quantitative RT-PCR[J]. Genome Res,1996,6:994-1001
[1]Wang X K, Li X, Currie R W, etal. Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1 β mRNA in ischemic brain tolerance[J]. Neurosci. Res,2000, 59(2):238-46
[2]朱芷葳,董常生.持家基因作为相对定量内标物的稳定性比较[J].生物技术通讯,2006,17(5)：807-809
[3]龙飞.基于活菌内标的单核细胞增生李斯特菌荧光定量PCR方法的建立[J].华中农业大学,2008
[4]Huggett J,Dheda K, Bustin S, et al. Real-time RT-PCR normalisation; strategies and considerations[J]. Genes and immunity,2005,6(4):279-284
[5]Chen C,Ridzon D.A,Broomer A J,et al.Real-time quantification of microRNAs by Stem-loop RT-PCR[J]. Nucleic Acids Research,2005,33(20):e179
[6]Heid C A, Stevens J, Livak K J, et al. Real time quantitative PCR[J]. Genome Research,1996,6(10): 986
[7]Arocho A, Chen B Y, Ladanyi M,et al.Validation of the 2-[DELTA][DELTA]Ct Calculation as an Alternate Method of Data Analysis for Quantitative PCR of BCR-ABL P210 Transcripts[J].Diagnostic Molecular Pathology,2006,15(1):56-61
[8]Bustin S A, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004; 15:155-166.
[9]Bustin S A. Quantification of mRNA using real-time reverse ranscription PCR (RT-PCR):trends and problems[J]. J Mol Endocrinol,2002,29:23-39
[10]Dheda K, Huggett JF, Bustin SA, et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques,2004,37:112-119
[11]Bas A, Forsberg G, Hammarstrom S,et al.Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes[J]. Scand J Immunol,2004,59:566-573
[12]Ullmannova V, Haskovec C. The use of housekeeping genes(HKG) as an internal control for the detection of gene expression by quantitative real-time RT-PCR[J]. Folia Biol (Praha),2003; 49: 211-216
[13]Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J]. Methods,2001,25:402-408
[14]Gibson UEM, Heid C A, Williams P M.A novel method for real time quantitative RT-PCR[J]. Genome Research,1996,6:994-1001
[15]Wang A, Doyle M V, Mark D F. Quantitation of mRNA by the polymerase chain reaction[J]. Proc. Natl.Acad. Sci. USA,1989,86:9717-9721
[16]Thiery R, Pannetierb C, Rziha H J, et al. A fluorescence-based quantitative PCR method for investigation of pseudorabies virus latency [J]. Journal of Virological Methods, 1996,61:79-87
[17]Chang H W,Cheng C A,Gu D L,et al. High-throughput avian molecular sexing by SYBR green-based real-time PCR combined with melting curve analysis[J]. BMC biotechnology,2008, 8(1):12
[18]Ponchel F, Toomes C, Bransfield K, et al. Real-time PCR based on SYBR-Green I fluorescence:an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions[J].BMC biotechnology,2003,3:18
[19]Simpson D A C, Feeney S, Boyle C, et al. Technical Brief:Retinal VEGF mRNA Measured by SYBR Green I Fluorescence:A Versatile Approach to Quantitative PCR[J]. Mol. Vis,2000,6: 178-183
[1]Xiao M, Xu P, Zhao J, et al. Oxidative stress-related responses of Bifidobacterium longum subsp. longum BBMN68 at the proteomic level after exposure to oxygen [J]. Microbiology,2011,157: 1573-1588
[2]Yuan J, Zhu L, Liu X, et al. A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705[J]. Mol Cell Proteomics,2006,5:1105-1108
[3]Kalupahana N S, Massiera F, Quignard-Boulange A, et al. Overproduction of Angiotensinogen From Adipose Tissue Induces Adipose Inflammation, Glucose Intolerance, and Insulin Resistance[J]. Obesity,2012,20:48-56
[4]Zhang H H, Feng L, Wang W, et al. Estrogen-responsive nitroso-proteome in uterine artery endothelial cells:Role of endothelial nitric oxide synthase and estrogen receptor-β[J]. Journal of Cellular Phyisiology,2012,227:146-159
[5]de Castro R O, Zhang J, Groves J R, et al. Tyrosines in the Carboxy-terminus of Syk once Phosphorylated Interact with Signaling Proteins including TULA-2, a Negative Regulator of Mast Cell Degranulation[J]. JBC,2012,287:8194-8204
[6]Yang Y, Primrose D A, Leung A C, et al. The PP1 phosphatase Flapwing regulates the activity of Merlin and Moesin in Drosophila[J]. Developmental Biology,2012,361:412-426
[7]Kohlgraf K G. Ackermann A R. Bumell K K. et al. Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay[J]. Archives of Oral Biology, 2012,57:197-204
[8]Ramsey A J, Milenkovic M, Oliveira A F, et al. Impaired NMDA receptor transmission alters striatal synapses and DISCI protein in an age-dependent manner[J]. PNAS USA,2011,108(14):5795-5800
[9]Huang Z, Hoffmann F K W, Norton R L, et al. Selenoprotein k is a novel target of m-calpain and cleavage is regulated by toll-like receptor-induced calpastatin in macrophages[J]. JBC,2011,286: 34830-34838
[10]Zhang H H, Wang Y P, Chen D B. Analysis of nitroso-Proteomes in Normotensive and Severe Preeclamptic Human Placentas[J]. Biol Reprod,2011,84:966-975
[11]Zhang L, Yu Z F, Jiang L, et al. Effect of post-harvest heat treatment on proteome change of peach fruit during ripening [J]. Journal of proteomics,2011,74:1135-1149
[12]Wang L, Zhu Y F, Guo X J, et al.A two-dimensional electrophoresis reference map of human ovary[J]. J Mol Med,2005,83:812-821
[13]肖曼.长双歧杆菌BBMN68可溶性蛋白质图谱的建立及其氧化胁迫反应的差异蛋白质组学分析[D].北京：中国农业大学,2011
[14]魏开华,应天翼.蛋白质组学实验技术精编[M].北京：化学工业出版社,2010
[15]席景会.低温胁迫下拟南芥差异蛋白质组学研究[D].吉林：吉林大学.2007
[16]徐永杰.猪肌肉组织差异蛋白质组学研究[D].武汉：华中农业大学.2010
[17]陈平,谢锦云,梁宋平,等.双向凝胶电泳银染蛋白质点的肽质谱指纹图分析[J].生物化学与生物物理学报,2000,32(4)：387-391
[18]盛京虎,解涛,丁达夫.串联质谱数据的从头解析与蛋白质的数据库搜索鉴定[J].生物化学与生物物理学报,2000,32(6)：595
[19]Chevalier F, Martin O, Rofidal V, et al. Proteomic investigation of natural variation between Arabidopsis ecotypes[J]. Proteomics,2004,4:1372-1381
[20]Setsuko K, Keiichi K, Kouji S, et al. Rice Proteome Database based on two-dimensional polyacrylamide gel electrophoresis:its status in 2003[J]. Nucleic Acids Res,2004,32:D388-D392
[21]Gorg A,Weiss W, Dunn M J. Current two-dimensional electrophoresis technology for proteomics[J]. Proteomics,2004,4:1-21
[22]Karas M,Bahr U,Ingendoh A, et al. Laser desorption/Ionization Mass Spectrometry of proteins of Mass 100000 to 250000 Dalton [J]. Angew. Chem. Int. Ed. Engl.,1989,28:760-761
[23]Fenn J B,Mann M.Meng C K, et al.Electrospray ionization for mass Spectrometry of large biololecules[J]. Science,1989,246:64-71
[24]陈主初,粱宋平,等.肿瘤蛋白质组学[M].长沙：湖南科学技术出版社,2002
[25]Marahiel M A, Nakano M M, Zuber P. Regulation of peptide antibiotic production in Bacillus[J]. Molecular Microbiology,1997,7(5):631-636
[26]Hahn J, Dubnau D J. Growth stage signal transduction and the requirements for srfA induction in development of competence[J]. Bacteriol,1991,173(22):7275-7282
[27]Hamoen L W, Venema G, Kuipers O P. Controlling competence in Bacillus subtilis:shared use of regulators [J]. Microbiology,2003,149:9-17
[28]Stein T. Bacillus subtilis antibiotics:structures, syntheses and specific functions[J]. Molecular Microbiology,2005,56(4):845-857
[29]Serror P, Sonenshein A L. CodY is required for nutritional repression of Bacillus subtilis genetic competence[J]. J. Bacteriol.1996,178:5910-5915
[30]Duitman E H, Wyczawski D, Boven L G, et al. Novel Methods for Genetic Transformation of Natural Bacillus subtilis Isolates Used To Study the Regulation of the Mycosubtilin and Surfactin Synthetases[J]. APPLIED AND ENVIRONMENTAL MICROBIOLOGY,2007,73(11):3490-3496
[31]王镜岩.生物化学[M].北京：高等教育出版版社,2002
[32]娄智勇.人源5-羧酸吡咯啉还原酶的结构与功能研究[J].生物物理学报,2009,25(4)： F0002-F0002
[33]Nicholson W L. The Bacillus subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/ 2,3-Butanediol Dehydrogenase [J]. Applied and environmental microbiology,2008,74(22): 6832-6838
[34]南芝润,范月仙.植物过氧化氢酶的研究进展[J].安徽农学通报,2008,14(5)：27-29
[35]Gibbons H S, Broomall S M, McNew LA, et al. Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaew var. globigii, a Historical Biowarfare Simulant[J]. PLoS ONE, 2011,6(3):e17836. doi:10.1371/journal.pone.0017836
[36]Mukhopadhyay A, Redding A M, Joachimiak M P, et al. Cell-wide responses to low-oxygen exposure in Desulfovibrio vulgaris Hildenborough[J]. J Bacteriol,2007,189:5996-6010
[37]Borriss R, Chen X, Rueckert C, et al. Relationship of Bacillus amyloliquefaciens clades associated with strains DSM7T and FZB42T:a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on their discriminating complete genome sequences[J]Int. J. Syst. Evol. Microbiol,2011,61: 1786-1801
[38]Chen X H, Koumoutsi A., Scholz R, et al.Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42[J].Nat. Biotechnol, 2007,25 (9):1007-1014