短小芽孢杆菌碱性蛋白酶基因启动子的功能研究
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摘要
短小芽孢杆菌UN-31-C-42是一株产生具有脱毛功能的碱性蛋白酶的菌株。该菌株的碱性蛋白酶基因编码区已被克隆，本研究通过TAIL—PCR(Thermalasymmetric interlaced PCR)扩增得到碱性蛋白酶基因编码区上游的启动子片段Papr，经测序、序列拼接及比对分析等对Papr进行了鉴定。该启动子片段长797bp，在片段的5’端存在一个开放阅读框，可能为磷酸甘油酸变位酶基因的部分编码区，故碱性蛋白酶基因启动子的长度为378 bp。通过对启动子序列的分析，推测出了该启动子的保守序列及转录起始位点。对启动子的顺序缺失研究证实基因起始密码子上游160bp的DNA片段包含完整的启动功能片段。
根据Papr序列设计引物，从短小芽孢杆菌UN-31-C-42基因组中扩增获得了包含启动子、信号肽、前肽、成熟肽及终止子的完整的碱性蛋白酶基因片段WAp。将WAp插入大肠杆菌—芽孢杆菌穿梭质粒载体pSUGV4中，构建了碱性蛋白酶基因表达质粒pSUBpWAp。将pSUBpWAp分别转入大肠杆菌JM109、枯草芽孢杆菌WB600以及短小芽孢杆菌UN-31-C-42中进行表达。包含了质粒pSUBpWAp的大肠杆菌JM109细胞内外均检测不到碱性蛋白酶活性。在枯草芽孢杆菌WB600中的表达则可在胞外检测到碱性蛋白酶活性，证明启动子Papr可在枯草芽孢杆菌中启动碱性蛋白酶基因的表达，产生的碱性蛋白酶活性最高达到466.5 U／ml，与包含Bp53启动子的碱性蛋白酶基因表达质粒pSUBpAp比较，酶活提高2倍。pSUBpWAp在短小芽孢杆菌UN-31-C-42中也能表达产生碱性蛋白酶活性，但宿主菌的蛋白酶产量并未提高。
利用细菌的16s rDNA通用引物从短小芽孢杆菌UN-31-C-42基因组中扩增到16s rDNA片段，将该片段插入载体pMD18-T中，构建了重组质粒pMD16s。从载体pSUGV4中扩增到卡那霉素抗性基因片段，与碱性蛋白酶基因一同插入质粒pMD16s的16s rDNA片段中，构建整合型质粒。拟将该质粒整合至短小芽孢杆菌基因组，提高其蛋白酶产量。
Bacillus pumilus UN-31-C-42 is a strain which produces alkaline protease with dehairing function. The native promoter of alkaline protease gene, Papr, was cloned by TAIL-PCR from B. pumilus UN-31-C-42. Through blast analysis, the sequence of promoter Papr was confirmed and the conserved sequences were deduced. The promoter was 378 bp in length, and a phosphoglycerate mutase gene was located at the upstream region of promoter Papr. Deletion analysis of Papr revealed that the promoter of 160 bp could initiate the transcription of alkaline protease gene.
The alkaline protease gene, including promoter, the coding region of prepeptide, propeptide and mature peptide, and terminator, was inserted into pSUGV4, an Escherichia coli-Bacillus subtilis shuttle vector. The recombinant plasmid pSUBpWAp was transformed into E. coli JM109, B. subtilis WB600 and B. pumilus UN-31-C-42, respectively.
Expression of alkaline protease gene in pSUBpWAp in E. coli JM109 did not result in detectable extracellular and intracellular protease activity. But it expressed active extracellular protease in B. subtilis WB600 and B. pumilus UN-31-C-42. The enzymatic activity of recombinants in B. subtilis WB600 was 466.5 U/ml. It was twice more than that of plasmid pSUBpAp which was under the control of promoter Bp53. The enzymatic activity of pSUBpWAp in B. pumilus UN-31-C-42 achieved 3960 U/ml, but it was still lower than the host strain.
The 16s rDNA was cloned from genome of B. pumilus UN-31-C-42 by PCR. The fragment of 16s rDNA was inserted into vector pMD18-T, constructing recombinant plasmid pMD16s. The fragments of alkaline protease gene and kanamycin-resistant gene were inserted into 16s rDNA in pMD 16s. The recombinant will be integrated in the chromosome of B. pumilus UN-31-C-42 to increase the yield of alkaline protease.
根据Papr序列设计引物，从短小芽孢杆菌UN-31-C-42基因组中扩增获得了包含启动子、信号肽、前肽、成熟肽及终止子的完整的碱性蛋白酶基因片段WAp。将WAp插入大肠杆菌—芽孢杆菌穿梭质粒载体pSUGV4中，构建了碱性蛋白酶基因表达质粒pSUBpWAp。将pSUBpWAp分别转入大肠杆菌JM109、枯草芽孢杆菌WB600以及短小芽孢杆菌UN-31-C-42中进行表达。包含了质粒pSUBpWAp的大肠杆菌JM109细胞内外均检测不到碱性蛋白酶活性。在枯草芽孢杆菌WB600中的表达则可在胞外检测到碱性蛋白酶活性，证明启动子Papr可在枯草芽孢杆菌中启动碱性蛋白酶基因的表达，产生的碱性蛋白酶活性最高达到466.5 U／ml，与包含Bp53启动子的碱性蛋白酶基因表达质粒pSUBpAp比较，酶活提高2倍。pSUBpWAp在短小芽孢杆菌UN-31-C-42中也能表达产生碱性蛋白酶活性，但宿主菌的蛋白酶产量并未提高。
利用细菌的16s rDNA通用引物从短小芽孢杆菌UN-31-C-42基因组中扩增到16s rDNA片段，将该片段插入载体pMD18-T中，构建了重组质粒pMD16s。从载体pSUGV4中扩增到卡那霉素抗性基因片段，与碱性蛋白酶基因一同插入质粒pMD16s的16s rDNA片段中，构建整合型质粒。拟将该质粒整合至短小芽孢杆菌基因组，提高其蛋白酶产量。
Bacillus pumilus UN-31-C-42 is a strain which produces alkaline protease with dehairing function. The native promoter of alkaline protease gene, Papr, was cloned by TAIL-PCR from B. pumilus UN-31-C-42. Through blast analysis, the sequence of promoter Papr was confirmed and the conserved sequences were deduced. The promoter was 378 bp in length, and a phosphoglycerate mutase gene was located at the upstream region of promoter Papr. Deletion analysis of Papr revealed that the promoter of 160 bp could initiate the transcription of alkaline protease gene.
The alkaline protease gene, including promoter, the coding region of prepeptide, propeptide and mature peptide, and terminator, was inserted into pSUGV4, an Escherichia coli-Bacillus subtilis shuttle vector. The recombinant plasmid pSUBpWAp was transformed into E. coli JM109, B. subtilis WB600 and B. pumilus UN-31-C-42, respectively.
Expression of alkaline protease gene in pSUBpWAp in E. coli JM109 did not result in detectable extracellular and intracellular protease activity. But it expressed active extracellular protease in B. subtilis WB600 and B. pumilus UN-31-C-42. The enzymatic activity of recombinants in B. subtilis WB600 was 466.5 U/ml. It was twice more than that of plasmid pSUBpAp which was under the control of promoter Bp53. The enzymatic activity of pSUBpWAp in B. pumilus UN-31-C-42 achieved 3960 U/ml, but it was still lower than the host strain.
The 16s rDNA was cloned from genome of B. pumilus UN-31-C-42 by PCR. The fragment of 16s rDNA was inserted into vector pMD18-T, constructing recombinant plasmid pMD16s. The fragments of alkaline protease gene and kanamycin-resistant gene were inserted into 16s rDNA in pMD 16s. The recombinant will be integrated in the chromosome of B. pumilus UN-31-C-42 to increase the yield of alkaline protease.
引文
1． 曹清玉．曲志才．万由衷2001短小芽孢杆菌启动子活性片段的克隆、表达及序列特征分析科学通报，46(2)：141-144
2． 杜俊岭，崔妍，韩继刚，赵刚勇，顾雅君，朱宝成．2004．脱毛碱性蛋白酶的纯化及其生化特性研究．河北农业大学学报，Vol．27 No．3．
3． 杜耀华，王正志．2005．原核启动子识别研究进展．生物技术，Vol．15，No．5：80-83
4． 黄庆，潘皎，彭勇，李昕，张义正．2004．短小芽孢杆菌(Bacillus pumilus)脱毛蛋白酶基因的克隆与序列分析．高技术通讯，14(158)：31-35．
5． 黄庆，胥成浩，张义正．1999．枯草杆菌强基因启动子DNA片段的序列测定． 四川大学学报 36(4)：759-763
6． 李志强．2000．酶法脱毛机理的研究．四川大学博士学位论文．
7． 刘成君，张义正．2001．大肠杆菌-枯草杆菌穿梭质粒载体pSUGV4的构建．四川大学学报(自然科学版)，38(2)：243-246．
8． 刘彦，何先祺，张义正．1998．碱性脱毛蛋白酶产生菌的选育．皮革科学与工程， 8(4)：6-13
9． 潘延云，周艳芳，张贺迎，吴宝东．2003．高产碱性蛋白酶工程菌的构建及鉴定．哈尔滨大学自然科学学报，Vol．19，No．4
10． 邱秀宝，袁影，戴宏，于颖．嗜碱性芽孢杆菌碱性蛋白酶的研究Ⅱ，诱变株选育及产酶条件．1990．微生物学报，30(2)：129-133
11． 唐雪明，邵蔚蓝，沈微，王正祥，方惠英，诸葛健．2002．地衣芽孢杆菌2709和6818碱性蛋白酶基因在大肠杆菌中的克隆，表达及序列分析．应用与环境生物学报， 8(2)：209-214
12． 肖怀秋，林亲录，李玉珍，赵谋明．2005．微生物碱性蛋白酶研进展．中国食品添加剂， No．5
13． 薛林贵．1997．我国碱性蛋白酶的应用及研究进展．微生物学通报，24(6)：370-371
14． 杨国泉．1992 酶制剂应用综述．中国皮革，21(8)：35-38
15． 张义正，刘德明，刘彦，程昌凤，黄庆．2002．脱毛蛋白酶、其制造方法、使用方法和产生此蛋白酶的微生物，中国专利：1361278A
16． 邹立扣，王红宁，潘欣．2003．枯草芽孢杆菌整合研究进展．生物技术通讯，Vol．14 No．16．
17. Aditi Kanhere and Manju Bansal. 2005. Structural properties of promoters: similarities and differences between prokaryotes and eukaryotes Nucleic Acids Research, Vol. 33, No. 10: 3165-3175
18. Aoyama M., C. Toma, M. Yasud. M. Iwanaga, 2000, Sequence of the gene encoding an alkaline serine proteinase of Bacillus pumilus TYO-67., Microbiol. Immunol., 44(5): 389-393
19. Ban-Yang Chang, Roy H. DOl. 1993. Conformational properties of Bacillus subtilis RNA polymerase σ~A factor during transcription initiation. Biochem. J. 294, 43-47
20. Ceglooski P, Luder G, Alonso JC.1990. Genetic analysis of rec E activities in Bacillus subtilis. Mol Gen Genet,(222):441
21. Craig Binnie, Mary Lampe, Richard Losick. 1986. Gene encoding the σ ~(37) species of RNA polymerase σ factor fromBacillus subtilis. Proc. Nati. Acad. Sci. Vol. 83, p: 5943-5947
22. Green G.H. 1952. Unhaidng by Means of Enzymes. J.S.L.T.C. 36:127-134
23. Ferrari. FA, Nguyen. A, Lang. D, et al. 1983.Construction and properties of and intergrable plasmid for Bacillus subtilis, Jbacteriiology,6:1513
24. Frohman, M. A., Dush, M. K., and Martin, G. R. 1988. Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Nutl. Acad. Sci. USA 85:8998-9002
25. Grill, E., and Somerville, C. 1991. Construction and characterization of a yeast artificial chromosomes library of Arabidopsis which is suitable for chromosome walking. Mol. Gen. Genet. 226:484-490
26. Hahn J, Dubnau D. 1985. Analysis of plasmid deletional instability in Bacillus subtilis Journal of bacterioligy, 1014-1023
27. Jane R. McLaughlin, Cheryl L. Murray, Jesse C. Rabinowitz. 1981. Unique Features in the Ribosome Binding Site Sequencoefthe Grampositive Staphylococcus aureus fl-Lactamase Gene. the Journal biological chemistry.Vol 256. No 21
28. Khasanov. FK, Zvingilla. DJ, Zainullin. AA, et al. 1986. Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology. Genetics, 112:441
29. Kunamneni Adinarayana, Poluri Elllaiah, Davuluri Siva Prasad et al. 2003. Purification and Partial Characterization of Thermostable Serine Alkaline Protease from a Newly Isolated Bacillus sublitis PE-11. AAPS PharmSciTech 4 (4)
30. Liu YG, Whittier RF. 1995.Thermal Asymmetric Interlaced PCR: Automatable Amplification and Sequencing of Insert End Fragments from PI and YAC Clones for Chromosome Walking. Genomics, 25, 674-681
31. Loh, E. Y., Elliot, J. F., Cwirla, S., Lanier, L., and Davis, M. M. 1989. Polymerase chain reaction with single-sided specificity: Analysis of T cell receptor 6 chain. Science 243: 217-220
32. Marc M. S. M. Wosten, Miranda Boeve, Mirjam. G.A. Koot, et al. 1998. Identification of Campylobacter jejuni Promoter Sequences Journal of bacteriology, Vol.180 No.3 p594-599
33. Marisa. M, Naomi. C, Takao. S, Takashi. H, Kimiko. Y. 1999. Cloning and characterization of the O-Methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of Demethylsterigmatocystin to Sterigmatocystin and Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin in Aflatoxin Biosynthesis. Microbiology, 4987-4994
34. Martin I. Voskuil and Glenn H. Chambliss. 1998. The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. Nucleic Acids Research, 1998, Vol. 26, No. 15: 3584-3590
35. Melville. SB, Labbe. R, Sonenshein. A L.1994. Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis. Infect Immun, 62(12): 5550-5558
36. Ochman, H, Gerber, A. S, and Hart, D. L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621-623
37. Pan J, Huang Q, Zhang YZ. 2004. Gene cloning and expression of an alkaline serine protease with dehairing function from Bacillus pumilus. Curr Microbiol 49:165-169
38. Parks, C. L., Chang, L.-S., and Shenk, T. 1991. A polymerase chain reaction mediated by a single primer: Cloning of genomic sequences adjacent to a serotonin receptor protein coding region.Nucleic Acids Res. 19: 7155-7160
39. Paul, R.G., Mohamed, I., Davighi, D., Addy, V.L. and Covington, A.D. 2001. The use of neutral protease in enzymatic unhairing. J Am Leather Chem Ass 96,180-185
40. Tsutomu. T, Terou. T, Itaya. 1996. M. A method to invert DNA segments of Bacillus subtilis 168 genome by recombination between two homologous sequences. Biotech Biotechem, 60(5):773
41. Tsuyoshi Nonaka, Masahim Fujihashi, AkikoKita. Katsuhisa etal. 2004. The crystal structure of an oxidatively-stable subtilisin alkaline serine protease, KP-43, with a C-terminal 8-barrel domain. J. Biol. Chem
42. XC Wu, W Lee, L Tran, and S L Wong. 1991. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellμLar proteases. JBacteriol, 173(16): 4952-4958
43. Yansura DG, Henner DJ (1984) Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. USA.i 1,439-443
1． 杜耀华，王正志．2005．原核启动子识别研究进展．生物技术，Vol．15，No．5：80-83
2． 邓晓东，费小雯，黄俊生等．2002．Isolation and Analysis of Rubber Hevein Gene and Its Promoter Sequence．植物学报，44(8)：936-940
3． 李维，张义正．2000．黄孢原毛平革菌基因启动子的分离与鉴定．生物工程学报，16(5)： 599-602．
4． 王新力，彭学贤，李宏．2000．香蕉果实特异性ACC合酶的cDN克隆及序列分析．生物工程学报，16(2)：134-136．
5． 吴乃虎．基因工程原理(下)．北京：科学出版社，2001：74-75
6. Berg, O.G, P.H. von Hippel. 1988. Selection of DNA binding sites by regulatory proteins. Trends Biochem. Sci, 13:207-211.
7. Bird, A.P. 1987. CpG islands as gene markers in the vertebrate nucleus. Trends Genet. 3: 342-347Brown T. Genomes. BIOS scientific Publishers Limited, 1999:199-213
8. Bolshoy A, Nero E. 2000. Ecologic genomic of DNA: upstream banding in prokaryotic promoters. Genome Research, 10(8):1185-1193.
9. Bucher, P. 1990. Weight matrix descriptions of four eukaryotic RNA polymerase Ⅱ promoter elements derived from 502 unrelated promoter sequences. J. Mol. Biol.212: 563-578.
10. Bucher, P, E.N. Trifonov. 1986. Compilation and analysis of eukaryotic POL II promoter sequences. Nucleic Acids Res. 14: 10009-10026.
11. Burley, S.K, R.G. Roeder. 1996. Biochemistry and structural biology of transcription factor IID (TFIID). Annu.Rev. Biochem. 65: 769-799.
12. Carcamo J, Buckbinder L, Reinberg D. 1991.The initiator directs the assembly of a transcription factor IID-dependent transcription complex. Proc Natl Acad Sci U S A. 88(18):8052-8056
13. Chen W, Provart NJ, Glazebrook J, Katagiri F, et al. 2002. Expression Profile Matrix of Arabidopsis Transcription Factor Genes Suggests Their Putative Functions in Response to Environment Stresses. Plant Cel, 14 (3):559-574
14. Claverie, J. M, I. Sauvaget. 1985. Assessing the biological significance of primary structure consensus patterns using sequence databanks. I. Heat-shock and glucocorticoid control elements in eukaryotic promoters.Comp. Appl. Biosci. 1:95-104.
15. David, s.m.A.B.stephen, H.M.Nicholas, A.H.David, et al.1990.Construction of lacZ promoter probe vectors for use in synechococcus: application to the identification of CO_2-regulated promoter. Gene, 90:43-49.
16. De Crombrugghe B, Busby S, Buc H. 1984. Cyclic AMP receptor protein: role in transcription activation. Science,224(4651):831-8.
17. Doi RH. 1982. Multiple RNA polymerase holoenzymes exert transcriptional specificity in Bacillus subtilis . Archives of Biochemistry and Biophysics, 214 : 772-776.
18. Donna, M.W, J.D.Elizabeth, S.L.Paul. 1981.Cloning restriction fragments that promote expression of gene in Bacillus subtilis J.Bacterol, 1146:1162-1165.
19. Estrem S, Gaal T, Ross w, et al. 1998. Identificadon of Up element consensus for bacterial promoters .Proc, Natl. Sci.USA, 95(17):9761-9766.
20. Fordor.I, O.V.Krasnikova, E.Berets.1990. Cloning, structure and features of a Saccharomyces cerevisia DNA fragment causing the expression of reporter gene. J.Mol.Biol, 24:1141-1418.
21. Grill E, Somerville. C. 1991. Construction and characterization of a yeast artificial chromosomes library of Arabidopsis which is suitable for chromosome walking. Mol. Gen. Genet. 226: 484-490
22. Gusmaroli G, Tonelli C, Mantovanir. 2002. Regulation of novel members of the Arabidopsisthaliana CCAAT-binding nuclear factor Y subunits.Gene, 283 (1- 2): 41 - 48
23. Hanne Jarmer, Thomas S. Larsen, Anders Krogh, et al. 2001. Sigma A recognition sites in the Bacillus subtilis genome . Microbiology, 147: 2417 - 2424
24. Harbury, P.A.B, K. Struhl. 1989. Functional distinctions between yeast TATA elements. Mol. Cell. Biol. 9: 5298-5304.
25. Hofmann, K, P. Bucher. 1995. The FHA domain: A putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem. Sci. 20: 347-349.
26. Horton P, Kanehlsa M. 1992. An assessment of neural network and statistical approaches for prediction of E.coil promoter sites. Nucleic Acids Research, 20(16): 4331-4338.
27. Javahery. R..A, Khachi. K. Lo, B. Zenzie-Gregoty.et al. 1994. DNA sequence requirements for transcriptional initiator activity in mammalian cells. Mol. Cell Biol. 14:116-127.
28. J. Don Chen, Ronald M, Evans. 2005. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature,377:454-457
29. Jeffrey T. Owens, Albert J. Chmura, Katsuhiko Murakami, et al. 1998. Mapping the Promoter DNA Sites Proximal to Conserved Regions of a70 in an Escherichia coli RNA Polymerase-lacUV5 Open Promoter Complex. Biochemistry, 37 (21): 7670 -7675
30. Johnson WC, Moran CP. Jr, Losick R. 1983. Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gene. Nature, 302 : 800-805.
31. Kanhere A, Bansal M. 2005. A novel method for prokaryotic promoter prediction based on DNA stability BMC Bioinformatics ,6:1-32.
32. Kanhere A, malnsal M. 2005. Structural properties of promoters: similarities and differences between prokaryotes and eukstyote. Nucleic Acids Research, 33(10):3165-3175.
33. Kozak, M. 1992. Regulation of translation in eukaryotic systems. Annu. Rev. Cell Biol. 8: 197-225.
34. Kuramohi M, Deshpand M, Karypis G, et al. 2001. Promoter Prediction for Prokaryotes. USA: Minnesota University TR-01-030.
35. Lewin B.Gene VI. Oxford University Press, 2004:457-465
36. Liu YG, Whittier RF. 1995. Thermal Asymmetric Interlaced PCR: Automatable Amplification and Sequencing of Insert End Fragments from PI and YAC Clones for Chromosome Walking. Genomics, 25,674-681
37. Losick R and Pero J. 1981. Cascades of sigma factors. Cell, 25: 582-585.
38. Meijer W, Salas M. 2004. Relevance of Up elements for three strong Bacillus subtilis phage φ29 promoters. Nucleic Acids Research, 32(3): 1166-1176.
39. Michael Fried, Donald M. Crothers. 1981. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis Nucleic Acids Research, Vol. 9, No. 23 6505-6525
40. Michell J, Zheng D, Busby S, et al. 2003. Identification and analysis of extended -10 promoters in Escherichia coli. Nucleic Acids Research, 31(16): 4689-4695
41. Michplep, Calos. 1978. DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation. Nature, 274: 762 - 765
42. Myrick KV, Gelbart WM. 2002. Universal Fast Walking for direct and versatile determination of flanking sequence. Gene, 284(1-2): 125-31.
43. Ochman, H., Gerber, A. S, Hart D. L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621-623
44. Ozoline O. N, Deev A. A, Arkhipova M. V, et al. 1999. Proximal transcribed regions of bacterial promoters have a non-random distribution of A/T tracts. Nucleic Acids Research, 27(24): 4768-4774.
45. Pedersen A, Baldi P, Brunak S, et al. 1996. Characterization of prokaryotic and eukaryotic promoters using hidden markov models: Proceedings of the Fourth International conference on Intelligent System for Molecular Biology, 182-191.
46. Rabwn T. Genomes. 1999. BIOS scientific Publishers Limited, 199-213
47. Rachael. L.N, W.W.Robert, L.R.Raymond.1979. Eukaryotic DNA fragments which act as promoters for a plasmid gene. Nature, 277:324-328.
48. Rosa. F.C. J.F.Martin, J.Gil. 1991. Construction and characterization of promoter-probe vectors for Corynbacteria using the kanamycin-resistance reporter gene. Gene, 98:117-121.
49. Samina Akbar, Tatiana A. Gaidenko, Choong Min Kang, et al. 2001. New Family of Regulators in the Environmental Signaling Pathway Which Activates the General Stress Transcription Factor σ~B of Bacillus subtilis. J. Bacteriol, 183:1329 -1338.
50. Sanchez A, Olmos J. 2004. Bacillus subtilis transcriptional regulators interaction. Biotechnol Lett, 26(5):403-407.
51. Stewart GC and Bott KF. 1983. DNA sequence of the tandem ribosomal RNA promoter for Bacillus subtilis opern rrnB. Nucleic Acids Research, 11:6289-6293.
52. Stormo G. 2000. DNA binding sites: representation and discovery. Bioinformatics, 16(1): 16-23.
53. Voskuil M, Chambliss G. 1998. The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. Nucleic Acids Research. 26 (15): 3584-3590
54. Wong SL , Price CW, Goldfarb DS .1984. The subtilisin E gene of Bacillus subtilis is transcribed from σ 37 promoter in vivo. Proc Natl Acad Sci USA, 81: 1184-1189.
2． 杜俊岭，崔妍，韩继刚，赵刚勇，顾雅君，朱宝成．2004．脱毛碱性蛋白酶的纯化及其生化特性研究．河北农业大学学报，Vol．27 No．3．
3． 杜耀华，王正志．2005．原核启动子识别研究进展．生物技术，Vol．15，No．5：80-83
4． 黄庆，潘皎，彭勇，李昕，张义正．2004．短小芽孢杆菌(Bacillus pumilus)脱毛蛋白酶基因的克隆与序列分析．高技术通讯，14(158)：31-35．
5． 黄庆，胥成浩，张义正．1999．枯草杆菌强基因启动子DNA片段的序列测定． 四川大学学报 36(4)：759-763
6． 李志强．2000．酶法脱毛机理的研究．四川大学博士学位论文．
7． 刘成君，张义正．2001．大肠杆菌-枯草杆菌穿梭质粒载体pSUGV4的构建．四川大学学报(自然科学版)，38(2)：243-246．
8． 刘彦，何先祺，张义正．1998．碱性脱毛蛋白酶产生菌的选育．皮革科学与工程， 8(4)：6-13
9． 潘延云，周艳芳，张贺迎，吴宝东．2003．高产碱性蛋白酶工程菌的构建及鉴定．哈尔滨大学自然科学学报，Vol．19，No．4
10． 邱秀宝，袁影，戴宏，于颖．嗜碱性芽孢杆菌碱性蛋白酶的研究Ⅱ，诱变株选育及产酶条件．1990．微生物学报，30(2)：129-133
11． 唐雪明，邵蔚蓝，沈微，王正祥，方惠英，诸葛健．2002．地衣芽孢杆菌2709和6818碱性蛋白酶基因在大肠杆菌中的克隆，表达及序列分析．应用与环境生物学报， 8(2)：209-214
12． 肖怀秋，林亲录，李玉珍，赵谋明．2005．微生物碱性蛋白酶研进展．中国食品添加剂， No．5
13． 薛林贵．1997．我国碱性蛋白酶的应用及研究进展．微生物学通报，24(6)：370-371
14． 杨国泉．1992 酶制剂应用综述．中国皮革，21(8)：35-38
15． 张义正，刘德明，刘彦，程昌凤，黄庆．2002．脱毛蛋白酶、其制造方法、使用方法和产生此蛋白酶的微生物，中国专利：1361278A
16． 邹立扣，王红宁，潘欣．2003．枯草芽孢杆菌整合研究进展．生物技术通讯，Vol．14 No．16．
17. Aditi Kanhere and Manju Bansal. 2005. Structural properties of promoters: similarities and differences between prokaryotes and eukaryotes Nucleic Acids Research, Vol. 33, No. 10: 3165-3175
18. Aoyama M., C. Toma, M. Yasud. M. Iwanaga, 2000, Sequence of the gene encoding an alkaline serine proteinase of Bacillus pumilus TYO-67., Microbiol. Immunol., 44(5): 389-393
19. Ban-Yang Chang, Roy H. DOl. 1993. Conformational properties of Bacillus subtilis RNA polymerase σ~A factor during transcription initiation. Biochem. J. 294, 43-47
20. Ceglooski P, Luder G, Alonso JC.1990. Genetic analysis of rec E activities in Bacillus subtilis. Mol Gen Genet,(222):441
21. Craig Binnie, Mary Lampe, Richard Losick. 1986. Gene encoding the σ ~(37) species of RNA polymerase σ factor fromBacillus subtilis. Proc. Nati. Acad. Sci. Vol. 83, p: 5943-5947
22. Green G.H. 1952. Unhaidng by Means of Enzymes. J.S.L.T.C. 36:127-134
23. Ferrari. FA, Nguyen. A, Lang. D, et al. 1983.Construction and properties of and intergrable plasmid for Bacillus subtilis, Jbacteriiology,6:1513
24. Frohman, M. A., Dush, M. K., and Martin, G. R. 1988. Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Nutl. Acad. Sci. USA 85:8998-9002
25. Grill, E., and Somerville, C. 1991. Construction and characterization of a yeast artificial chromosomes library of Arabidopsis which is suitable for chromosome walking. Mol. Gen. Genet. 226:484-490
26. Hahn J, Dubnau D. 1985. Analysis of plasmid deletional instability in Bacillus subtilis Journal of bacterioligy, 1014-1023
27. Jane R. McLaughlin, Cheryl L. Murray, Jesse C. Rabinowitz. 1981. Unique Features in the Ribosome Binding Site Sequencoefthe Grampositive Staphylococcus aureus fl-Lactamase Gene. the Journal biological chemistry.Vol 256. No 21
28. Khasanov. FK, Zvingilla. DJ, Zainullin. AA, et al. 1986. Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology. Genetics, 112:441
29. Kunamneni Adinarayana, Poluri Elllaiah, Davuluri Siva Prasad et al. 2003. Purification and Partial Characterization of Thermostable Serine Alkaline Protease from a Newly Isolated Bacillus sublitis PE-11. AAPS PharmSciTech 4 (4)
30. Liu YG, Whittier RF. 1995.Thermal Asymmetric Interlaced PCR: Automatable Amplification and Sequencing of Insert End Fragments from PI and YAC Clones for Chromosome Walking. Genomics, 25, 674-681
31. Loh, E. Y., Elliot, J. F., Cwirla, S., Lanier, L., and Davis, M. M. 1989. Polymerase chain reaction with single-sided specificity: Analysis of T cell receptor 6 chain. Science 243: 217-220
32. Marc M. S. M. Wosten, Miranda Boeve, Mirjam. G.A. Koot, et al. 1998. Identification of Campylobacter jejuni Promoter Sequences Journal of bacteriology, Vol.180 No.3 p594-599
33. Marisa. M, Naomi. C, Takao. S, Takashi. H, Kimiko. Y. 1999. Cloning and characterization of the O-Methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of Demethylsterigmatocystin to Sterigmatocystin and Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin in Aflatoxin Biosynthesis. Microbiology, 4987-4994
34. Martin I. Voskuil and Glenn H. Chambliss. 1998. The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. Nucleic Acids Research, 1998, Vol. 26, No. 15: 3584-3590
35. Melville. SB, Labbe. R, Sonenshein. A L.1994. Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis. Infect Immun, 62(12): 5550-5558
36. Ochman, H, Gerber, A. S, and Hart, D. L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621-623
37. Pan J, Huang Q, Zhang YZ. 2004. Gene cloning and expression of an alkaline serine protease with dehairing function from Bacillus pumilus. Curr Microbiol 49:165-169
38. Parks, C. L., Chang, L.-S., and Shenk, T. 1991. A polymerase chain reaction mediated by a single primer: Cloning of genomic sequences adjacent to a serotonin receptor protein coding region.Nucleic Acids Res. 19: 7155-7160
39. Paul, R.G., Mohamed, I., Davighi, D., Addy, V.L. and Covington, A.D. 2001. The use of neutral protease in enzymatic unhairing. J Am Leather Chem Ass 96,180-185
40. Tsutomu. T, Terou. T, Itaya. 1996. M. A method to invert DNA segments of Bacillus subtilis 168 genome by recombination between two homologous sequences. Biotech Biotechem, 60(5):773
41. Tsuyoshi Nonaka, Masahim Fujihashi, AkikoKita. Katsuhisa etal. 2004. The crystal structure of an oxidatively-stable subtilisin alkaline serine protease, KP-43, with a C-terminal 8-barrel domain. J. Biol. Chem
42. XC Wu, W Lee, L Tran, and S L Wong. 1991. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellμLar proteases. JBacteriol, 173(16): 4952-4958
43. Yansura DG, Henner DJ (1984) Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. USA.i 1,439-443
1． 杜耀华，王正志．2005．原核启动子识别研究进展．生物技术，Vol．15，No．5：80-83
2． 邓晓东，费小雯，黄俊生等．2002．Isolation and Analysis of Rubber Hevein Gene and Its Promoter Sequence．植物学报，44(8)：936-940
3． 李维，张义正．2000．黄孢原毛平革菌基因启动子的分离与鉴定．生物工程学报，16(5)： 599-602．
4． 王新力，彭学贤，李宏．2000．香蕉果实特异性ACC合酶的cDN克隆及序列分析．生物工程学报，16(2)：134-136．
5． 吴乃虎．基因工程原理(下)．北京：科学出版社，2001：74-75
6. Berg, O.G, P.H. von Hippel. 1988. Selection of DNA binding sites by regulatory proteins. Trends Biochem. Sci, 13:207-211.
7. Bird, A.P. 1987. CpG islands as gene markers in the vertebrate nucleus. Trends Genet. 3: 342-347Brown T. Genomes. BIOS scientific Publishers Limited, 1999:199-213
8. Bolshoy A, Nero E. 2000. Ecologic genomic of DNA: upstream banding in prokaryotic promoters. Genome Research, 10(8):1185-1193.
9. Bucher, P. 1990. Weight matrix descriptions of four eukaryotic RNA polymerase Ⅱ promoter elements derived from 502 unrelated promoter sequences. J. Mol. Biol.212: 563-578.
10. Bucher, P, E.N. Trifonov. 1986. Compilation and analysis of eukaryotic POL II promoter sequences. Nucleic Acids Res. 14: 10009-10026.
11. Burley, S.K, R.G. Roeder. 1996. Biochemistry and structural biology of transcription factor IID (TFIID). Annu.Rev. Biochem. 65: 769-799.
12. Carcamo J, Buckbinder L, Reinberg D. 1991.The initiator directs the assembly of a transcription factor IID-dependent transcription complex. Proc Natl Acad Sci U S A. 88(18):8052-8056
13. Chen W, Provart NJ, Glazebrook J, Katagiri F, et al. 2002. Expression Profile Matrix of Arabidopsis Transcription Factor Genes Suggests Their Putative Functions in Response to Environment Stresses. Plant Cel, 14 (3):559-574
14. Claverie, J. M, I. Sauvaget. 1985. Assessing the biological significance of primary structure consensus patterns using sequence databanks. I. Heat-shock and glucocorticoid control elements in eukaryotic promoters.Comp. Appl. Biosci. 1:95-104.
15. David, s.m.A.B.stephen, H.M.Nicholas, A.H.David, et al.1990.Construction of lacZ promoter probe vectors for use in synechococcus: application to the identification of CO_2-regulated promoter. Gene, 90:43-49.
16. De Crombrugghe B, Busby S, Buc H. 1984. Cyclic AMP receptor protein: role in transcription activation. Science,224(4651):831-8.
17. Doi RH. 1982. Multiple RNA polymerase holoenzymes exert transcriptional specificity in Bacillus subtilis . Archives of Biochemistry and Biophysics, 214 : 772-776.
18. Donna, M.W, J.D.Elizabeth, S.L.Paul. 1981.Cloning restriction fragments that promote expression of gene in Bacillus subtilis J.Bacterol, 1146:1162-1165.
19. Estrem S, Gaal T, Ross w, et al. 1998. Identificadon of Up element consensus for bacterial promoters .Proc, Natl. Sci.USA, 95(17):9761-9766.
20. Fordor.I, O.V.Krasnikova, E.Berets.1990. Cloning, structure and features of a Saccharomyces cerevisia DNA fragment causing the expression of reporter gene. J.Mol.Biol, 24:1141-1418.
21. Grill E, Somerville. C. 1991. Construction and characterization of a yeast artificial chromosomes library of Arabidopsis which is suitable for chromosome walking. Mol. Gen. Genet. 226: 484-490
22. Gusmaroli G, Tonelli C, Mantovanir. 2002. Regulation of novel members of the Arabidopsisthaliana CCAAT-binding nuclear factor Y subunits.Gene, 283 (1- 2): 41 - 48
23. Hanne Jarmer, Thomas S. Larsen, Anders Krogh, et al. 2001. Sigma A recognition sites in the Bacillus subtilis genome . Microbiology, 147: 2417 - 2424
24. Harbury, P.A.B, K. Struhl. 1989. Functional distinctions between yeast TATA elements. Mol. Cell. Biol. 9: 5298-5304.
25. Hofmann, K, P. Bucher. 1995. The FHA domain: A putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem. Sci. 20: 347-349.
26. Horton P, Kanehlsa M. 1992. An assessment of neural network and statistical approaches for prediction of E.coil promoter sites. Nucleic Acids Research, 20(16): 4331-4338.
27. Javahery. R..A, Khachi. K. Lo, B. Zenzie-Gregoty.et al. 1994. DNA sequence requirements for transcriptional initiator activity in mammalian cells. Mol. Cell Biol. 14:116-127.
28. J. Don Chen, Ronald M, Evans. 2005. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature,377:454-457
29. Jeffrey T. Owens, Albert J. Chmura, Katsuhiko Murakami, et al. 1998. Mapping the Promoter DNA Sites Proximal to Conserved Regions of a70 in an Escherichia coli RNA Polymerase-lacUV5 Open Promoter Complex. Biochemistry, 37 (21): 7670 -7675
30. Johnson WC, Moran CP. Jr, Losick R. 1983. Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gene. Nature, 302 : 800-805.
31. Kanhere A, Bansal M. 2005. A novel method for prokaryotic promoter prediction based on DNA stability BMC Bioinformatics ,6:1-32.
32. Kanhere A, malnsal M. 2005. Structural properties of promoters: similarities and differences between prokaryotes and eukstyote. Nucleic Acids Research, 33(10):3165-3175.
33. Kozak, M. 1992. Regulation of translation in eukaryotic systems. Annu. Rev. Cell Biol. 8: 197-225.
34. Kuramohi M, Deshpand M, Karypis G, et al. 2001. Promoter Prediction for Prokaryotes. USA: Minnesota University TR-01-030.
35. Lewin B.Gene VI. Oxford University Press, 2004:457-465
36. Liu YG, Whittier RF. 1995. Thermal Asymmetric Interlaced PCR: Automatable Amplification and Sequencing of Insert End Fragments from PI and YAC Clones for Chromosome Walking. Genomics, 25,674-681
37. Losick R and Pero J. 1981. Cascades of sigma factors. Cell, 25: 582-585.
38. Meijer W, Salas M. 2004. Relevance of Up elements for three strong Bacillus subtilis phage φ29 promoters. Nucleic Acids Research, 32(3): 1166-1176.
39. Michael Fried, Donald M. Crothers. 1981. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis Nucleic Acids Research, Vol. 9, No. 23 6505-6525
40. Michell J, Zheng D, Busby S, et al. 2003. Identification and analysis of extended -10 promoters in Escherichia coli. Nucleic Acids Research, 31(16): 4689-4695
41. Michplep, Calos. 1978. DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation. Nature, 274: 762 - 765
42. Myrick KV, Gelbart WM. 2002. Universal Fast Walking for direct and versatile determination of flanking sequence. Gene, 284(1-2): 125-31.
43. Ochman, H., Gerber, A. S, Hart D. L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621-623
44. Ozoline O. N, Deev A. A, Arkhipova M. V, et al. 1999. Proximal transcribed regions of bacterial promoters have a non-random distribution of A/T tracts. Nucleic Acids Research, 27(24): 4768-4774.
45. Pedersen A, Baldi P, Brunak S, et al. 1996. Characterization of prokaryotic and eukaryotic promoters using hidden markov models: Proceedings of the Fourth International conference on Intelligent System for Molecular Biology, 182-191.
46. Rabwn T. Genomes. 1999. BIOS scientific Publishers Limited, 199-213
47. Rachael. L.N, W.W.Robert, L.R.Raymond.1979. Eukaryotic DNA fragments which act as promoters for a plasmid gene. Nature, 277:324-328.
48. Rosa. F.C. J.F.Martin, J.Gil. 1991. Construction and characterization of promoter-probe vectors for Corynbacteria using the kanamycin-resistance reporter gene. Gene, 98:117-121.
49. Samina Akbar, Tatiana A. Gaidenko, Choong Min Kang, et al. 2001. New Family of Regulators in the Environmental Signaling Pathway Which Activates the General Stress Transcription Factor σ~B of Bacillus subtilis. J. Bacteriol, 183:1329 -1338.
50. Sanchez A, Olmos J. 2004. Bacillus subtilis transcriptional regulators interaction. Biotechnol Lett, 26(5):403-407.
51. Stewart GC and Bott KF. 1983. DNA sequence of the tandem ribosomal RNA promoter for Bacillus subtilis opern rrnB. Nucleic Acids Research, 11:6289-6293.
52. Stormo G. 2000. DNA binding sites: representation and discovery. Bioinformatics, 16(1): 16-23.
53. Voskuil M, Chambliss G. 1998. The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. Nucleic Acids Research. 26 (15): 3584-3590
54. Wong SL , Price CW, Goldfarb DS .1984. The subtilisin E gene of Bacillus subtilis is transcribed from σ 37 promoter in vivo. Proc Natl Acad Sci USA, 81: 1184-1189.