WO2020042292A1 - 启动子、其载体及其应用 - Google Patents

启动子、其载体及其应用 Download PDF

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WO2020042292A1
WO2020042292A1 PCT/CN2018/110217 CN2018110217W WO2020042292A1 WO 2020042292 A1 WO2020042292 A1 WO 2020042292A1 CN 2018110217 W CN2018110217 W CN 2018110217W WO 2020042292 A1 WO2020042292 A1 WO 2020042292A1
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promoter
sequence
atg
seq
gene
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French (fr)
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刘秀霞
董贵彬
赵子豪
司雅楠
王新月
杨艳坤
白仲虎
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element

Definitions

  • the invention belongs to the field of biological engineering technology, and particularly relates to a promoter, a vector thereof and an application thereof.
  • Promoter is a key DNA sequence that controls the initiation of gene transcription and plays an important role in regulating gene expression. Promoters mainly control the transcription level of genes. A large number of studies have proved that the influence of promoter regions in various sequence regions that control gene expression exceeds 45%. Therefore, studies on gene expression were screened for highly active promoters. Is of great significance.
  • the existing technology has the problems of low protein expression efficiency and few available genetic tools, and the development of efficient promoters for foreign protein expression is an important way to solve these problems.
  • the inducible promoters commonly used in recent years include the tac promoter (Ptac), arabinose promoter, maltose-inducible promoter, propionic acid-inducible promoter, temperature-sensitive promoter, etc., but the expression activity of these promoters is very limited and can be There are few choices. In view of the importance of inducible promoters in protein expression and metabolic engineering research, it is very necessary to develop a highly active inducible promoter of Corynebacterium glutamicum.
  • the present invention overcomes the shortcomings in the prior art and provides a promoter.
  • a promoter including: a promoter core region sequence, the promoter core region sequence including at least the Corynebacterium glutamicum SEQ ID NO: 6 gene coding sequence ATG Sequences upstream from 91bp to 180bp.
  • the promoter includes at least a promoter core region sequence, and the 3 'end of the promoter core region sequence is located in the Corynebacterium glutamicum SEQ ID NO: 6 gene encoding
  • the upstream of the sequence ATG is 91 bp, and the 5 ′ end is located at any base of 181 bp to 200 bp upstream of the ATG glutamic acid SEQ ID ID: 6 gene coding sequence.
  • the sequence interval of the promoter includes at least the 3 'end located at the 91 bp and 5' end upstream of the C. glutamicum SEQ ID NO: 6 gene coding sequence ATG. It is located at any base between 201bp and 257bp upstream of the ATG glutamic acid SEQ ID ID: 6 gene coding sequence ATG.
  • the sequence interval of the promoter includes at least the 3 ′ end located at the 91 bp and 5 ′ end upstream of the C. glutamicum SEQ ID ID: 6 gene coding sequence ATG. It is located at any base between 201bp and 345bp upstream of the ATG glutamic acid SEQ ID ID: 6 gene coding sequence ATG.
  • a 5'UTR sequence is further included.
  • the length of the 5'UTR sequence is 90bp.
  • the 5'UTR sequence is a 1 bp to 90 bp interval sequence upstream of the ATG coding sequence of the Corynebacterium glutamicum SEQ ID ID: 6 gene.
  • the method further includes a source gene sequence, and the source gene sequence includes a SEQ ID NO: 6 gene coding sequence ATG and a base downstream of the ATG.
  • the source gene sequence includes a C. glutamicum SEQ ID ID: 6 gene coding sequence ATG and a 59 bp base downstream of the ATG.
  • the complementary sequence of the promoter sequence is included.
  • a source gene sequence is included, and the source gene sequence is shown in SEQ ID NO: 2.
  • the nucleotide sequence of the promoter is shown in SEQ ID NO: 3.
  • the promoter can be induced by any one or more of benzyl alcohol, benzaldehyde, benzoic acid, and catechol.
  • the induced concentration range of the benzyl alcohol is 1 mM to 50 mM.
  • the induced concentration of the benzyl alcohol is 10 mM.
  • the present invention overcomes the shortcomings in the prior art and provides a carrier.
  • the present invention provides the following technical solution: a vector, wherein the vector is loaded with the promoter according to any one of claims 1 to 15.
  • the vector includes a protein expression vector, and its nucleotide sequence is as shown in SEQ ID NO: 4.
  • the present invention overcomes the shortcomings in the prior art and provides the use of the promoter in protein expression.
  • the present invention provides the following technical solution: the application of the promoter in protein expression.
  • the promoter is used for expression of an endogenous or exogenous protein of Corynebacterium glutamicum.
  • the promoter and gene fragments of the present invention are constructed to form an expression frame, which can express a large amount of protein in prokaryotes, and especially after being induced by an inducer, the protein expression activity is significantly higher than that of traditional Ptac. Is a super efficient promoter.
  • the promoter of the present invention can be used for efficient expression of prokaryotic endogenous or exogenous proteins.
  • Figure 1 shows the sequence of the expression vector p19-0-ZDA.
  • Figure 2 is a comparison of the induction activity of P ZDA with different lengths
  • Figure 3 is a comparison of P ZDA expression activity under different inducers
  • Figure 4 is a comparison of P ZDA expression activity induced by benzyl alcohol at different concentrations and its activity compared with Ptac activity;
  • FIG. 5 is an SDS-PAGE experimental analysis diagram of recombinant green-protein expressing by Corynebacterium glutamicum;
  • Lane M protein Marker26610; 1: blank control; 2: Ptac without IPTG; 3: Ptac with IPTG (final concentration 100 ⁇ M); 4: P ZDA without benzyl alcohol; 5, 6, 7, 8, 9 and 10: P ZDA plus different amounts of benzyl alcohol (final concentrations are 1 mM, 10 mM, 15 mM, 20 mM, 25 mM, and 50 mM, respectively).
  • FIG. 6 is an example of application of recombinant Z. glutamicum using P ZDA to express human gastric factor protein
  • Lane M protein Marker26610; 1: blank control; 2: benzyl alcohol (final concentration 10 mM) induced expression of human gastric factors.
  • FIG. 7 SDS-PAGE experimental analysis of recombinant B. glutamicum expressing BNP
  • M protein Marker26610; 1: Ptac without IPTG 2: Ptac plus IPTG (final concentration 100 ⁇ M) expresses BNP; 3: P ZDA does not induce BNP expression; 4: P ZDA plus benzyl alcohol (final concentration 10 mM) induces BNP expression.
  • Figure 8 ELASA quantitative analysis of recombinant B. glutamicum expressing BNP
  • Ptac does not add IPTG
  • 2 Ptac plus IPTG (100 ⁇ M) expresses BNP
  • 3 P ZDA does not induce BNP expression
  • 4 P ZDA plus benzyl alcohol (final concentration 10 mM) induces BNP expression.
  • an embodiment or “an embodiment” referred to herein refers to a particular feature, structure, or characteristic that can be included in at least one implementation of the present invention.
  • the appearances of "in one embodiment” in various places in this specification do not all refer to the same embodiment, nor are they separate or selectively mutually exclusive embodiments.
  • the plasmid backbone used in the present invention includes a detection vector p19-0, which has the shuttle ability of E. coli and Corynebacterium glutamicum and can stably exist in two kinds of bacteria; two sides of the multi-cloning site of the detection vector are two Terminators, a T7 terminator and a rrnB terminator, can avoid the effect of the vector's own promoter on the expression box.
  • the construction method of the detection vector p19-0 is as follows:
  • PXMJ19 was used as a template, p19-MUT-F and p19-MUT-R were used as primers to amplify the mutant linear plasmid.
  • the product was treated with Dpn at 37 ° C for 2 hours and transformed directly into E. coli DH5 ⁇ .
  • the correct mutant plasmid was named p19. -mut;
  • the reaction system was 10 ⁇ Q5Buffer 5 ⁇ L, dNTPs (2.5 mmol / L) 4 ⁇ L, upstream and downstream primers (10 ⁇ mol / L) each 1 ⁇ L, Q5 DNA polymerase (2U / ⁇ L) 0.5 ⁇ L, template 1 ⁇ L, and water was added to 50 ⁇ L;
  • PCR amplification conditions are: 98 ° C for 3 min; 98 ° C for 15s; 72 ° C 3.25min, 30 cycles; 72 ° C for 2min;
  • the plasmid backbone vector of the present invention can be replaced with other prior art vectors.
  • the bacterial genome extraction kit of Tiangen Biochemical Technology Co., Ltd. was used to extract the genome of Corynebacterium glutamicum (taking Corynebacterium glutamicum CGMCC1.15647 as an example) for the promoter cloning template; the glutamic acid used in the present invention Corynebacterium CGMCC1.15647, whose Latin name is C.glutamicumBZH001, was donated by Zhangjiagang Huachang Pharmaceutical Co., Ltd. in 2013.
  • the gene C.glutamicumBZH001 of SEQ ID ID NO: 6 gene coding sequence ATG upstream 355bp to downstream 62bp sequence region design promoter clone primers 355ZDA-F and ZDA-R (SEQ ID ID NO: 6
  • the gene coding sequence ATG starts from the 501bp base of the SEQ ID NO: 6 sequence
  • the SEQ ID NO: 6 gene coding sequence is the 501bp to 1355bp of the sequence listing SEQ ID ID NO: 6
  • the cloned promoter sequence and its downstream The 62bp sequence (including ATG) of the gene ATG, and a 15bp RBS sequence (AAAGGAGGACAACTA) and two reverse BsaI restriction sites and one BamH restriction site were introduced into the 5 'end of the downstream primer ZDA-R.
  • the BamH I restriction site coincides with the second Bsa I restriction site nick;
  • the reaction system was 10 ⁇ Q5Buffer, 5 ⁇ L, dNTPs (2.5 mmol / L) 4 ⁇ L, upstream and downstream primers (10 ⁇ mol / L) each 1 ⁇ L, Q5 DNA polymerase (2U / ⁇ L) 0.5 ⁇ L, template 1 ⁇ L, and water was added to 50 ⁇ L.
  • PCR amplification conditions were: 98 ° C for 3 minutes; 98 ° C for 15s; 60 ° C for 15s; 72 ° C for 15s; 30 cycles; 72 ° C for 45s.
  • the aforementioned P ZDA promoter fragment and p19-0 detection vector cloned by PCR were double-digested with restriction enzymes BamH I and Hind III of TAKALA, and the two fragments were digested with T4 DNA ligase from TAKALA. Ligation gave the expression vector p19-0-ZDA.
  • the expression plasmid contains the promoter P ZDA , and two reverse Bsa I restriction sites are inserted immediately downstream of the promoter to facilitate the seamless insertion of the gene.
  • the sequence of the expression vector p19-0-ZDA is shown in SEQ ID NO: 4.
  • EGFP was used as a reporter gene to test the activity of the promoter of the present invention.
  • egfp-F and egfp-R egfp fluorescent protein EGFP gene was cloned by Bsa I restriction sites for cloning into an expression vector downstream of a promoter p19-0-ZDA P ZDA obtained EGFP expression vector p19-0 -ZDA-egfp for expression of fluorescent protein EGFP.
  • the gene sequence of egfp is shown in SEQ ID NO: 5.
  • the reaction system was 10 ⁇ Q5Buffer, 5 ⁇ L, dNTPs (2.5 mmol / L) 4 ⁇ L, upstream and downstream primers (10 ⁇ mol / L) each 1 ⁇ L, Q5 DNA polymerase (2U / ⁇ L) 0.5 ⁇ L, template 1 ⁇ L, and water was added to 50 ⁇ L.
  • the PCR amplification conditions were: 98 ° C for 3 min; 98 ° C for 15s; 60 ° C for 15s; 72 ° C for 30s; 30 cycles; 72 ° C for 1 min and 15s.
  • EGFP is used as one of the "reporter genes" to test the activity of the promoter of the present invention.
  • EGFP can also be replaced with other endogenous genes, foreign genes, and other target genes.
  • the upstream primers 98ZDA-F (referring to the 5 'end of the promoter located at 98 bp upstream of the ATG coding sequence of SEQ ID NO: 6), 180ZDA-F (referring to the 5' end of the promoter located at the SEQ ID NO: 6 gene encoding 180bp upstream of the sequence ATG), 200ZDA-F (referring to the 5 'end of the promoter is located 200bp upstream of the coding sequence of the ATG gene), 257ZDA-F (referring to the 5' end of the promoter is located in the gene encoding the SEQ ID NO: 6 gene) 257bp upstream of the sequence ATG), 308ZDA-F (referring to the 5 'end of the promoter is located at the SEQ ID NO: 6 gene encoding sequence 308bp upstream) and 345ZDA-F (referring to the 5' end of the promoter is located at the gene encoding SEQ ID NO: 6)
  • a 62 bp sequence region downstream of the ATG coding sequence of the SEQ ID NO: 6 gene is selected as the source gene, that is, the 3 'end of the promoter of the present invention is located at the ATG and 59 bp downstream of the coding sequence of the ID NO: 6 gene
  • the source gene sequence is shown in SEQ ID NO: 2.
  • the SEQ ID NO: 6 gene coding sequence ATG and 59 bp downstream of ATG are only one of the implementation methods of the present invention. The research of the present invention found that the degree of fragmentation of the source gene can be adjusted while still inducing the promoter of the present invention. Express activity.
  • the operation method is to use the above primers and primers egfp-R respectively to perform PCR amplification using the p19-0-ZDA-egfp plasmid as a template (the PCR system and the amplification program are the same as above), and the amplification
  • the amplified fragment was digested with Hind III and BamH I, and then ligated into the detection vector p19-0 to construct the corresponding EGFP expression vector.
  • the constructed vector was cloned by E. coli DH5 ⁇ and transformed into Corynebacterium glutamicum by electric shock.
  • Recombinant Corynebacterium glutamicum containing the expression vector was inoculated into 5 mL of LBB liquid medium containing chloramphenicol at a concentration of 10 ⁇ g / L, and cultured at 30 ° C and 230 rpm overnight to prepare seed liquids, and the OD 600 of each sub-liquid was measured separately; Various sub-liquids were transferred to fresh 20mL / 100mL (100mL triangle flasks containing 20mL of medium) LB medium, so that the initial OD 600 was 0.2, and shake flask culture was performed at 230 ° C and 230rpm, and each of the recombinant bacteria was cultured for 2h.
  • the inducer was added; and after 20 hours of culture, the OD 600 and the fluorescence intensity of the bacteria were measured by a UV spectrophotometer and a fluorescence spectrophotometer respectively, and the fluorescence intensity of the unit OD 600 of each strain was calculated for expression activity Comparison.
  • the present invention continues to select promoters of fragments of different lengths to study the activity of the promoter, and further truncates the length of the promoter located at the upstream sequence of SEQ ID NO: 6ATG to 308bp.
  • the promoter-induced expression activity is reduced compared to 345bp, However, its promoter expression activity is still significantly higher than that of the prior art Ptac promoter (in conjunction with Figures 2, 5, and 8 of the present invention, it can be seen that the 257P ZDA promoter of the present invention does not undergo expression induced by an inducer, Its promoter expression activity is already equivalent to or even higher than the expression activity of the prior art Ptac under the inducer-induced expression state.)
  • the promoter has good expression although it does not have inducible expression activity.
  • the P ZDA promoter of the present invention has the best inducible expression activity when the length is 345 bp upstream of SEQ ID NO: 6ATG, and the fluorescence intensity of the unit OD 600 reaches 16272. This structure is selected for subsequent experiments.
  • the sequence of the operon region is a C. glutamicum SEQ ID ID ID: 6 gene upstream sequence of 201 bp to 345 bp of the ATG coding sequence, of which the C. glutamicum SEQ ID ID NO: 6 gene encoding sequence ATG upstream of 345 bp length
  • the sequence is shown in SEQ ID NO: 1;
  • the sequence of the core region of the promoter is Corynebacterium glutamicum SEQ ID ID: 6 gene ATG upstream sequence of 91bp to 200bp;
  • the 5'UTR sequence is Corynebacterium glutamicum SEQ ID ID: 6 gene upstream 1 bp to 90 bp sequence of ATG; part of the source gene sequence includes SEQ ID NO: 6 gene encoding sequence ATG and 59 bp downstream of ATG, such as SEQ ID ID NO : 2 as shown.
  • the nucleotide sequence of the promoter is shown in SEQ ID NO: 3.
  • the 345P ZDA promoter has inductive activity over a wide range of benzyl alcohol concentrations (1 mM to 50 mM). However, the expression activity was highest in LB medium with a final concentration of 10 mM, which was much higher than that of the control Ptac. The fluorescence intensity of the unit OD600 reached 16,740.
  • Ptac is a positive control plasmid constructed with the above RBS sequence (AAAGGAGGACAACTA), a foreign gene fragment and plasmid pXMJ19, and uses the plasmid pXMJ19's own Ptac promoter.
  • This classic positive control represents the efficient expression of recombinant proteins in Corynebacterium glutamicum Level, its construction includes the following steps:
  • the clone consists of a RBS sequence and an egfp gene to form a gene fragment.
  • the egfp gene is shown in SEQ ID NO: 5, and the required primers are: SD-EGFP-F and SD-EGFP-R.
  • the PCR reaction system and procedures used are the same as above;
  • the primers GIF-F and GIF-R were used to express human gastric factor protein (GIF, apparent molecular weight of about 45 kDa, GeneID: 2694) as the expression gene, and it was successfully detected in the ultrasonic disrupted pellet of the bacteria To a large number of protein expression, the results are shown in Figure 6.
  • GIF human gastric factor protein
  • GIF-F AA GGTCTC AAATGGCCTGGTTTGCCCTG (underlined is the restriction site Bsa I)
  • GIF-R AA GGTCTC GGATCCTTAATACTGGGTGAAATTGGCG (the restriction site is Bsa I);
  • NT-ProBNP-F and NT-ProBNP-R were used to replace the expressed gene with HIS-SUMO-N-terminal natriuretic peptide precursor (NT-proBNP, apparent molecular weight of about 25kDa, GeneID: 4879) was expressed, and a Ptac vector containing Ptac and induced by IPTG to express a foreign gene was used as a control.
  • NT-proBNP HIS-SUMO-N-terminal natriuretic peptide precursor
  • Ptac vector containing Ptac and induced by IPTG to express a foreign gene was used as a control.
  • the results are shown in Figures 7 and 8.
  • the SDS-PAGE analysis results were consistent with the ELASA quantitative analysis results.
  • NT-ProBNP-F AA GGTCTC AAATGGGTCATCATCACCACCACCAC (the restriction site is Bsa I)
  • NT-ProBNP-R AA GGTCTC GGATCCTTAGCGCGGGGCACGCAG (the restriction site is Bsa I);
  • the inducible promoter 345P ZDA derived from Corynebacterium glutamicum according to the present invention has significantly higher EGFP and HIS-SUMO-N-terminal brain natriuretic peptide precursor expression under the induction of benzyl alcohol than IPTG-induced Ptac, binding
  • FIG. 2, FIG. 5, and FIG. 8 that under the condition that the 257P ZDA promoter of the present invention is not induced by the inducer, its promoter expression activity is already equivalent to or even higher than that of the prior art Ptac induced by the inducer.
  • the promoter of the present invention is of great significance in the expression of foreign proteins in C. glutamicum and in metabolic engineering.

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Abstract

启动子、其载体及其应用。启动子包括启动子核心区序列，该启动子核心区序列至少包含谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游91bp～180bp区间序列。将该启动子与基因片段进行构建组成表达框，能够在原核生物中大量表达蛋白，特别是被诱导剂诱导后，蛋白表达活性显著高于传统Ptac的诱导表达活性。

Description

启动子、其载体及其应用 技术领域
本发明属于生物工程技术领域，具体涉及启动子、其载体及其应用。
背景技术
启动子是一段控制着基因转录起始的关键DNA序列，对基因的表达具有重要的调控作用。启动子主要控制基因的转录水平，有研究通过大量的分析证明在控制基因表达的各种序列区域中，启动子区域的影响力超过45％，因此，筛选高活性的启动子对基因表达的研究具有重要意义。
目前现有技术存在蛋白表达效率低，可用遗传工具少等问题，而开发可用于外源蛋白表达的高效启动子是解决这些问题的重要途径。近年来常用的诱导型启动子有tac启动子(Ptac)、阿拉伯糖启动子、麦芽糖诱导启动子、丙酸诱导启动子、温度敏感启动子等，但是这些启动子的表达活力很有限，且可选择的种类少。鉴于诱导型启动子在蛋白表达和代谢工程研究中的重要意义，非常有必要开发一种谷氨酸棒杆菌的高活力的诱导型启动子。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。
作为本发明其中一个方面，本发明克服现有技术中存在的不足，提供启动子。
为解决上述技术问题，本发明提供了如下技术方案：启动子，其中：包括，启动子核心区序列，所述启动子核心区序列至少包含谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游91bp～180bp区间序列。
作为本发明所述的启动子的一种优选方案：所述启动子至少包括启动子核心区序列，所述启动子核心区序列的3’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游第91bp，5’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游181bp～200bp中任一碱基。
作为本发明所述的启动子的一种优选方案：所述启动子的序列区间至少包括，3’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游第91bp， 5’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游201bp～257bp中任一碱基。
作为本发明所述的启动子的一种优选方案：所述启动子的序列区间至少包括，3’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游第91bp，5’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游201bp～345bp中任一碱基。
作为本发明所述的启动子的一种优选方案：还包括5’UTR序列。
作为本发明所述的启动子的一种优选方案：所述5’UTR序列的长度为90bp。
作为本发明所述的启动子的一种优选方案：所述5’UTR序列为谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG上游1bp～90bp区间序列。
作为本发明所述的启动子的一种优选方案：还包括，源基因序列，所述源基因序列包括SEQ ID NO：6基因编码序列ATG以及ATG下游的碱基。
作为本发明所述的启动子的一种优选方案：所述源基因序列包括谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG以及ATG下游的59bp碱基。
作为本发明所述的启动子的一种优选方案：包括所述启动子序列的互补序列。
作为本发明所述的启动子的一种优选方案：包括源基因序列，所述源基因序列如SEQ ID NO：2所示。
作为本发明所述的启动子的一种优选方案：所述启动子的核苷酸序列如SEQ ID NO：3所示。
作为本发明所述的启动子的一种优选方案：所述启动子能够被包括苯甲醇、苯甲醛、苯甲酸、儿茶酚的任一种或几种诱导。
作为本发明所述的启动子的一种优选方案：所述苯甲醇的诱导浓度范围为1mM～50mM。
作为本发明所述的启动子的一种优选方案：所述苯甲醇的诱导浓度为10mM。
作为本发明的另一个方面，本发明克服现有技术中存在的不足，提供载体。
为解决上述技术问题，本发明提供了如下技术方案：载体，其中：所述载体装载权利要求1～15任一所述的启动子。
作为本发明所述的载体的一种优选方案：所述载体包括蛋白表达载体，其核苷酸序列如SEQ ID NO：4。
作为本发明的另一个方面，本发明克服现有技术中存在的不足，提供所述的启动子在蛋白表达中的应用。
为解决上述技术问题，本发明提供了如下技术方案：所述的启动子在蛋白表达中的应用。
作为本发明所述的启动子在蛋白表达中的应用的一种优选方案：所述启动子用于谷氨酸棒杆菌内源或外源蛋白的表达。
本发明的有益效果：将本发明启动子与基因片段进行构建组成表达框，能够在原核生物中大量表达蛋白，特别是被诱导剂诱导后，蛋白表达活性显著高于传统Ptac的诱导表达活性，是一种超高效启动子。本发明启动子能够用于原核生物内源或外源蛋白的高效表达。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：
图1为表达载体p19-0-ZDA的序列图谱。
图2为P ZDA不同长度诱导活性对比；
1：空质粒对照；2：P ZDA-98bp不加苯甲醇；3：P ZDA-98bp加苯甲醇(终浓度10mM)；4：P ZDA-180bp不加苯甲醇；5：P ZDA-180bp加苯甲醇(终浓度10mM)；6：P ZDA-200bp不加苯甲醇；7：P ZDA-200bp加苯甲醇(终浓度10mM)；8：P ZDA-257bp不加苯甲醇；9：P ZDA-257bp加苯甲醇(终浓度10mM)；10：P ZDA-308bp不加苯甲醇；11：P ZDA-308bp加苯甲醇(终浓度10mM)；12：P ZDA-345bp不加苯甲醇；13：P ZDA-345bp加苯甲醇(终浓度10mM)；14：P ZDA-355bp不加苯甲醇；15：P ZDA-355bp加苯甲醇(终浓度10mM)；其中，P ZDA后的长度(98bp、180bp、200bp、257bp、308bp、345bp、355bp)代表P ZDA位于SEQ ID NO：6ATG上游序列的长度。
图3为P ZDA在不同诱导剂下的表达活性对比；
1：P ZDA不加诱导剂；2：P ZDA加苯甲醇(终浓度10mM)；3：P ZDA加苯甲 醛(终浓度10mM)；4：P ZDA加苯甲酸(终浓度10mM)；5：P ZDA加儿茶酚(终浓度10mM)。
图4为P ZDA在不同浓度苯甲醇诱导下表达活性对比及其活性与Ptac活性对比；
1：Ptac不加IPTG；2：Ptac加IPTG(终浓度100μM)；3：P ZDA不加苯甲醇；4、5、6、7、8和9：P ZDA加不同量的苯甲醇(终浓度依次为1mM、10mM、15mM、20mM、25mM和50mM)。
图5为重组谷氨酸棒杆菌表达绿色荧光蛋白的SDS-PAGE实验分析图；
泳道M：蛋白Marker26610；1：空白对照；2：Ptac不加IPTG；3：Ptac加IPTG(终浓度100μM)；4：P ZDA不加苯甲醇；5、6、7、8、9和10：分别表示P ZDA加不同量的苯甲醇(终浓度依次为1mM、10mM、15mM、20mM、25mM和50mM)。
图6为重组谷氨酸棒杆菌应用P ZDA表达人胃内因素蛋白应用实例；
泳道M：蛋白Marker26610；1：空白对照；2：苯甲醇(终浓度10mM)诱导表达人胃内因子。
图7重组谷氨酸棒杆菌表达BNP的SDS-PAGE实验分析图；
M：蛋白Marker26610；1：Ptac不加IPTG 2：Ptac加IPTG(终浓度100μM)表达BNP；3：P ZDA不诱导表达BNP；4：P ZDA加苯甲醇(终浓度10mM)诱导表达BNP。
图8重组谷氨酸棒杆菌表达BNP的ELASA定量分析图；
1：Ptac不加IPTG；2：Ptac加IPTG(100μM)表达BNP；3：P ZDA不诱导表达BNP；4：P ZDA加苯甲醇(终浓度10mM)诱导表达BNP。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少 一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
载体p19-0的构建：
本发明采用的质粒骨架包括探测载体p19-0，该载体具有大肠杆菌和谷氨酸棒杆菌的穿梭能力，能够在两种菌中稳定存在；该探测载体多克隆位点两侧分别有两个终止子，一个T7终止子和一个rrnB终止子，可以避免载体自带启动子对该表达框的影响。
探测载体p19-0的构建方法如下：
将pXMJ19质粒上的干扰性Bsa I酶切位点GGTCTC突变为GGTCAC；
以pXMJ19为模版，p19-MUT-F、p19-MUT-R为引物扩增突变后的线性质粒，产物用Dpn I 37℃处理2h后直接转化到大肠杆菌DH5α，得到正确突变的质粒命名为p19-mut；
p19-MUT-F GATGGTAGTGTGGGGTCACCCCATGCGAGAGTAG
p19-MUT-R CTACTCTCGCATGGGGTGACCCCACACTACCATC
反应体系为10×Q5Buffer 5μL、dNTPs(2.5mmol/L)4μL、上下游引物(10μmol/L)各1μL、Q5DNA聚合酶(2U/μL)0.5μL、模板1μL，加水至50μL；
PCR扩增条件为：98℃3min；98℃15s；72℃3.25min，30个循环；72℃2min；
使用EcoRV和Hind III对质粒p19-mut进行双酶切，去掉质粒中的lac I和tac启动子片段；使用引物p19-0-F、p19-0-R将两片段中间的骨架部分用PCR克隆出来，并在下游引物中加入T7终止子，得到的质粒为p19-0；
p19-0-F ATCCTATCATGCCATACCGCG
p19-0-R
CCC AAGCTTCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTA(下划线为酶切位点Hind III)。
本发明质粒骨架载体可以替换为其他现有技术载体。
启动子的克隆(本发明的启动子命名为P ZDA)：
使用天根生化科技有限公司的细菌基因组提取试剂盒，提取谷氨酸棒杆菌 (以谷氨酸棒杆菌CGMCC1.15647为例)的基因组，用于启动子克隆模板；本发明使用的谷氨酸棒杆菌CGMCC1.15647，其拉丁文命名为C.glutamicumBZH001，于2013年受赠于张家港市华昌药业有限公司。
作为本发明其中一种实施方式：选取C.glutamicumBZH 001的基因SEQ ID NO：6基因编码序列ATG上游355bp至下游62bp序列区域设计启动子克隆引物355ZDA-F和ZDA-R(SEQ ID NO：6基因编码序列ATG起始于SEQ ID NO：6序列的第501bp碱基，SEQ ID NO：6基因编码序列为序列表SEQ ID NO：6的第501bp～第1355bp)，克隆启动子序列及其下游基因ATG后62bp序列(包含ATG)，并在下游引物ZDA-R的5’端依次引入15bp的RBS序列(AAAGGAGGACAACTA)和两个反向的Bsa I酶切位点以及一个BamH I酶切位点，且BamH I酶切位点与第二个Bsa I酶切位点切口重合；
ZDA-F TTCG AAGCTTCTTTGAAGCGGTGTGTGAC(下划线为酶切位点Hind III)
ZDA-R
CCGG GGATCCGAGACCAAAAAATTTTTT GGTCTCACATTAGTTGTCCTCCTTTTCAGTTGCCTTGTTGCC(下划线酶切位点依次为BamH I、Bsa I和Bsa I)。
反应体系为10×Q5Buffer 5μL、dNTPs(2.5mmol/L)4μL、上下游引物(10μmol/L)各1μL、Q5DNA聚合酶(2U/μL)0.5μL、模板1μL，加水至50μL。
PCR扩增条件为：98℃3min；98℃15s；60℃15s；72℃15s，30个循环；72℃45s。
表达载体p19-0-ZDA的构建：
将前述通过PCR克隆的P ZDA启动子片段和p19-0探测载体使用TAKALA公司的限制性核酸内切酶BamH I和Hind III分别双酶切，酶切后两片段使用TAKALA公司的T4DNA连接酶进行连接，得到表达载体p19-0-ZDA。该表达质粒含有启动子P ZDA，并在紧接启动子下游插入两个反向的Bsa I酶切位点，方便基因的无痕插入。表达载体p19-0-ZDA的序列如SEQ ID NO：4。
EGFP表达载体p19-0-ZDA-egfp的构建：
以EGFP为例，将EGFP作为报告基因用来测试本发明启动子的活性。
采用引物egfp-F和egfp-R对荧光蛋白EGFP的基因egfp进行克隆，并通 过Bsa I酶切位点克隆到表达载体p19-0-ZDA的启动子P ZDA的下游得到EGFP表达载体p19-0-ZDA-egfp，进行荧光蛋白EGFP的表达。egfp的基因序列如SEQ ID NO：5所示。
egfp-F AA GGTCTCAAATGGTGAGCAAGGGCGA(下划线酶切位点为Bsa I)
egfp-R AA GGTCTCGGATCCTTACTTGTACAGCTCGTCCATG(酶切位点Bsa I)
反应体系为10×Q5Buffer 5μL、dNTPs(2.5mmol/L)4μL、上下游引物(10μmol/L)各1μL、Q5DNA聚合酶(2U/μL)0.5μL、模板1μL，加水至50μL。
PCR扩增条件为：98℃3min；98℃15s；60℃15s；72℃30s，30个循环；72℃1min15s。
本发明以EGFP作为其中一种“报告基因”用来测试本发明启动子的活性，EGFP也可以替换为其他内源基因、外源基因等目的基因。
启动子鉴定：
首先对本发明启动子P ZDA进行5’race实验，确定该启动子的转录起始位点在SEQ ID NO：6基因ATG上游91bp处。方法为使用5’race试剂盒
Figure PCTCN2018110217-appb-000001
RACE 5’/3’Kit(TaKaRa，中国)，严格按照说明书操作，确定该启动子的转录起始位点为SEQ ID NO：6ATG上游91bp处。初步判定启动子核心区在ATG上游91bp之前，5’UTR为ATG上游1-90bp。
然后，分别设计上游引物98ZDA-F(指启动子的5’端位于SEQ ID NO：6基因编码序列ATG上游98bp)、180ZDA-F(指启动子的5’端位于SEQ ID NO：6基因编码序列ATG上游180bp)、200ZDA-F(指启动子的5’端位于SEQ ID NO：6基因编码序列ATG上游200bp)、257ZDA-F(指启动子的5’端位于SEQ ID NO：6基因编码序列ATG上游257bp)、308ZDA-F(指启动子的5’端位于SEQ ID NO：6基因编码序列ATG上游308bp)和345ZDA-F(指启动子的5’端位于SEQ ID NO：6基因编码序列ATG上游345bp)对P ZDA的5’端进行截短。
作为本发明其中一种实施方式，选取SEQ ID NO：6基因编码序列ATG下游62bp序列区域作为源基因，即本发明启动子3’端位于SEQ ID NO：6 基因编码序列ATG及ATG下游59bp，源基因序列如SEQ ID NO：2所示。作为源基因，SEQ ID NO：6基因编码序列ATG及ATG下游59bp仅为本发明其中一种实施方法，本发明研究发现，源基因的片段程度可以进行调整，而仍然使本发明启动子具备诱导表达活性。
作为本发明其中一种实施方式，操作方法为分别用上述引物与引物egfp-R组合，以p19-0-ZDA-egfp质粒为模板进行PCR扩增(PCR体系和扩增程序同上)，将扩增得到的片段分别用Hind III和BamH I双酶切后连入探测载体p19-0构建相应的EGFP表达载体，将构建好的载体通过大肠杆菌DH5α克隆后分别电击转化谷氨酸棒杆菌，氯霉素抗性平板进行筛选，并通过菌落PCR验证，即可得到含有表达载体并能够表达荧光蛋白的重组谷氨酸棒杆菌；通过测量单位菌体OD 600的荧光强度可以反应荧光蛋白EGFP的表达量，间接测定启动子的强度。
98ZDA-F TTCG AAGCTTTGTTGTTATCGAGTTCAAC(下划线为酶切位点Hind III)
180ZDA-F TTCG AAGCTTAGAAATATTTATGCTTCTAAAG(下划线为酶切位点Hind III)
200ZDA-F TTCG AAGCTTTGCTTGAAGTGGGGTTA(下划线为酶切位点Hind III)
257ZDA-F TTCG AAGCTTCCTAGGGTTTGCTCG(下划线为酶切位点Hind III)
308ZDA-F TTCG AAGCTTGCGTATGACCAACAGTG(下划线为酶切位点Hind III)
345ZDA-F TTCG AAGCTTGTGTGTGACATTTGCGAG(下划线为酶切位点Hind III)
将含表达载体的重组谷氨酸棒杆菌接种到5mL含氯霉素浓度为10μg/L的LBB液体培养基，于30℃230rpm过夜培养制备种子液，分别测定各种子液的OD 600；将各种子液分别转接于新鲜的20mL/100mL(100mL三角瓶装有20mL的培养基)LB培养基，使初始OD 600均为0.2，30℃230rpm进行摇瓶培养，其中各个重组菌于培养2h后加入诱导剂；并于培养20h后，分别用紫外分光光度计和荧光分光光度计测菌体OD 600和菌体荧光强度，并计算得出各菌株的 单位OD 600的荧光强度用于表达活性的比较。
不同长度的P ZDA在苯甲醇(终浓度10mM)诱导下的表达活性，结果如图2；当启动子位于SEQ ID NO：6基因ATG上游序列的长度截短为345bp时启动子诱导表达活性与355bp长度对应的启动子诱导表达活性相当，因此说明本发明启动子可以包括所述SEQ ID NO：6基因ATG上游345bp的5’端更长的序列，也可以不包括。
本发明继续选择不同长度片段的启动子用以研究启动子的活性，继续截短启动子位于SEQ ID NO：6ATG上游序列的长度为308bp，启动子诱导表达活性虽然相较于345bp有所下降，但其启动子表达活性仍然明显高于现有技术的Ptac启动子(结合本发明图2、图5、图8可以看出，本发明257P ZDA启动子在不经过诱导剂诱导表达的条件下，其启动子表达活性已经相当于甚至高于现有技术Ptac在诱导剂诱导表达状态下的表达活性)，当截短到200bp时，该启动子虽然不具有诱导表达活性，但仍具有良好的表达活性，当截短到180bp时，该启动子虽然不具有诱导表达活性，但仍具有良好的表达活性，以上实验表明P ZDA的操纵子序列位于SEQ ID NO：6基因编码序列ATG上游201bp～345bp之间，核心区序列位于ATG上游200bp之后；当启动子位于SEQ ID NO：6ATG上游序列的长度截短到98bp时，该启动子诱导表达活性完全丧失，与空质粒对照单位OD600荧光强度相当。本发明P ZDA启动子长度为SEQ ID NO：6ATG上游345bp时诱导表达活性最好，单位OD 600的荧光强度达到16272，选取该结构进行后续实验。
因此，作为本发明启动子的其中一种优选方案：
本发明启动子中，操纵子区序列为谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG上游201bp～345bp序列，其中，谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG上游345bp长度序列如SEQ ID NO：1所示；启动子核心区序列为谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG上游91bp～200bp序列；
5’UTR序列为谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG上游1bp～90bp序列；部分源基因序列包括SEQ ID NO：6基因编码序列ATG和ATG下游的59bp序列，如SEQ ID NO：2所示。优选地，该启动子的核苷酸序列如SEQ ID NO：3所示。
启动子诱导活性鉴定：
分别使用苯甲醇、苯甲醛、苯甲酸和儿茶酚作为诱导剂进行了P ZDA诱导活性实验，实验结果如图3所示，可见苯甲醇、苯甲醛、苯甲酸和儿茶酚对P ZDA均有明显诱导活性，但相同浓度下(10mM)，苯甲醇的诱导效果最好，单位OD600的荧光强度达到16601。
以苯甲醇作为诱导剂，设置不同的浓度梯度比较P ZDA活性的变化，对比结果如图4所示，345P ZDA启动子在一个很宽的苯甲醇浓度范围(1mM～50mM)都有诱导活性，但在终浓度为10mM的LB培养基中表达活性最高，且远高于对照Ptac的表达活性，单位OD600的荧光强度达到16740。
同时对各菌株表达外源蛋白情况进行SDS-PAGE分析：取PBS洗三次的等量菌体，分别用等量的PBS重悬菌体，并加入等量的细玻璃珠，用高通量匀质器破菌提蛋白；将匀质液离心，取溶有蛋白的上清跑蛋白胶，蛋白胶上层为4％的浓缩胶，下层为12％的分离胶；结果如图5所示，可见各菌株表达EGFP(表观分子量约30kDa)的结果与单位OD600菌体荧光强度结果相一致。
Ptac是以上述RBS序列(AAAGGAGGACAACTA)、外源基因片段与质粒pXMJ19共同构建的阳性对照质粒，应用质粒pXMJ19自身Ptac启动子，这一经典的阳性对照代表了的谷氨酸棒杆菌高效表达重组蛋白的水平，其构建包括以下步骤：
克隆由RBS序列与egfp基因构成基因片段，egfp基因如SEQ ID NO：5所示，所需的引物为：SD-EGFP-F和SD-EGFP-R。所用的PCR反应体系和程序同上；
SD-EGFP-F CCC AAGCTTAAAGGAGGACAACTAATGGTGAGCAAGGGCG(酶切位点Hind III)
SD-EGFP-R CCC GGATCCTTACTTGTACAGCTCGTCCATG(酶切位点BamH I)
Hind III和BamH I双酶切；
T4DNA连接酶连接。
启动子强弱鉴定：
采用如上所说实验方案，使用引物GIF-F和GIF-R将人胃内因子蛋白(GIF，表观分子量约45kDa，GeneID：2694)作为表达基因进行表达，成功在菌体超声破碎沉淀中检测到大量蛋白表达，结果如图6所示。
GIF-F：AA GGTCTCAAATGGCCTGGTTTGCCCTG(下划线为酶切位点为Bsa I)
GIF-R：AA GGTCTCGGATCCTTAATACTGGGTGAAATTGGCG(酶切位点为Bsa I)；
采用如上所说实验方案，使用引物：NT-ProBNP-F和NT-ProBNP-R将表达基因替换为HIS-SUMO-N端脑钠肽前体(NT-proBNP，表观分子量约25kDa,GeneID：4879)进行表达，并将含有Ptac，受IPTG诱导表达外源基因的Ptac载体做对照，结果如图7、图8所示，SDS-PAGE分析结果与ELASA定量分析结果一致，成功在菌体超声破碎的上清中检测到大量蛋白表达，并且本申请启动子345P ZDA在苯甲醇诱导下比IPTG诱导的Ptac表达活性更高，HIS-SUMO-N端脑钠肽前体的表达量达到440.43mg/L。
NT-ProBNP-F：AA GGTCTCAAATGGGTCATCATCACCACCACCAC(酶切位点为Bsa I)
NT-ProBNP-R：AA GGTCTCGGATCCTTAGCGCGGGGCACGCAG(酶切位点为Bsa I)；
结果证明，本发明的来源于谷氨酸棒杆菌的诱导型启动子345P ZDA在苯甲醇诱导下比IPTG诱导的Ptac表达EGFP、HIS-SUMO-N端脑钠肽前体活性明显更高，结合本发明图2、图5、图8可以看出，本发明257P ZDA启动子在不经过诱导剂诱导表达的条件下，其启动子表达活性已经相当于甚至高于现有技术Ptac在诱导剂诱导表达状态下的表达活性，本发明启动子在谷氨酸棒杆菌表达外源蛋白以及代谢工程改造中具有重要意义。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (19)

  1. 启动子，其特征在于：包括，启动子核心区序列，所述启动子核心区序列至少包含谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游91bp～180bp区间序列。
  2. 如权利要求1所述的启动子，其特征在于：所述启动子至少包括启动子核心区序列，所述启动子核心区序列的3’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游第91bp，5’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游181bp～200bp中任一碱基。
  3. 如权利要求2所述的启动子，其特征在于：所述启动子的序列区间至少包括，3’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游第91bp，5’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游201bp～257bp中任一碱基。
  4. 如权利要求2所述的启动子，其特征在于：所述启动子的序列区间至少包括，3’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游第91bp，5’端位于谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG的上游201bp～345bp中任一碱基。
  5. 如权利要求1～4中任一所述的启动子，其特征在于：还包括5’UTR序列。
  6. 如权利要求5所述的启动子，其特征在于：所述5’UTR序列的长度为90bp。
  7. 如权利要求6所述的启动子，其特征在于：所述5’UTR序列为谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG上游1bp～90bp区间序列。
  8. 如权利要求1～4、6、7中任一所述的启动子，其特征在于：还包括，源基因序列，所述源基因序列包括SEQ ID NO：6基因编码序列ATG以及ATG下游的碱基。
  9. 如权利要求8所述的启动子，其特征在于：所述源基因序列包括谷氨酸棒杆菌SEQ ID NO：6基因编码序列ATG以及ATG下游的59bp碱基。
  10. 如权利要求1～4、6、7、9中任一所述的启动子，其特征在于：包括所述启动子序列的互补序列。
  11. 如权利要求1～4、6、7、9中任一所述的启动子，其特征在于：包括源基因序列，所述源基因序列如SEQ ID NO：2所示。
  12. 如权利要求1～4、6、7、9中任一所述的启动子，其特征在于：所述启动子的核苷酸序列如SEQ ID NO：3所示。
  13. 如权利要求3、4、6、7、9任一所述的启动子，其特征在于：所述启动子能够被包括苯甲醇、苯甲醛、苯甲酸、儿茶酚的任一种或几种诱导。
  14. 如权利要求13所述的启动子，其特征在于：所述苯甲醇的诱导浓度范围为1mM～50mM。
  15. 根据权利要求14所述的启动子，其特征在于：所述苯甲醇的诱导浓度为10mM。
  16. 载体，其特征在于：所述载体装载权利要求1～15任一所述的启动子。
  17. 如权利要求16所述的载体，其特征在于：所述载体包括蛋白表达载体，其核苷酸序列如SEQ ID NO：4。
  18. 权利要求1～17任一所述的启动子在蛋白表达中的应用。
  19. 如权利要求18所述的应用，其特征在于：所述启动子用于谷氨酸棒杆菌内源或外源蛋白的表达。
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