CN103966215B - Be derived from the methyl n-undecyl ketone inducible promoter of bollworm - Google Patents
Be derived from the methyl n-undecyl ketone inducible promoter of bollworm Download PDFInfo
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Abstract
The present invention discloses a kind of the methyl n-undecyl ketone inducible promoter sequence and the 5 ' non-translational region that are derived from bolworm cell pigment CYP6B7 gene, and contains eukaryotic expression vector and the clone of above-mentioned sequence.Promotor of the present invention and 5 ' non-translational region, can be used for the high expression of eukaryotic gene, for obtaining low abundance gene studies, be that research insect and the mutual work of plant secondary substance and the pest-resistant new variety of development of new lay the foundation to the research of bollworm Plant secondary material inducible promoter.
Description
Technical field
The present invention relates to plant secondary substance inducible promoter field, specifically, relate to the methyl n-undecyl ketone inducible promoter being derived from bolworm cell pigment CYP6B7 gene.
Background technology
Bollworm (Helicoverpaarmigera) is the important pests causing the agriculture underproduction, and its damaging range is quite extensive, comprises more than 60 kind of raise crop and 67 kinds of wild plants such as cotton, tomato, corn, wheat, soybean, peanut, paddy rice.
Plant-feed insect obtains nutrition with various predation strategy from host plant.But plant is not passive victim, can produce Plant secondary material or Buchner's bodies for plant-feed insect, plant also can discharge volatile matter and lure predator to take food insect.Methyl n-undecyl ketone is the defensive plant secondary substance of the one be extensively present in plant of Solanaceae, is the highest with content in wild-type tomato.Tomato is as the host plant of bollworm, early-stage Study finds that the plant secondary substance methyl n-undecyl ketone contained in tomato can induce the high expression level of the detoxifying gene CYP6B7 in bollworm, and this may be the important physiological function that bollworm utilizes methyl n-undecyl ketone and coerces to start its defense mechanism and then reply methyl n-undecyl ketone as chemical co-ordination signal.
The regulation and control of genetic expression all likely occur in several link, and relate to the regulation and control etc. of the rear level of DNA level, transcriptional level, post-transcriptional level, translation skill and translation, wherein the regulation and control of transcriptional level are the most general control methods.Research about the regulation and control of Plant secondary material induction insect P450 gene transcription level mainly concentrates on the research in its cis-regulating element and trans-acting factor, cis-regulating element in promotor is considered to play an important role on genetic transcription regulates, the expression of the cis-regulating element interaction regulatory gene in trans-acting factor and promotor.XRE-AhR(foreign peoples's aryl hydrocarbon receptor compound response element as in CYP6B family gene promotor), AhR, XRE-Xan(xanthotoxin response element) cis-regulating element has higher homology, this class component is relevant to toxic substance and Plant secondary material metabolism, and the Metabolism regulation of the expression and Plant secondary material that further illustrate CYP6B family gene has substantial connection.EcRE/ARE/XRE-xan element is deposited in the P450 gene C YP6B4 of metabolism furocoumarin(e) and CYP6B1 in the yellow swallowtail butterfly of North America secret note, these transcriptional regulatory elements regulate and control the overexpression (McdonneC.M. of CYP6B4 gene under xanthotoxin or benzopyrene induction, BrownaB.R., BerenbaumaM.R., SchulerM.A., 2004.Conservedregulatoryelementsinthepromotersoftwoallel ochemical-induciblecytochromeP450genesdifferentiallyregu latetranscription.InsectBiochemistryandMolecularBiology, 34 (10): 1129-1139.).
Therefore, goal gene overexpression is by the adjustment of Gene Transcription in vitro, its cis-regulating element may regulate and control the expression of goal gene, this promotor also demonstrating bollworm CYP6B7 gene has higher methyl n-undecyl ketone induced activity most probably, this kind of inducible promoter is under the stimulation of some specific physics or chemical signal, can fast and effeciently the transcribing of induced gene, resist the impact of coercing.At present, the bollworm Plant secondary material inducible promoter of separation andpreconcentration is also few.The clone promoter related for new highly active bollworm plant secondary substance and the qualification of induced activity, to determining that the research of transcriptional elements relevant with plant secondary substance methyl n-undecyl ketone in bollworm and transcription factor is significant further, because Plant secondary material determines that can insect one of the Main Factors taking food certain kind of plant, be therefore that the mutual work of research insect and plant secondary substance and the pest-resistant new variety of development of new lay the foundation to the research of bollworm Plant secondary material inducible promoter.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of plant secondary substance methyl n-undecyl ketone inducible promoter sequence deriving from bolworm cell cytochrome p 450 CYP6B7 gene, this promoter sequence can induce goal gene high level expression.
In order to realize the object of the invention, first the present invention provides a kind of methyl n-undecyl ketone inducible promoter, and described promoter sequence comprises-1bp relative to the transcription initiation site of SEQIDNo.1 to the DNA nucleotide sequence in-1540bp district.
Further, described promoter sequence is derived from bolworm cell pigment CYP6B7 gene.
The present invention also provides a kind of 5 ' non-translational region of bolworm cell pigment CYP6B7 gene, and described 5 ' non-translational region comprises+1bp relative to the transcription initiation site of SEQIDNo.1 to the DNA nucleotide sequence in+50bp district.
The present invention also provides the eukaryotic expression vector containing aforementioned promoter sequence and aforementioned 5 ' non-translational region sequence.
Further, described carrier is the inducible vector for eukaryotic cell transfection.
In embodiments of the present invention, it is pGL4.1-proCYP6B7 that the present invention is used for the inducible vector of eukaryotic cell transfection, and the promotor of described bollworm CYP6B7 and 5' non-translational region to be positioned on carrier before external source reporter gene in expression vector.The invention provides the promotor by inserting bollworm CYP6B7 gene of the present invention and the pGL4.1-proCYP6B7 of 5' non-translational region extremely containing structure in the carrier (pGL4.1basic) of Luciferase reporter gene.But described Luciferase reporter gene is foreign gene, and expection can replace with other useful foreign gene any.
The present invention also provides the clone containing aforementioned bearer.
As preferably, described cell is the bollworm fat body cells system of plant secondary substance induction type transient expression.Also can replace with other similar insect cell lines.
The present invention also provides a kind of gene order containing methyl n-undecyl ketone inducible promoter, and described gene order is as shown in SEQIDNo.1.
The present invention is also provided for increasing the PCR primer pair of the DNA fragmentation comprising SEQIDNo.1 sequence, and the nucleotide sequence of described primer pair is respectively as shown in SEQIDNo.2 and SEQIDNo.3.
The application that the present invention also provides aforementioned promotor or forementioned gene sequence to carry out in methyl n-undecyl ketone abduction delivering in eukaryotic cell.
Beneficial effect of the present invention is:
Promoter sequence of the present invention can be used for the high expression of eukaryotic gene, for obtaining low abundance gene studies, be that research insect and the mutual work of plant secondary substance and the pest-resistant new variety of development of new lay the foundation to the research of bollworm Plant secondary material inducible promoter.
Accompanying drawing explanation
Fig. 1 is the methyl n-undecyl ketone inducible promoter of bolworm cell pigment CYP6B7 gene of the present invention and the DNA sequence dna of 5 ' non-translational region.
Fig. 2 is CYP6B7-proPCR electrophorogram of the present invention;
Wherein, M:DL15000+2000marker; 5 ' flanking region sequence of 1-2:CYP6B7 gene.
Fig. 3 is pGL4-CYP6B7-pro vector construction schematic diagram of the present invention.
Fig. 4 is that CYP6B7-pro of the present invention connects pGL4.1basic enzyme and cuts qualification electrophorogram;
Wherein, M:DL15000+2000marker; 1-2:pGL4.1-CYP6B7Pro recombinant plasmid is through MluI and XhoI double digestion electrophoresis result.
Fig. 5 is the change that after the present invention adds methyl n-undecyl ketone, CYP6B7-pro starts reporter gene Luciferase activity.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The clone of the methyl n-undecyl ketone inducible promoter of embodiment 1 bollworm CYP6B7 gene
The promotor of bollworm CYP6B7 (promoter sequence comprises-1bp relative to the transcription initiation site of SEQIDNO:1 to the DNA nucleotide sequence in-1540bp district), is identified in the 5' flanking region sequence of bollworm CYP6B7 gene.
Bollworm CYP6B7 gene logs in NCBIGenBank(accession number: DQ497428.2), and sequence table shows the Plant secondary material methyl n-undecyl ketone inducible promoter of said gene of the present invention and the DNA sequence dna of 5' non-translational region.(see figure 1) in promoter sequence of the present invention, indicate with '+1 ' above transcription initiation site (analyses and prediction website http://www.fruitfly.org/seq_tools/promoter.html) the base T inferred, ATG double underline marks, and TATA box marks with an undulating line.And analyze the Binding site for transcription factor of promotor with promoter Analysis website (http://www.gene-regulation.com/pub/programs.html#pmatch), the Binding site for transcription factor (Nrf2:MafK that some of them are important; Nrf2; AREB6) mark with underscore.
With bollworm genomic dna for template, the amplification of chromosome walking method obtains 5 ' the flanking region sequence of goal gene CYP6B7, through 1% agarose gel electrophoresis analysis, amplifies the band (Fig. 2) that length is about 1800bp, and reclaims.Reclaim fragment be connected with pMD18-T carrier obtain recombinant plasmid pMD18-T-CYP6B7Proh after check order, sequencing result and CYP6B7 gene order are compared, remove and CYP6B7 gene order lap, obtain CYP6B7 gene 5 ' flanking sequence, use analysis of biological information software analysis to show that CYP6B7 gene 5 ' flanking sequence comprises the 5' non-translational region that total length is the promoter sequence of the CYP6B7 gene of 1540bp and the CYP6B7 gene of 50bp.Fig. 1 shows the methyl n-undecyl ketone inducible promoter of bolworm cell pigment CYP6B7 gene and the DNA sequence dna of 5 ' non-translational region, in FIG, the initiator codon ' ATG ' of albumen synthesis marks with double underline, TATA box marks with an undulating line, the base ' T ' of transcription initiation site marks with '+1 ', and adds black expression.
More particularly, by chromosome walking method pcr amplification, clone obtains the promoter sequence of the bollworm CYP6B7 gene of 1540bp and the 5' non-translational region sequence of 50bp, and the primer needed for above-mentioned PCR primer that increases shows in Table 1 in detail.For pcr amplification, response procedures: 95 DEG C of 3min; 95 DEG C of 30S, 52 DEG C of 30S, 72 DEG C of 2min, 32 circulations; 72 DEG C of 10min.
Table 1: primer
| Upstream primer | 5'-TTTTGAGGAGCTGTTTACAC-3' |
| Downstream primer | 5'-TTCTCAATAACTTTAACATCACA-3' |
The structure of embodiment 2 eukaryotic cell methyl n-undecyl ketone inducible expression vector
The sequence of bollworm CYP6B7 gene methyl n-undecyl ketone inducible promoter cloned in embodiment 1 and 5' non-translational region is inserted in carrier jointly, thus builds eukaryotic cell methyl n-undecyl ketone inducible expression vector.
More particularly, be eukaryotic expression vector pGL4.1 and recombinant plasmid pMD18-T-CYP6B7Pro XhoI and MluI is carried out enzyme respectively cut, then use T4 ligase enzyme to connect them.This carrier is referred to as pGL4.1-CYP6B7Pro, and Fig. 3 is shown in by schematic diagram, for driving the expression of the Luciferase reporter gene on expression vector, cuts qualification (Fig. 4) and order-checking confirms have successfully been obtained the recombinant plasmid of promotor and carrier through XhoI and MluI enzyme.
In the diagram, with the gene Luciferase of Photinus pyralis LUC of encoding for reporter gene, selective marker is ammonia benzyl resistant gene.In addition CYP6B7Pro is inserted in the expression of upstream as its promotor startup firefly luciferase gene of Luciferase gene.
Embodiment 3 identifies the activity of methyl n-undecyl ketone inducible promoter of the present invention
Pass through lipofection, the internal reference plasmid pRL-TK corotation of the carrier pGL4.1-CYP6B7Pro built in example 2 and expression renilla luciferase is moved in bollworm fat body cells, for identifying the induced activity of the methyl n-undecyl ketone of promotor, detect the activity of Photinus pyralis LUC activity and renilla luciferase before and after methyl n-undecyl ketone induction.
More particularly, by recombinant plasmid pGL4.1-CYP6B7Pro (3000ng) and internal reference plasmid pRL-TK(300ng) namely with the ratio of 10:1, by lipofection transient transfection plasmid to completing in 12 orifice plates of bollworm fat body cells in advance, final concentration is used to be that the methyl n-undecyl ketone of 0.126mM then induces process 24 hours after transfection 24h, collecting cell, uses the two fluorescence detection reagent kit of promega company to detect plant secondary substance methyl n-undecyl ketone starts reporter gene activity impact on CYP6B7 gene promoter.The result display of Fig. 5 is compared and the control group only adding etoh solvent, adds methyl n-undecyl ketone and the promotor of CYP6B7 gene can be induced significantly to start the activity of reporter gene Luciferase.
As mentioned above, the invention provides the methyl n-undecyl ketone inducible promoter sequence of bollworm CYP6B7 gene and the 5' non-translational region of CYP6B7 gene.
The invention provides the carrier for expression of eukaryon that the promotor of bollworm CYP6B7 gene and the 5' non-translational region of CYP6B7 gene are inserted into pGL4.1 and obtain.
Through using described carrier for expression of eukaryon to identify, the promotor of CYP6B7 gene of the present invention and the 5' non-translational region of CYP6B7 gene can start the expression of its downstream goal gene after the induction of plant secondary substance methyl n-undecyl ketone.
Therefore, the present invention is to determining that the research of transcriptional elements relevant with plant secondary substance methyl n-undecyl ketone in bollworm and transcription factor is significant further, because Plant secondary material determines that can insect one of the Main Factors taking food certain kind of plant, be therefore that the mutual work of research insect and plant secondary substance and the pest-resistant new variety of development of new lay the foundation to the research of bollworm plant secondary substance inducible promoter.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (9)
1. a methyl n-undecyl ketone inducible promoter, is characterized in that, described promoter sequence is to the DNA nucleotide sequence in-1540bp district relative to the-1bp of the transcription initiation site of SEQIDNo.1; Described transcription initiation site is the 1541bp in SEQIDNo.1.
2. promotor according to claim 1, is characterized in that, described promoter sequence is derived from bolworm cell pigment CYP6B7 gene.
3. contain the eukaryotic expression vector of 5 ' non-translational region sequence of promoter sequence and bolworm cell pigment CYP6B7 gene described in claim 1; Described 5 ' non-translational region comprises+1bp relative to the transcription initiation site of SEQIDNo.1 to the DNA nucleotide sequence in+50bp district.
4. carrier according to claim 3, is characterized in that, described carrier is the inducible vector for eukaryotic cell transfection.
5. the clone containing carrier described in claim 3 or 4.
6. clone according to claim 5, is characterized in that, described cell is bollworm fat body cells system.
7. the gene order containing methyl n-undecyl ketone inducible promoter according to claim 1, it is characterized in that, described gene order is as shown in SEQIDNo.1.
8. comprising the PCR primer pair of the DNA fragmentation of SEQIDNo.1 sequence for increasing, it is characterized in that, the nucleotide sequence of described primer pair is respectively as shown in SEQIDNo.2 and SEQIDNo.3.
9. the promotor described in claim 1 or 2 or gene order according to claim 7 carry out the application in methyl n-undecyl ketone abduction delivering in eukaryotic cell.
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| WO2009100433A1 (en) * | 2008-02-10 | 2009-08-13 | Monsanto Technology Llc | Methods and compositions for plant pest control |
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| WO2009100433A1 (en) * | 2008-02-10 | 2009-08-13 | Monsanto Technology Llc | Methods and compositions for plant pest control |
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| 昆虫基因启动子及细胞色素P450基因启动子研究进展;李芬等;《生命科学》;20120531;第24卷(第5期);470-474页 * |
| 棉铃虫中肠P450 CYP6B6 基因5"上游序列的克隆及序列分析;张雷;《生物技术通报》;20111231(第7期);摘要、第130页2.2.1部分 * |
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