CN104195138B - A kind of cold-inducible promoter and application thereof - Google Patents

Abstract

One cold-inducible promoter of the present invention and application thereof belong to genetic engineering field, are specifically related to the non-coding nucleic acid that in DNA recombinant technique, a kind of regulator gene is expressed.The promoter that the present invention provides, the nucleotides sequence of this promoter is classified as SEQ ID NO:1.Promoter of the present invention can provide experiment and technical support for the research and development of plant bioreactor.Cold-inducible promoter described in utilization, using plant as bioreactor, expresses vaccine, antibody and other pharmaceutical proteins etc., can break through traditional agriculture category, extend to medical domain.

Description

A kind of cold-inducible promoter and application thereof

Technical field

The invention belongs to genetic engineering field, be specifically related to the non-coding that in DNA recombinant technique, a kind of regulator gene is expressed Nucleic acid.

Background technology

In plant genetic engineering is studied, external source genes of interest is made to express in receptor, to improve crop to arid, low The resistance of the environment stresses such as warm, saline and alkaline and pest and disease damage, or improve crop quality and Crop Improvement economical character etc., it has also become mesh A kind of important means of preceding crop breeding.Eukaryotic gene expression regulation includes pretranscriptional control, transcriptional control, transcribe after Regulation and control, translational control and post-translational control.Wherein, the regulation and control of transcriptional level are coordinated to make by cis acting element and transcription factor With, it is the key link of expression regulation.Therefore, separation and research about promoter become one of domestic and international focus studied.

Use constitutive promoter to order about exogenous gene expression, not only cause the wasting of resources, also directly affect the life of plant Long growth and normal physiological metabolism, and use inducible promoter, not only do not result in the waste of various resource, can strengthen again Plant resistance to external world, reduces growth and development of plants and the impact of other metabolic pathways.Therefore, external source base how is controlled Because of high efficient expression at regular time and quantity, it it is the key point effectively applied in terms of crop genetic improvement of technique for gene engineering.Except Finding outside effective genes of interest, finding that the promoter of effectively control exogenous gene expression becomes in genetic engineering improvement needs The major issue solved.Promoter is roughly divided into constitutive promoter according to its function and model of action, tissue specificity starts Son and inducible promoter three kinds.The gene expression of constitutive promoter regulation and control is not affected by external environmental condition, Ji Hu All plant different tissues can be expressed.Typically cauliflower mosaic virus (cauli-flower mosaic virus, CaMV) 35S promoter, be widely used in dicotyledon transgenic engineering (Benfey etc., 1990, Science 250, 959-966) .Other unifacial leaf have maize ubiquitin Ubiquitin promoter (Streatfield etc., 2004, Transgenic Res 13,299-312) and rice actin (Actin) promoter.Such promoter activity is higher, but outside causing due to it Source gene is expressed at the different parts of the whole growth period of transgenic plant, affects growth promoter and the normal physiological generation of plant Thanking, some product is likely to result in toxic action, is unfavorable for the raising of quality and yield, results even in the death of plant. Kasuga etc. utilize induction type rd29A promoter to replace CaMV35S promoter arabidopsis thaliana transformation, improve transgenic and intend south The drought resistance of mustard, decrease simultaneously the plant strain growth that composing type process LAN causes stagnate or dwarfism generation (Kasuga, 2004, Plant and Cell Physiology 45,346-350).Therefore, in order to preferably regulate and control the table of plant gene Reaching, tissue specific promoter and the research of inducible promoter and application are increasingly paid attention to by people.

Tissue specific promoter is that regulation and control genes of interest is only expressed in certain organs or tissue.Such promoter is general There are some specific motifs relevant to tissue specificity.The isolated from arabidopsis such as Nitz encodes in root and hypocotyl The myrosin gene Pyk10 promoter sequence of specifically expressing, this promoter can regulate and control genes of interest at transgenic arabidopsis root Portion's high level specifically expressing (Nitz etc., 2001, Plant Science, 161,337-346).Bate etc. pass through deletion mutation The promoter analyzing Rhizoma Solani tuber osi specific expression gene LAT25 finds, regulation and control pollen-specific Expression element is positioned at this gene promoter Son-492 to-52 sections (Bate etc., 1998,37,859-869).Maize leaf specificity PPCA1 promoter (Gowik Deng, 2004, Plant Cell 16,1077-1090), strawberry fruit specificity GalUR promoter (Agius etc., 2005, J Exp Bot 56,37-46) etc..The further investigation of such promoter contributes to illustrating growth and development of plants and physiological metabolism Rationale, and be with a wide range of applications.Inducible promoter refers to not express under normal condition or low expression, is subject to The stimulation of some physically or chemically signal, can be significantly increased the expression one class promoter of gene.According to induction because of Element, inducible promoter can be divided into Light-inducible promoter, temperature inducible promoter, wound inducement type promoter, hormone to lure Conductivity type promoter and fungus induction type promoter etc..It is characterized in: induced by physically or chemically factor；Containing several functions Element, the collaborative activity strengthened or reduce promoter；Part inducible promoter has the spy of tissue-specific promoter simultaneously Point.Other inducible promoters such as Oryza sativa L. plasma membrane CaATPase promoter is by arid, cold, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and abscisic acid Induction (Huda etc., 2013, PLoS One 8).Arabidopsis RBCS-1A promoter is Light-inducible promoter, has simultaneously Specificity (practising rain beautiful jade etc., 2012, Acta Agronomica Sinica 38,1561-1569) in a organized way；For inducible promoter, purpose base Because only just expressing after accepting inducement signal, not only do not result in the waste of various resource, plant can be strengthened again external The resistance on boundary, reduces growth and development of plants and the impact of other metabolic pathways.Therefore, study and utilize inducible promoter, Adversity gene engineering for the research various physiological Mechanism coerced of plant responding and research crops has important meaning, Also have broad application prospects in terms of basic research and Plant Biotechnology.But up to the present, can apply to turn base Because the inducible promoter of research is the most little.Therefore, for new degeneration-resistant promoter related clone, cis acting element Interaction between the determination of particular sequence, each element, and remain with the research of the transcription factor of these element interactions The emphasis of promoter research from now on.

Summary of the invention

It is an object of the invention to provide a kind of promoter and application thereof that can start destination gene expression when low temperature induction. One aspect of the present invention provides a kind of promoter, and the nucleotides sequence of this promoter is classified as SEQ ID NO:1.

Present invention also offers the recombinant vector containing above-mentioned promoter, expression cassette, transgenic cell or recombinant bacterium.

Described recombinant vector be insert at the multiple clone site of carrier described in promoter after the recombiant plasmid that obtains.

Described expression cassette is by described promoter, the genes of interest of this promoter expression, and transcription terminator composition； Described promoter is connected with described genes of interest with functional way, and described genes of interest is with described transcription terminator even Connect.

The present invention have an aspect there is provided described promoter start genes of interest in plant express in application. Preferably when temperature-induced, particularly preferably when low temperature induction.

Described low temperature induction, refers to that some plant genes are cold environmental conditions (referring generally to more than 0 DEG C, less than 15 DEG C) Under, start the mode expressed.

A further aspect of the invention there is provided a kind of method utilizing described promoter to cultivate transgenic plant, bag Include following steps:

1) genes of interest recombinant expression carrier is built: genes of interest is inserted into the recombinant vector containing described promoter In, low temperature induction makes described promoter start described destination gene expression, obtains genes of interest recombinant expression carrier；

2) being imported in purpose plant by the genes of interest recombinant expression carrier that step 1) builds, low temperature induction makes described opening Mover starts described destination gene expression, obtains expressing the transgenic plant of described genes of interest.

A further aspect of the invention there is provided a kind of method making plant express genes of interest when low temperature induction, its It is genes of interest to be inserted in the recombinant vector containing described promoter, and this recombinant vector is imported in purpose plant, so Rear low temperature induction genes of interest is expressed in plant.

Promoter of the present invention can provide experiment and technical support for the research and development of plant bioreactor.Utilize institute The cold-inducible promoter stated, using plant as bioreactor, expresses vaccine, antibody and other pharmaceutical proteins etc., can dash forward Broken traditional agriculture category, extends to medical domain.

Accompanying drawing explanation

Fig. 1: promoter of the present inventionpCD1Build PCR electrophoretogram.

The promoter of Fig. 2: the present inventionpCD1Carrier T plasmid enzyme restriction identifies electrophoretogram.

The promoter of Fig. 3: the present inventionpCD1Connect pC0390GUS enzyme action and identify electrophoretogram.

Fig. 4: under the conditions of low temperature induction, promoterpCD1The gus gene histochemical stain of transformation of tobacco blade.

Fig. 5: under the conditions of low temperature induction, promoterpCD1With the GUS expression activitiy compareed.

Detailed description of the invention

Below in conjunction with the accompanying drawings the method for the present invention is described in detail, but purpose described below is exemplary Rather than limit the scope of the present invention.

Embodiment 1: cold-inducible promoter of the present inventionpCD1Separation

The preparation of 1.1 sweet buckwheat genomic DNAs

Being 8000LUX by the sweet buckwheat Seedling of band culture matrix at illumination condition, temperature is 25^oGrow 3 weeks under the conditions of C.Then Proceeding to illumination condition is 8000LUX, and plant genes group DNA then using Ai Delai bio tech ltd, Beijing is fast Speed is extracted test kit (catalog number (Cat.No.) DN15) and is extracted DNA.

1.2 promoterpCD1Cloned culturing

With the DNA that extracts above as template, expand FeDREB1 upstream region of gene promoter sequence by PCR method.Its amplification is drawn Thing is as follows, introduces Hind III restriction enzyme site in its forward primer, introducing Bamh I restriction enzyme site in downstream primer:

Forward primer: 5'-CCCAAGCTTATAGACCTAGTCAGGATCA-3'

Downstream primer: 5'-GCGGATCCGATCGATCCTGAATATAT-3'

PCR reaction system

PCR response procedures: 94^OC degeneration 5 min；94^OC degeneration 30 s, 60^OC anneals 30 s, and 72^OC extends 2 min, and 29 Individual circulation；72^OC 10 min,4^OC is incubated.

Detect PCR result with the agarose gel electrophoresis of 1.0 %, see Fig. 1.

Genes of interest DNA fragment recovery from agarose gel Tian Gen biochemical technology company limited DNA reclaims reagent Box reclaims, and operation illustrates to carry out according to test kit.

PMD19-T Simple Vector) coupled reaction

Coupled reaction operates, even according to the PMD19-T Simple Vector support agent box explanation of takara company Connect reaction system as follows:

Mixing reactant, of short duration centrifugal, 4 DEG C overnight.

Qualification with positive colony

Escherichia coli for converting are that DH5a(is purchased from Tian Gen biochemical technology company limited).The preparation of competent cell and The conversion connecting product the most aseptically operates.Picking converts the white colony on flat board, uses plasmid to extract examination on a small quantity Agent box extracts plasmid (be purchased from Tian Gen biochemical technology company limited) as template, PCR primer as described above and amplification bar Part carries out PCR amplification, produces the positive colony of 270bp gene through agarose gel electrophoresis detection, and positive colony is namedpCD1。

4. sequence verification

Through the positive colony identified, Sangon (Shanghai Sheng Gong Bioisystech Co., Ltd) is sent to carry out DNA sequencing, its core Nucleotide sequence is as shown in sequence table 1.

Embodiment 2: promoterpCD1The structure of plant transient expression vector

Utilize Bam HI andHind III Cobra venom endonuclease enzyme action recombinant cloning vector (T+ pCD1) andpC0390::GUS Carrier, then by promoterpCD1WithpC0390::GUS The gus reporter gene of carrier connects, and builds plant transient expression vector (Fig. 2, Fig. 3).The plant transient expression vector built is namedpCD1::GUS 。pC0390::GUSCarrier andpC35S:: GUSCarrier is provided by Ningxia University xuwei doctor Rong, concrete building process reference literature (Xu etc., 2010, Planta, 231: 475 487).Use electric shocking method, by buildpCD1::GUSCarrier,pC0390::GUSCarrier (negative control) andpC35S:: GUSCarrier (positive control) is directed respectively in Agrobacterium tumefaciems competence bacterial strain GV3101 with standby.

Embodiment 3: identify under cryogenic conditions, promoterpCD1Activity

3.1

3.1.1 Nicotiana tabacum L. is cultivated and instantaneous conversion

Ben Shi tobacco seed, after 4 DEG C of K cryogenic treatment 1 week, is seeded in the sterilization matrix of nutritive cube, and covers fresh-keeping Film, moves into artificial intelligence's weather incubator after seeding room grows 3 weeks and emerges.The tobacco plant of 5 weeks is cultivated in incubator For agriculture bacillus mediated instantaneous conversion analysis.Concrete method for transformation is as follows: 1. inoculation comprises what promoter and GUS constructed Agrobacterium 20 μ l is in LB fluid medium fresh for 20mL, additional rifampicin 50mg L^-1, Kan 50mg L^-1, at 28 DEG C of constant temperature Shaking table 240rpm, incubated overnight；2. the Agrobacterium bacterium solution of incubated overnight is transferred in the sterile centrifugation tube of 50mL, in room temperature 6000rpm, centrifugal 10min；3. supernatant is abandoned, and with permeabilization buffer (the 10 mM MES, 10 mM MgCl of 10mL₂, 100 μMs AS pH, 5.7) resuspended thalline；4. take resuspended after bacterium solution 2mL, measure OD600 value, be diluted to permeabilization buffer The bacterium solution of OD600 value about 0.6 is used for converting；5. before injecting tobacco leaf epidermal cells by the Agrobacterium bacterium solution after dilution, will Nicotiana tabacum L. is taken out from climate box, is placed in 1h under incandescent light, makes pore open being beneficial to completely inject infiltration.6. with 1mL's The syringe of needleless draws ready Agrobacterium bacterium solution, at the injection point chosen, is propped up by syringe needle, slowly bacterium solution is noted Enter the intercellular substance of tobacco leaf, make bacterium solution penetrate into whole blade as far as possible.7. the tobacco plant after infiltration covers preservative film, Put back to weather incubator, renewal cultivation 1d；8. after cultivating 1 d, transformation of tobacco plant is put into the low temperature incubator of 4 DEG C and carries out low temperature Induction processes；The most above-mentioned each transformation of tobacco plant processes and after normal condition cultivates 1 d at low temperature stress respectively, clip pair immediately The blade processing plant according to plant and low temperature stress uses with further analysis.

3.1.2 transformation of tobacco plant leaf GUS expression activity is analyzed

GUS histochemical stain is with reference to the scheme of Jefferson (1987).Tobacco leaf material is placed in sterilizing plate In, add GUS dyeing liquor (10 mM Na₂EDTA, 100 mM NaH₂PO₄, 0.5 Mm K₄Fe(CN)₆·3H₂O, 0.1% Triton X-100 and 0.5 mg L^-1X-Gluc, pH 8.0) cover material；Plate is placed in the constant incubator of 37 DEG C Middle cultivation 24h；Reclaim dyeing liquor；To plate adds 70% ethanol, in 37 DEG C of incubation 5-6h；Outwell the ethanol in plate, add 90% ethanol, continues incubation 10h at 37 DEG C；Outwell ethanol, carry out GUS dyeing and observe and photograph.Take comparison and the process of about 1g Tissues of Tobacco is placed in mortar, powdered by liquid nitrogen grinding, adds Extraction buffer (containing 50 mM L^-1Sodium phosphate, 10 mM L^-1EDTA, 0.1% TritonX-100,1% Sarcosyl, 10 mmol L^-1Beta-mercaptoethanol), mixing, 4 DEG C, 5000 After rpm is centrifuged 10min, takes supernatant and obtain GUS soluble protein crude extract.Taking-up part adds 1 mM L^-1GUS reaction Substrate 4-MUG, after 37 DEG C of insulation 30 min, adds 0.2 m M L^-1 Na₂CO₃Reaction terminating liquid terminates reaction.Utilize Hitachi 850 type fluorescent spectrophotometer assay fluorescent values, exciting light 365 nm, launches light 455 nm.Soluble protein content reference Bradford(1976) method measures, and measures GUS protein content, excitation wavelength 595 with UV-2102C ultraviolet spectrophotometer nm.The required enzyme amount generating 1 pM 4-MU with 1 min hydrolysis 4-MUG is an enzyme activity unit, with every milligram of albumen Enzyme activity represents GUS activity.Above-mentioned experiment is in triplicate.

Fig. 4 shows, what GUS activity was the strongest demonstrates the deepest blueness, and light blue expression activity is relatively low, represents without blue GUS activity is relatively low.Wild type (WT) and conversionpC0390::GUS The tobacco leaf of plant expression vector (negative control) exists Blueness can not be dyed by X-Gluc solution under the conditions of normal growth, low temperature induction, but convertpC35S::GUSPlant express The tobacco leaf of carrier the most all can be dyed blueness, explanation by X-Gluc solutionpC0390::GUS Plant Expression vector can be used for the activity analysis of the promoter fragment of this experiment.Compare normal growing conditions in contrast, convertpCD1::GUS Tobacco leaf under the conditions of low temperature induction, dyed blueness by X-Gluc, illustratepCD1(-270bp) is low temperature Inducible promoter.The fluorescent quantitation result of the GUS in Fig. 5 shows,pCD1(-270bp) promoter section activity is positive right According to activity 32.6%, illustrate that this promoter is the promoter of a kind of high efficient cryogenic induction type.

3.2

Select arabidopsis, Oryza sativa L. and sweet buckwheat respectively, use and 3.1 similar approach, analyze promoter pCD1 activity at low temperature Under the conditions of activity.It was found that compare normal growing conditions in contrast, convertpCD1::GUS Arabidopsis, water Rice and sweet buckwheat blade are all dyed blueness by X-Gluc under the conditions of low temperature induction, illustratepCD1(-270bp) is cold-inducible Promoter.The fluorescent quantitation of GUS shows, in three kinds of plantspCD1(-270bp) promoter section activity is positive control respectively Activity 36.2 %, 32.5% and 37.8%, illustrate that this promoter is the promoter of a kind of high efficient cryogenic induction type.

Sequence table

<110>Changjiang University

<120>a kind of cold-inducible promoter and application thereof

<160> 3

<170> PatentIn version 3.5

<210>1

<211> 270

<212> DNA

<213>sweet buckwheat

<220>

<223>promoter pCD4

<400> 1

atagacctag tcaggatcac gactgtactc ccttacccag aaacctcctt acgaacactt 60

gcgccagctc cctgaatatc caatccgcca gaccgccacc agctgtctcc tccgtgttaa 120

ccaccaccac actcttccca cactctattt cctcgtcccc aattcaacca acactatata 180

aaatcacacc atccatctta caacttaatt agattaaatt cttaaatctt aatcaactca 240

aaattacttc atatatattc aggatcgatc 270

<210>2

<211> 28

<212> DNA

<213>artificial sequence

<220>

<223> primer 1

<400> 2

cccaagctta tagacctagt caggatca 28

<210>3

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223> primer 2

<400> 3

gcggatccga tcgatcctga atatat 26

Claims (4)

1. a promoter, it is characterised in that the nucleotides sequence of this promoter is classified as SEQ ID NO:1.

2. contain the recombinant vector of promoter as claimed in claim 1, expression cassette or recombinant bacterium.

3. promoter as claimed in claim 1 starts genes of interest in Nicotiana tabacum L., arabidopsis, Oryza sativa L. or sweet buckwheat when low temperature induction The application of middle expression.

4. one kind makes the method that plant expresses genes of interest when low temperature induction, it is characterised in that the method is to be inserted by genes of interest Enter in the recombinant vector containing promoter as claimed in claim 1, and this recombinant vector is imported in purpose plant, the lowest Temperature induction genes of interest is expressed in plant, and described plant is Nicotiana tabacum L., arabidopsis, Oryza sativa L. or sweet buckwheat.