CN102094021B - Corn callus specific promoter and cloning method and application thereof - Google Patents
Corn callus specific promoter and cloning method and application thereof Download PDFInfo
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- CN102094021B CN102094021B CN 201010587043 CN201010587043A CN102094021B CN 102094021 B CN102094021 B CN 102094021B CN 201010587043 CN201010587043 CN 201010587043 CN 201010587043 A CN201010587043 A CN 201010587043A CN 102094021 B CN102094021 B CN 102094021B
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Abstract
The invention discloses a corn callus specific promoter and a cloning method and application thereof. A WRKY gene is obtained by utilizing bioinformatics analysis, the sequence comparison of the gene and the genome is performed, a primer is designed to perform polymerase chain reaction (PCR) and obtain the promoter region of the gene 5', the promoter is 2880bp and has the sequence shown in the (400)1 item of the sequence table; the analysis of the promoter shows that the promoter contains a plurality of acting elements; and the promoter is subcloned to the vector with GUS gene, expression of the GUS gene is started, and the vector is transformed in rice. The staining examine of each tissue of rice shows that the specificity of the promoter can be expressed in callus, thus the promoter has important application value in genetic engineering and the gene function researches.
Description
Technical field
The present invention relates to plant promoter, its cloning process and application thereof, particularly relate to a kind of maize calli specificity promoter, its cloning process and the application in cultivating the security transgenic plant thereof.
Background technology
The selection markers gene has vital role in plant genetic engineering, utilize the selection markers gene in the plant transgene process, from a large amount of unconverted cells, to filter out transformant.Yet; Screening-gene in the transgenic plant has caused the worry of people to a series of Biosafeties aspect again; Like selectable marker gene whether encode deleterious material and allergen; Whether be unfavorable for the change of plant metabolism, whether can reduce the curative effect of curative drug, and whether can between close species and pathogenic agent, move etc.How the rejecting screening marker gene has become the problem that people are concerned about from transgenic plant.At present; Mainly contain two kinds of technology with rejecting screening marker gene of potential using value: a kind of is goal gene and selection markers gene cotransformation technology, and it can make goal gene and selection markers gene through the free isolating principle of gene rejecting screening token-based thereby reservation goal gene in the offspring; Another kind is to recombinate through locus specificity to wipe out the technology of selection markers gene; In addition, the selection markers gene knochout technique etc. that also has the mediation of transposon-mediated selection markers gene knochout technique and homologous recombination.But above-mentioned from the gene level the rejecting screening marker gene have very complicated, the shortcoming that the transformation period is long.
Summary of the invention
The invention provides a kind of promotor specific expressed in maize calli.Maize calli specificity promoter provided by the present invention, name is called ZMW, derives from Zea corn (Zea mays L.).This promotor size 2880bp.
Plan involved in the present invention does; A kind of maize calli specificity promoter; Method through information biology is tentatively definite; This promotor of amplification in corn B73 self-mating system through checking specific expressed promotor in callus in paddy rice, has in the sequence table sequence under < 400>1.
A kind of cloning process of above-mentioned maize calli specificity promoter; Utilize information biology screening candidate gene; Compare with genome sequence then; The design primer, upstream primer: 5 '-CCG AAG CTT ATG ACA AGA GAA GTT GGA CAA G-3 ', downstream primer: 5 '-GCG AGA TCT TCA GAT CGA CCT CCT AGC TCT A-3 ' this upstream region of gene 5 ' regional DNA sequence that increases; Be connected with gus gene then, through transgenic paddy rice checking promoter function.
According to the application of above-mentioned maize calli specificity promoter in plant callus gene functional research and gene expression regulation.
A maize calli specificity promoter ZMW provided by the invention specifically expressing in the callus of paddy rice; Can instruct the expression of foreign gene specific space-time in the development of plants process; Have interim strong, highly sensitive advantage with this method screening transgenic paddy rice, and, can not express in other periods because screening-gene only can be expressed in the callus stage; It can not only make the expression product of goal gene accumulate at certain space; Increase regional expression amount, and avoided the unnecessary waste of plant nutrition, have bigger actual application value.
Description of drawings
Fig. 1 is the GUS coloration result (this promotor is specific to be blue in callus) of transfer-gen plant.
A is a callus; B is a root; C is a leaf; D is a pollen; E is a stem; F is clever shell; G is immature seed; H is sophisticated seed; I is an ovary.
Coloration result shows that gus gene is specific expressed in callus, and does not all express at other positions, and the blue portion among the A figure is catches the position.
Embodiment
Below in conjunction with specific embodiment the present invention is further specified.
Method therefor is ordinary method if no special instructions among the following embodiment; The primer is synthetic by Shanghai Jierui Biology Engineering Co., Ltd; Order-checking is undertaken by Invitrogen company; The PCR test kit, connect endonuclease in test kit and the vector construction process all available from from precious biotechnology ltd, plasmid extraction kit and glue recovery test kit are all purchased the Bioisystech Co., Ltd in full Shi Jin, and the equal reference reagent box of method specification sheets carries out.
One, the specific expressed gene of maize calli
WrkyAcquisition
At first in NCBI website (http://www.ncbi.nlm.nih.gov) with keyword " wrky " muca gene, obtain the protein sequence of Arabidopis thaliana wrky gene.Set up the zein local data base, use " DNATOOLS 6.0 " software that the protein sequence of the Arabidopis thaliana wrky gene of acquisition is carried out BlastP (E-value=0.001) sequence alignment, filter out the highest corn candidate albumen sequence of homology.Then use " DNATOOLS 6.0 " software that the corn complete genome DNA sequence data that obtains is set up local data base, carry out BlastN (E-value=0.001) sequence alignment, filter out the highest candidate gene of homology with the zein sequence that obtains.
Transfer-gen plant is carried out the GUS tissue staining, but in the callus of Arabidopis thaliana, detect the expression of GUS, infer that GUS possibly express in the callus of paddy rice.
Two, the clone of maize calli specificity promoter wrky
To start wrky expression promoter called after ZMW, be this segmental primer of nucleotide sequence design pcr amplification of 2812bp according to wrky upper reaches length, and the upper reaches add HindIII (AAGCTT) restriction enzyme site, and downstream add BglII (AGATCT) restriction enzyme site
Primer sequence is following:
Primer 1 (upstream primer): 5 '-CCG AAG CTT ATG ACA AGA GAA GTT GGA CAA G-3 '
Primer 2 (downstream primer): 5 '-GCG AGA TCT TCA GAT CGA CCT CCT AGC TCT A-3 '
Extract corn gene group DNA and as template, under the guiding of primer 1 and primer 2, carry out pcr amplification, the PCR reaction system is:
ddH
2O： 39.6μL
10X?PCR?Buffer： 5μL；
10mM?dNTP： 1μL；
10uM primer 1:1 μ L;
10uM primer 2: 1 μ L;
Corn B73 genomic dna: 10pg;
DNA?Polymerase(2U/ul)：0.4μL；
The PCR reaction conditions is:
After reaction finishes; The PCR product is carried out 1% agarose gel electrophoresis detect, reclaim the also purpose fragment of purifying 2812bp, it is cloned into carrier pMD18-T Vector (precious biotech firm); Be transformed into again in the Trans5 α chemoreception attitude cell (full Shi Jin Bioisystech Co., Ltd); The PCR screening positive clone checks order, and sequencing result shows that wrky has SEQ ID № in the sequence table: 1 dna sequence dna; SEQ ID № in the sequence table: 1 by 2812 based compositions, will contain the recombinant plasmid vector called after PMD18-ZMW of ZMW dna sequence dna.
Three, the foundation of WRKY gene promoter ZMW expression vector
To scale off with Hind III and Bgl II enzyme with the wrky fragment that pcr amplification obtains and clones on the PMD18-T carrier, be connected carrier construction p-ZMW with the carrier of cutting pCAMBIA1301 with same enzyme.Its vector construction figure is specifically: hpt: hygromycin gene has a no promotor at this upstream region of gene; Wrky: promotor ZMW; Gus:glucoronidase gene, i.e. gus reporter gene; Nos: terminator is gus reporter genes in its downstream.
Get that the overexpression vector plasmid DNA of 1 μ g joins in the 200 μ L EH105 competent cells mixing behind the purifying; Ice bath changed in the liquid nitrogen quick-frozen over to 1 minute after 5 minutes, then move to 37 ℃ of water-baths 5 minutes, added 1 mL YEP liquid nutrient medium, and 28 ℃, 250 rpm prefigurations reach 4-5 hour; Centrifugal 30 seconds of 10000 rpm abandon supernatant, add 0.1mL YEP liquid nutrient medium, again suspension cell; Coat on the YEP solid plate that contains 100 μ g/mL Kan and 50 μ g/mL Rifampins, cultivated about 48 hours for 28 ℃.The single bacterium colony that grows on the picking flat board is inoculated in the YEP liquid nutrient medium that contains 100 μ g/mL Kan and 50 μ g/mL Rifampins, extracts plasmid at last.Earlier with PCR method preliminary evaluation (primer is primer 1 and primer 2); Then cut checking with Hind III and Bgl II enzyme.
Four, agriculture bacillus mediated rice conversion
The ZMW expression vector p-ZMW plasmid that will contain goal gene through the competent method of Agrobacterium imports among the agrobacterium strains EH105; Filter out positive colony through PCR, then with being total to the Agrobacterium importing plant acceptor material rice transformation that culture method will contain goal gene.
(1) inducing culture of paddy rice mature embryo callus and succeeding transfer culture
The paddy rice mature seed that shells is used ddH earlier
20 washes 4-5 time, uses 70% alcohol immersion 1-2 minute again, soaks 30 minutes with 20% Youxiaolin that contains 1/1000 tween then; That does not stop during this time rocks; Thereby carry out surface sterilization, use aseptic water washing 3-4 time afterwards, again mature seed is placed on the aseptic filter paper behind the suck dry moisture; Be placed on the callus of induce substratum, 28 ℃ of low light levels are cultivated.After about 10-15 days, callus is changed on the subculture medium, under the same conditions succeeding transfer culture.Per two all succeeding transfer culture are once selected succeeding transfer culture 5-7 days behind twice of the subculture, and the callus that color and luster is yellowish is used for common cultivation.
(2) be used for the preparation of the Agrobacterium bacterium liquid of rice transformation
The Agrobacterium that will contain plant expression vector is inoculated in the YEP liquid nutrient medium (containing 100 μ g/mL Kan and 50 μ g/mL Rifampins), and 28 ℃ of shaking culture are to OD
600=0.6-1.0; 5000 rpm collected thalline in centrifugal 5 minutes under the room temperature, subsequently it were suspended in liquid and were total in the substratum, and the adjustment cell concentration is to OD
600=0.4, be the agrobacterium suspension that common culture transformation paddy rice is used.
(3) Agrobacterium is infected the rice callus tissue
Select state better the callus of (succeeding transfer culture 5-7 days, color and luster yellowish) put into the aseptic triangular flask of 25 mL, add an amount of agrobacterium suspension to guarantee having enough bacterium liquid to contact with material, cultivation is 20-30 minute on 28 ℃ of 150 rpm shaking table.Outwell bacterium liquid afterwards, callus is placed on to inhale on the aseptic filter paper removes unnecessary bacterium liquid, transfer to the solid that is covered with one deck aseptic filter paper immediately and be total on the substratum, 22 ℃ of dark cultivations 2-3 days.
(4) antibacterial processing
Use ddH
2It is limpid to scavenging solution that O cleans callus 3-4 time; Discard scavenging solution; Blot with aseptic filter paper; Changing in the solution that contains cephamycin (500mg/L) and Pyocianil (200mg/L) more than vibration sterilization half hour, blotting, going in the petridish of the two-layer aseptic filter paper in shop drying treatment 24-72 hour with aseptic filter paper; Callus is transferred to is added with on cephamycin (500mg/L) and Totomycin (50mg/L) screening culture medium, about 30 days of 28 ℃ of illumination cultivation.
(5) differentiation of resistant calli
The resistant calli that after screening, grows; Selecting the fine and close hygromycin resistance callus of milk yellow goes on the division culture medium that contains 50mg/L Totomycin (or 20mg/L PPT); Secretly cultivated 3 days for 28 ℃ earlier; Go to then under 30 ℃ of full exposure conditions and cultivate, generally pass through about 15-20 days, have green point to occur.Further differentiate seedling behind the 30-40d.
(6) take root, strong sprout and transplanting
The seedling height that resistant calli differentiates moves on on the root media during greater than 3cm, cultivates 2-3 week.Select that high 15cm is above, the seedling of well developed root system,, in the greenhouse, transplant and bury with warm water flush away substratum.The water surface is not to flood seedling degree of being, fine day need shade and become (being as the criterion with guttation) alive to seedling.Treat to move into Tanaka's growth after the transgenic seedling robust growth.
Five, transgenic paddy rice evaluation
In order to detect transgenic paddy rice, be template generally, with the upstream primer and the downstream primer pairing amplification corresponding gene of intermediate carrier resistance screening gene Totomycin, preliminary evaluation transfer-gen plant with the total DNA of transgenic paddy rice that extracts.
Be used to detect the primer sequence of hygromycin gene:
Primer 3 (upstream primer): 5 '-ACTCACCGCGACGTCTGT-3 '
Primer 4 (downstream primer): 5 '-TTTCTTTGCCCTCGGACG-3 ';
After preliminary evaluation is transfer-gen plant, then transfer-gen plant is carried out the GUS tissue staining.Get stem, root, leaf, pollen, the clever shell of transgenic paddy rice; In the plant tissue and little centrifuge tube to be detected such as callus, immature seed, sophisticated seed and ovary; Add GUS damping fluid submergence tissue block; The GUS staining fluid that adds 5% (v/v) consumption was again preserved 16-24 hour down for 37 ℃ behind the mixing.Green materials such as blade change in 70% ethanolic soln and decolour 2-3 time, are white in color until the negative control material.Naked eyes or microscopically are observed, and the blue point under the white background is the GUS expression vector, and as shown in the figure (A is a callus; B is a root; C is a leaf; D is a pollen; E is a stem; F is clever shell; G is immature seed; H is sophisticated seed; I is an ovary).In the normal organ of transfer-gen plant, all do not detect the expression of gus gene, and only in callus, detect the expression of gus gene, thereby proof wrky is only specific expressed in the rice callus tissue.
Claims (3)
1. maize calli specificity promoter; It is characterized in that: the method through information biology is tentatively definite; This promotor of amplification in corn B73 self-mating system; Through in paddy rice, verifying promotor specific expressed in callus, its dna sequence dna is the sequence shown in the SEQ ID NO:1 in the sequence table.
2. the cloning process of a maize calli specificity promoter as claimed in claim 1; It is characterized in that: utilize information biology screening candidate gene, compare with genome sequence then, the design primer; Upstream primer: 5 '-CCG AAG CTT ATG ACA AGA GAA GTT GGA CAA G-3 '; Downstream primer: 5 '-GCG AGA TCT TCA GAT CGA CCT CCT AGC TCT A-3 ' is a template with corn B73 self-mating system genomic dna, and this candidate gene upper reaches 5 ' regional DNA sequence increases; Be connected with gus gene then, through transgenic paddy rice checking promoter function.
3. the application of maize calli specificity promoter as claimed in claim 1 in plant callus gene functional research and gene expression regulation.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268443B (en) * | 2011-07-25 | 2013-01-30 | 安徽农业大学 | Application of corn WRKY gene in enhancing plant stress tolerance |
| CN102787116B (en) * | 2012-03-12 | 2013-10-02 | 安徽农业大学 | Corn callus specific promoter and application |
| CN104651360B (en) * | 2013-11-25 | 2017-07-11 | 中国农业科学院生物技术研究所 | Corn tissue's specificity promoter and its application |
| CN103789312A (en) * | 2014-02-13 | 2014-05-14 | 安徽农业大学 | Corn endosperm tissue specificity promoter and application thereof |
| CN107988226A (en) * | 2017-12-11 | 2018-05-04 | 浙江省农业科学院 | A kind of identification and application of the special High-expression promoter of Rice Callus |
| CN110055342A (en) * | 2019-05-28 | 2019-07-26 | 广西大学 | Molecular labeling relevant to Guangxi fiber crops chicken egg productivity and its application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993005164A1 (en) * | 1991-09-02 | 1993-03-18 | The University Of Leicester | Callus-specific promoters |
| JP2005143338A (en) * | 2003-11-12 | 2005-06-09 | National Institute Of Agrobiological Sciences | Promoter having callus- and seed embryo-specific expression activity |
| CN100457906C (en) * | 2006-04-06 | 2009-02-04 | 北京凯拓迪恩生物技术研发中心有限责任公司 | Specificity start factor of paddy rice traumatic tissue and uses |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002097085A2 (en) * | 2001-05-31 | 2002-12-05 | Syngenta Limited. | Callus specific plant promoters |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993005164A1 (en) * | 1991-09-02 | 1993-03-18 | The University Of Leicester | Callus-specific promoters |
| JP2005143338A (en) * | 2003-11-12 | 2005-06-09 | National Institute Of Agrobiological Sciences | Promoter having callus- and seed embryo-specific expression activity |
| CN100457906C (en) * | 2006-04-06 | 2009-02-04 | 北京凯拓迪恩生物技术研发中心有限责任公司 | Specificity start factor of paddy rice traumatic tissue and uses |
Non-Patent Citations (4)
| Title |
|---|
| Jacob Bade1 et al..T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes.《Plant Molecular Biology》.2003,第52卷(第1期),53-68. * |
| Yuhya Wakasa et al..Higher-level accumulation of foreign gene products in transgenic rice seeds by the callus-specific selection system.《Journal of Bioscience and Bioengineering》.2009,第107卷(第1期),78-83. * |
| 朱苏文等.玉米淀粉分支酶基因启动子的克隆及生物信息学分析.《安徽农业科学》.2007,第35卷(第14期),4126-4127,4129. * |
| 王颖等.高等植物启动子的研究进展.《西北植物学报》.2003,第23卷(第11期),2040-2048. * |
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