CN103952414A - Tobacco glycosyltransferase inducible promoter and application thereof - Google Patents
Tobacco glycosyltransferase inducible promoter and application thereof Download PDFInfo
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- CN103952414A CN103952414A CN201410217346.7A CN201410217346A CN103952414A CN 103952414 A CN103952414 A CN 103952414A CN 201410217346 A CN201410217346 A CN 201410217346A CN 103952414 A CN103952414 A CN 103952414A
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Abstract
The invention provides a tobacco glycosyltransferase inducible promoter Sm-NgtP. The promoter Sm-NgtP is separated into a tobacco glycosyltransferase gene, and has the nucleotide sequence shown in SEQ ID NO. 1. The promoter Sm-NgtP is amplified by adopting a primer to obtain five promoter fragments with missing 5' ends namely the promoter fragments GTPA, GTPB, GTPC, GTPD and GTPE; the five promoter fragments are used for constructing plant expression vectors, and the plant expression vectors pGTPA, pGTPB, pGTPC, pGTPD and pGTPE are obtained. Study on the activities of the plant expression vectors shows that GTPC is a promoter dually induced by methyl jasmonic acid and salicylic acid; GTPD is a constitutive strong expression promoter; the nucleotide sequence of the GTPC promoter fragment is shown in SEQ ID NO.2; the nucleotide sequence of the GTPD promoter fragment is shown in SEQ ID NO.3.
Description
Technical field
The present invention relates to a kind of isolation identification and application of the tobacco native gene super promoter that is subject to jasmonic and the dual induction of Whitfield's ointment, by separating and identify this promotor, can be applied in the genetically engineered of plant, to reach abduction delivering and the functional study of genetic improvement of some gene of plant, belong to plant gene engineering technology field.
Background technology
Along with molecular biological development, transgenosis has become the effective means of research functional genomics, is just progressively obtained feasibility study and is generally accepted in recent years by transgenosis approach improvement plant economical character.Promotor is the DNA sequence dna that gene 5 ' end is combined with RNA polymerase and some trans-acting factors.In eukaryote, there are three kinds of RNA polymerase, are respectively combined with dissimilar promotor.RNA polymerase I is only transcribed rRNA, and RNA polymerase III is responsible for transcribing tRNA and 5S rRNA.RNA polymerase II is responsible for transcribing of protein gene and part snRNA, and its promoter structure is the most complicated.Function, the structure of this class promotor can be divided into two portions: ubiquitous, transcribe essential core promoter (core promoter) district and specific promoter district.Promotor contains a series of sites of being combined with differential protein, conventionally claims that these sites are cis-acting elements (cis-element); These differential proteins are called trans-acting factor (trans-factor).Transcription initiation site, TATA box etc. belong to core promoter district; The cis-acting elements of regulate gene expression activity is classified as specific promoter district.
Whitfield's ointment (salicylic acid, SA) is the endogenous Small Phenolic Molecule compound of one that plant materials intensive amount is lower.SA can induce various plants to produce resistance to virus, fungi and Micobial Disease.SA is one of signaling molecule bringing out systemic acquired resistance, relates to also anaphylaxis and the SAR reaction of involved in plant, in the SAR of plant signal transduction, plays keying action.SA is as plant hormone and signaling molecule, the directly or indirectly expression of genes involved in regulating plant body growth course.Isolate at present the cis-acting elements of the several SA of being subject to indirect adjustments and controlss.As-1 (Activation Sequence-1) type Whitfield's ointment response element.SA induction detects several controlling elements that are derived from cauliflower mosaic virus CaMV35S promotor, confirms that SA response element is to be positioned at-90 and-46 liang of as-1 elements of locating.With after bigcatkin willow acid treatment tobacco, the inducible factor ASF-1 (Activation Sequence Factor-1) of confirmation SA and as-1 combination of elements are in two TGACG.LS7 element (TCTACGTCAC) is the SA induced element separating from Arabidopis thaliana PR-1 (pathogenesis related gene 1) gene promoter with LS5 element (ACGTCATAGA).SA is by the NPR1 in activated path, and the NPR1 activating transcription factor TGA2 having activated is to promote TGA2 transcription factor and LS7, LS5 combination of elements, thus activation downstream PR-1 genetic expression.W-box (TTGAC) is found in Arabidopsis thaliana NPR1 gene promoter, is the specific recognition site of WRKY DBP, and the expression of WRKY gene is subject to the just regulation and control of SA.W-box sudden change can blocking-up WRKY DBP specific recognition, cause promotor cannot activate the expression of downstream NPR1 gene, thereby affect the expression of the Analysis of Defence Genes Involved that SA induces by NPR1.TCA-1 (tobacco nuclear protein1) binding site is a 10bp sequence (TCATCTTCTT) by Induced by Salicylic Acid TCA-1 combination.This element repeats for several times in barley β-1-3-glucanase gene 5 ' end non-coding region, existing known have exceed 30 and be subject to the promotor of various stress inducible genes to contain this element.
Jasmonic (Jasmonic Acid, JA) and methyl jasmonate (methyl jasmonate, MeJA) are the derivative compounds with cyclopentanone group of linolenic acid.Along with the molecular biological rise eighties in 20th century, the biosynthesizing of people to them and the molecular regulation mechanism of metabolism have been done detailed research, find that jasmonic regulation and control are permitted polygenic expression.Two aspects are summed up: a part of gene is relevant with development of plants; Another part gene is relevant with self-defense system, show to adverse circumstances such as fungi infestation, disease and pest, arid, physical abuse and osmotic stresses stress (Wu Jingsong, plant health. the molecular biology research of jasmonic effect. BULLETIN OF BOTANY Vol., 2002,19 (2): 164-170).By the analysis to jasmonic induced gene promoter, had been found that the response element of many jasmonic inductions, but these researchs focus mostly on the gene promoter relevant with plant self-defense system at present.T/G-box (AACGTG), is the binding site of a bHLH-leucine zipper JAMYC2 and JAMYC10 albumen, and the expression of JAMYC2 and JAMYC10 is subject to the induction of JAs.JAMYC overexpression can strengthen the expression of the Analysis of Defence Genes Involved of JAs induction in potato, but does not cause the accumulation of this genoid transcription product.JERE (jasmonate-and elicitor-responsive element) is that a class is responsible for the MeJA that confirms in a synthetic gene Str (strictosidine synthase) promotor of secondary metabolite and the element of ethylene responses as far back as Vinca plant.JERE with other MeJA response element different be to comprise a GCC-box-like element.JASE1 (CGTCAATGAA) and JASE2 (CATACGTCGTCAA), be found in Arabidopis thaliana 12-O-phytodienoic acid-10, the promotor of 11-reductase enzyme (OPR1) gene-179 and-170 between.Promotor connects research and the RNA hybridization analysis of gus reporter gene and shows, OPR1 gene is subject to the upper regulation and control of old and feeble and JA.Promoter deletion and linking scanning mutation analysis show that JASE1 and JASE2 are cis-acting elements old and feeble and that SA replys.Stress response element (Stress responsive element, SRE) is the cis-acting elements of a long 13bp.SRE in the confirmation tobacco long terminal repeat retrotransposon Tto1 promotors such as Takeda is the cis-regulating element of response tissue culture, damage and MeJA induction.
Summary of the invention
The invention provides a kind of promotor in tobacco sugar based transferase with induction type and superpower activity that derives from, this promotor hard to bear methyl jasmonic acid of energy and the dual induction of Whitfield's ointment, in the present invention, also utilize this promotor construction of expression vector, utilize methyl jasmonic acid and Determination of Salicylic Acid control, and then reach the object of all kinds of destination gene expressions of induction by genetic transforming method.
Realizing the technical scheme that above-mentioned purpose of the present invention adopts is:
A kind of tobacco sugar based transferase inducible promoter Sm-NgtP, this promotor Sm-NgtP is located away from tobacco glycosyltransferase gene, and its nucleotide sequence is as shown in SEQ ID NO.1.
Gene order to above-mentioned promotor Sm-NgtP is analyzed, and finds that promoter element concentrates between-800~-50bp.Adopt primer pair promotor Sm-NgtP to increase, amplification obtains the promotor segment of 55 ' end disappearances, be respectively promoter fragment GTPA, GTPB, GTPC, GTPD and GTPE, utilize above-mentioned five promoter fragments to build plant expression vector, obtain plant expression vector pGTPA, pGTPB, pGTPC, pGTPD and pGTPE, studying the activity of above-mentioned plant expression vector finds, promoter fragment GTPC is the promotor that is subject to methyl jasmonic acid and the dual induction of Whitfield's ointment, GTPD is composing type strongly expressed promotor, the nucleotide sequence of GTPC promoter fragment is as shown in SEQ ID NO.2, be subject to the nucleotide sequence of GTPD promoter fragment of methyl jasmonic acid and the dual induction of Whitfield's ointment as shown in SEQ ID NO.3.
A kind of method of utilizing vegetable cell to express goal gene is also provided in the present invention, comprise the following steps: with carrier for expression of eukaryon pGTPC or carrier for expression of eukaryon pGTPD transformed plant cells cultivation, then adopt methyl jasmonic acid and Induced by Salicylic Acid vegetable cell to express goal gene; Described plant is cereal grass, or is any in cotton, tobacco, rape and eggplant class plant.
Brief description of the drawings
Fig. 1 is the nucleotide sequence figure of promotor Sm-NgtP provided by the invention;
Fig. 2 is the expression vector pCAMBIA1301-Sm-NgtP expression vector that the present invention builds;
Fig. 3 is the expression activity figures of five kinds of gus genes under promoter fragment control under jasmonic and Whitfield's ointment processing.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to detailed specific description, but protection scope of the present invention is not limited to following examples.
In the present embodiment, choose GTs gene as candidate gene, the about 3kb of GTs complete sequence gene, according to the sequential analysis design primer of upstream, GT gene coding region, isolate the promotor of the candidate gene that is subject to jasmonic and the dual induction of Whitfield's ointment, by this promotor called after Sm-NgtP, the nucleotide sequence of this promotor is as shown in Fig. 1 and SEQ ID NO.1, in Fig. 1, underscore part is amplification Sm-NgtP promotor primer sequence used, what shade showed is basic promoter element sequence, and promoter element core sequence represents with wavy line.
Design primer amplification obtains five promoter fragments, be respectively promoter fragment GTPA, GTPB, GTPC, GTPD and GTPE, wherein the nucleotide sequence of GTPC promoter fragment is as shown in SEQ ID NO.2, and the nucleotide sequence of GTPD promoter fragment is as shown in SEQ ID NO.3.With restriction enzyme Hind III, Nco I double digestion carrier, reclaim purifying lengthy motion picture disconnected, couple together with promotor segment disconnected the lengthy motion picture of carrier pCAMBIA1301 double digestion with T4 ligase enzyme again, after transforming, screen, identifying, obtain plant expression vector pGTPA, pGTPB, pGTPC, pGTPD, pGTPE, as shown in Figure 2, the expression vector pCAMBIA1301-Sm-NgtP expression vector that the present invention builds.This carrier is containing hygromycin resistance screening-gene (HRG), and the each fragment in promotor Sm-NgtP is fused to gus gene 5 ' end non-translational region.
The increase promoter fragment of five different lengthss obtaining of this promotor replaces pCAMBIA35S promotor and GUS to construct five expression vectors, transforms W38 type tobacco, acquisition transformed plant.Below by the abduction delivering that transformed plant is carried out to GT promotor, and floral organ tissue site Coloration experiment, the tissue expression of this gene and the space-time characterisation of being induced studied.
Concrete operations are as follows:
1, experiment reagent
1) X-Gluc staining fluid (10mgmL
-1): 20mg X-Gluc is dissolved in 2mL2, in methonal.
2) phosphate buffer solution (2molL
-1): claim 31.6g K
2hPO
4, 2.5g KH
2pO
4be dissolved in 100mL intermediate water, be adjusted to pH7.8.
3) Tripotassium iron hexacyanide (0.05molL
-1): the 0.1646g Tripotassium iron hexacyanide is dissolved in 10mL intermediate water.
4) yellow prussiate of potash (0.05molL
-1): 0.2112g yellow prussiate of potash is dissolved in 10mL intermediate water.
5) 0.5molL
-1dTT: with 0.01molL
-1sodium-acetate is solvent, and after preparing, 200 μ L/ prop up and point install to eppendorf and manage ,-70 DEG C of preservations.
6) GUS reaction solution (20 DEG C of preservations): protein extract (no DTT) 25mL; DTT (0.5M) 200 μ L; MUG22mg.
7) the blank reaction solution of GUS: protein extract (no DTT) 25mL; DTT (0.5M) 200 μ L.
8) reaction terminating liquid: 0.2molL
-1na
2cO
3: 10.6g Na
2cO
3be dissolved in 500mL intermediate water.
9) GUS dye liquor
2, transgenosis culture medium prescription
1) molysite (Fe
2eDTA) preparation of stock solution (100X)
Prepare 800ml distilled water and be heated to 70 DEG C, adding b diammonium disodium edta (Na
2eDTA2H
2o) 3.73 grams, after fully dissolving, in 70 DEG C of water-baths, keep 2 hours, be settled to 1000ml, 4 DEG C save backup.
2) VITAMIN stock solution (100X) preparation
Add water and be settled to 1000ml, 4 DEG C save backup.
3) preparation of MS substratum macroelement mother liquor (10X)
Under room temperature, dissolve and be settled to 1000ml.
4) preparation of MS substratum trace element mother liquor (100X)
Under room temperature, dissolve and be settled to 1000ml.
5) callus induction substratum MS+2mg L
-1bA+0.2mg L
-1nAA+30g L
-1sucrose+8.0g L
-1agar powder (pH5.8)
6) screening culture medium MS+2mg L
-1bA+0.2mg L
-1nAA+30g L
-1sucrose+8.0g L
-1agar powder+600mg L
-1amp+50mg L
-1hpt II (pH5.8)
7) root media 1/2MS+600mg L
-1amp+30g L
-1sucrose+8.0g L
-1agar powder (pH5.8)
8) 2,4-D stock solution (1mg L
-1) preparation: weigh 2,4-D100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, then add after 10ml distilled water dissolves completely and be settled to 100ml, under room temperature, preserve.
9) 6-BA stock solution (1mg L
-1) preparation: weigh 6-BA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, then add after 10ml distilled water dissolves completely and be settled to 100ml, room temperature preservation.
10) naphthylacetic acid (NAA) stock solution (1mg L
-1) preparation: weigh NAA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, then add after 10ml distilled water dissolves completely and be settled to 100ml, 4 DEG C save backup.
3, Transgenic Tobacco step of converting
1) vegetable material: the seed of tobacco bred W38 is through 75% alcohol immersion 1min, 0.1%HgCl
2sterilizing 15min, sterilized water washs after 5 times, is laid on 1/2MS substratum, in 28 DEG C (16h daytime/8h nights) lower sprouting, gets the blade of growing 1~2 month after sprouting, as the starting materials of genetic transformation.
2) agrobacterium tumefaciens is cultivated: the single bacterium colony of picking agrobacterium tumefaciens, and in 1.5ml LB liquid nutrient medium, 28 DEG C of overnight incubation; Get the above culture of 1mL, add in 100mL LB liquid nutrient medium, 28 DEG C are cultured to OD600 is 0.6~1.0; By centrifugal 100mL culture, collect thalline, resuspended with liquid MS medium.
3) cultivate altogether: get tender, the healthy and strong blade of children of tobacco aseptic seedling, remove master pulse, blade is cut into the leaf dish of 0.8cm × 0.8cm left and right, be placed on callus induction substratum, anti-uppermost leaf dish dehydration; About 100 leaf dishes are put into the substratum of 100mL resuspension Agrobacterium, and 190rpm, cultivates 10min on 28 DEG C of shaking tables altogether; Medication spoon is pulled leaf dish out, is placed on the thieving paper of sterilizing, blots the unnecessary bacterium liquid of Ye Panshang; The leaf dish blotting is lain on the callus induction substratum that adds lid layer filter paper, seal culture dish with sealed membrane, 28 DEG C of left and right, cultivate altogether in dark place approximately 60 hours.
4) induction is sprouted: after cultivating altogether 60 hours, according to the following steps leaf panel surface Agrobacterium is washed off, shake frequently, makes leaf dish fully contact solution: sterilized water 15min therebetween; Sterilized water+Amp (600mg L
-1) 15min; MS+Amp (600mg L
-1) 20min.Medication spoon is pulled leaf dish out, is placed on thieving paper, blots excessive moisture; By downward the leaf dish back side, be placed in screening culture medium, leaf plate edge is gently pressed in substratum; Seal culture dish with sealed membrane, 28 DEG C of left and right, 16 hours every days illumination cultivation; After approximately two weeks, cut the leaf plate edge with bud, insert in screening culture medium and continue to cultivate.
5) root induction: induce in screening culture medium after approximately 4 weeks, the young shoot that leaf plate edge is grown cuts from base portion and leaf dish, and young shoot is inserted to root induction in root media, each seedling is numbered to 28 DEG C of left and right, 16 hours illumination cultivation.
4, transgene tobacco Whitfield's ointment (SA), methyl jasmonate (MeJA) are processed
The transgenic tobacco plant blade of five promoter fragments in tobacco gene promotor Sm-NgtP carries out respectively SA (1mmolL
-1), MeJA (1mmolL
-1) induction processing, and establish control group (distilled water).
5, GUS determination of activity
The fluoroscopic examination of GUS activity is with reference to Jefferson method.
1) QB method is extracted protein: mill is even rapidly in the grinding that adds 500 μ L QB extracting solutions for cut≤0.1g sample, move in ice bath 1.5mL ep pipe, then 4000rpm4 DEG C of centrifugal 10min, get supernatant, 4 DEG C of preservations.
2) GUS enzyme is lived and is reacted: 4 μ L protein supernatants add in 100 μ LGUS reaction solutions, mix, and are statically placed in 37 DEG C of water-baths and react 15min; Then add 900 μ L reaction terminating termination reactions at night, mix rapidly rear mensuration fluorescent value.
3) mensuration of GUS enzyme activity: the content of Xylene Brilliant Cyanine G method working sample 4-MU; GUS enzyme activity unit is defined as: the enzyme amount that every milligram of protein, every min hydrolysis 4-MUG generate 1pmol4-MU is a unit of activity, obtains the enzyme activity of each sample according to definition, and GUS activity unit is pmol4-MU mg
-1protein min-1.
Gus gene under final five kinds of promoter fragment controls and the control group expression activity figure under jasmonic and Whitfield's ointment processing as shown in Figure 3, as can be seen from Figure 3, promoter fragment GTPC is the promotor that is subject to methyl jasmonic acid and the dual induction of Whitfield's ointment, and GTPD is composing type strongly expressed promotor.
Above embodiment is only the utilization of the present invention in tobacco, and those skilled in the art can utilize Sm-NgtP promotor provided by the present invention and each promoter fragment structure to be subject to the expression vector conversion of plant of methyl jasmonic acid (MeJA) and the dual induction of Whitfield's ointment (SA) to improve the abduction delivering of goal gene.Recipient plant can be other Important Economic crop of cereal crop including paddy rice, wheat, corn etc. and some, for example cereal grass, cotton, tobacco, rape and eggplant class plant.
Claims (6)
1. a tobacco sugar based transferase inducible promoter Sm-NgtP, is characterized in that: the nucleotide sequence of this promotor Sm-NgtP is as shown in SEQ ID NO.1.
2. Accessory Right requires described in 1 an isolated promoter fragment GTPC in promotor Sm-NgtP, it is characterized in that: the nucleotide sequence of this promoter fragment is as shown in SEQ ID NO.2.
3. a carrier for expression of eukaryon pGTPC, is characterized in that: the promotor in described carrier for expression of eukaryon is promoter fragment GTPC described in claim 2.
4. Accessory Right requires described in 1 an isolated promoter fragment GTPD in promotor Sm-NgtP, it is characterized in that: the nucleotide sequence of this promoter fragment is as shown in SEQ ID NO.3.
5. a carrier for expression of eukaryon pGTPD, is characterized in that: the promotor in described carrier for expression of eukaryon is promoter fragment GTPD described in claim 2.
6. a method of utilizing vegetable cell to express goal gene, it is characterized in that comprising the following steps: with the carrier for expression of eukaryon pGTPC described in claim 3 or the carrier for expression of eukaryon pGTPD transformed plant cells described in claim 5 cultivation, then adopt methyl jasmonic acid and Induced by Salicylic Acid vegetable cell to express goal gene; Described plant is cereal grass, or is any in cotton, tobacco, rape and eggplant class plant.
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CN104818258A (en) * | 2015-03-04 | 2015-08-05 | 中国农业科学院棉花研究所 | Gossypium hirsutum glycosyltransferase GhUGT85O1, coding gene and applications thereof |
CN107254469A (en) * | 2017-06-06 | 2017-10-17 | 中南民族大学 | The promoter SmP of one plant vasular beam specifically expressing clone and application |
KR20170142222A (en) * | 2016-06-16 | 2017-12-28 | 주식회사 바이오에프디엔씨 | Recombinant Protein Expression Vector in Plant cell and the Method for Preparing the Protein |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104818258A (en) * | 2015-03-04 | 2015-08-05 | 中国农业科学院棉花研究所 | Gossypium hirsutum glycosyltransferase GhUGT85O1, coding gene and applications thereof |
CN104818258B (en) * | 2015-03-04 | 2018-01-30 | 中国农业科学院棉花研究所 | Upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application |
KR20170142222A (en) * | 2016-06-16 | 2017-12-28 | 주식회사 바이오에프디엔씨 | Recombinant Protein Expression Vector in Plant cell and the Method for Preparing the Protein |
KR102649733B1 (en) | 2016-06-16 | 2024-03-21 | 주식회사 바이오에프디엔씨 | Recombinant Protein Expression Vector in Plant cell and the Method for Preparing the Protein |
CN107254469A (en) * | 2017-06-06 | 2017-10-17 | 中南民族大学 | The promoter SmP of one plant vasular beam specifically expressing clone and application |
CN107254469B (en) * | 2017-06-06 | 2020-10-16 | 中南民族大学 | Cloning and application of promoter SmP specifically expressed by plant vascular bundle |
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