CN102134568B - Promoter for MADS-box gene in maize and application thereof - Google Patents
Promoter for MADS-box gene in maize and application thereof Download PDFInfo
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Abstract
The invention discloses a promoter for MADS-box gene in maize and application thereof. The promoter for the MADS-box gene in maize can respond to various environmental stress signals, and has the nucleotide sequence shown as a sequence 1 in a sequence table, or a DNA sequence of a promoter of a gene having over 85 percent of consistence with the sequence 1; and the promoter is used for constructing vectors for inducing response to one or more of drought or deaquation, abscisic acid (ABA, ABscisic Acid), potassium ion (K+) inflow, coldness, salt, germs, light, sugar, heavy metals and wounds, so as to be applied to breeding transgenic stress-resistant plants.
Description
Technical field
The present invention relates to the promotor of plant MADS-box gene, relate in particular to the promotor and the application thereof of corn MADS-box gene.
Background technology
Corn is one of global of paramount importance food crop.Arid is the essential environmental factors of the global Maize Production of restriction.According to conservative estimation, only in the torrid areas, because the corn that arid causes loss reaches 2,000 ten thousand tons every year at least, rate of loss is at least 17%.According to another conservative estimation, corn accounts for 21.1% of China total output of grain state food crop output formation.For many years, in national Program for Tackling Key Problems, maize breeding is always stressed high-quality, high yield, multiresistance, but reality is to be unalterable quota with the corn yield when examination, and this objectively will induce the breeding expert to ignore the quality and the flexibility of corn breeding.In addition, the drought-resistant character of corn is the quantitative character of a controlled by multiple genes, and drought resisting mechanism is very complicated, and is therefore limited to its understanding, in the difficulty that has objectively caused the drought-resistant maize breeding again.
For a long time, place hope on drought both at home and abroad and coerce the evaluation of responsive genes and expression study is resolved corn as the breach drought resisting mechanism thereof.Development and application along with DNA chip or microarray technology; Found that a large amount of plant droughts coerce responsive genes and range gene express spectra; These expression of gene have when tangible, empty specificity, tissue specificity and condition specificity; Form the network regulation system of a complicacy, still do not make proper explanations but express regulation mechanism for these responsive genes.
Big quantity research shows, genetic expression the time, empty specificity, tissue specificity and condition specificity depend on the promotor of gene to a great extent.The formation of heterogeneic promotor changes greatly, for example it is generally acknowledged that the promotor by the identification of Yeast Nucleic Acid (RNA, Ribonucleic Acid) polymerase II contains a TATA box and or initial startup factor of control specific transcriptional.For many years; The TATA box is considered to control the main core parts of basal transcription mechanism; Contain one but the promotor of many other genes lacks the TATA box element and start the factor; Even same promotor because the variation (epigenetic alteration) of the inner back life of promotor, for example methylates or the supermethylation meeting causes the heterogeneic effect of transcribing.In addition, many genes, particularly some family genes do not have significant difference on domain; But its transcription is but very different, the thermal excited transcryption factor HSF1 of for example human and muroid, HSF2; HSF3 and HSF4; Wherein HSF1 is relevant with the expression of HSP with HSF3, and HSF2 does not have any reaction to the stresser of classics, though HSF4 is similar with HSF2 with HSF1; But express with the tissue specificity mode, one of reason is that the heat shock element in these thermal excited transcryption factors HSFs promotor separately is different.
In addition, because the for example degeneration-resistant proterties of many proterties of corn receives controlled by multiple genes, rely on to transform the systemic resistance that term single gene is difficult to realize the corn complete stool.The research proof; Transform a spot of gene and be difficult to give the resistance trait that the crop accords with production needs, also attempt in the world transforming the gene more than 2 simultaneously, in addition; Owing to be subject to present transgenic technology, the winner that trial transforms 2 above genes simultaneously is very few.
The research to the promotor of corn gene has both at home and abroad obtained certain progress.On January 7th, 2009, the plant promoter of including in (The Eukaryotic Promoter Database) in the promotor private database has 198, and wherein the promotor of corn has 21.21 corn promotors having reported have comprised 11 alcohol soluble protein gene promotors; The promotor of 2 light harvesting protein genes; 1 sucrose synthase gene promotor, 1 uridine diphosphate(UDP) (UDP, Uridine DiphosPhate)-glucose-starch Transglucosylase gene promoter; 3 cyanidin(e) synthesis related gene promotors, 2 alcohol dehydrogenase gene promotors and 1 HSP memory promotor.
At present, pay attention to as yet for the promotor research of the plant transcription factor of coercing response (TFs, Transcription Factors) gene both at home and abroad, do not coerce the research report of promotor of the TFs gene of response about the drought of corn.In the research in early days; EST (the ESTs of corn expressing gene is coerced down in this laboratory to drought; Expressed Sequence Tags) carried out the large scale sequencing analysis, made the cDNA microarray of 11855 genes; With this microarray large scale analysis drought coerce the genetic expression of corn in following seedling stage, found that drought coerces down the gene that the TFs with remarkable up-regulated expression characteristic is one type of MADS-box (data not shown).The gene of MADS-box is proved in the Eukaryotic growth control of nearly all research and signal conduction and works.Therefore, the clone of the gene promoter of corn MADS-box provides theoretical foundation for further illustrating corn adversity gene expression network adjustment and drought resisting mechanism, in transgenic corns drought resisting breeding, also has its using value.
Reference:
Li Wanchen, Rong Tingzhao (2000) China 21 century maize genetic breeding engineering prospect, corn science 8:10-14.
Xu Yawei, Chai Xiaojie, Wang Piwu, Zuo Yanting, the clone and the expression vector establishment of Lv Pin (2006) corn starch branching enzyme sbe II b gene promoter, corn science, 14:84-87.
Zhang Shihuang, Hu Ruifa (2000) maize breeding induce innovation factor, corn science, 8:3-7.
Becker?A，Theissen?G(2003)The?major?clades?of?MADS-box?genes?and?theirrole?in?the?development?and?evolution?of?flowering?plants.Mol?Phyl?Evol?29，464-489.
Bray EA，Bailey-Serres J，Weretilnyk?E?(2000)Responses?to?abioticstresses.Chapter?22.In?W?Gruissem，B?Buchannan，R?Jones，eds，Responses?toAbiotic?Stresses.American?Society?of?Plant?Physiologists，Rockville，MD，pp1158-1249.
Bruce?WB，Edmeades?GO，Barker?TC(2002)Molecular?and?physiologicalapproaches?to?maize?improvement?for?drought?tolerance.J?Exp?Bot?53：13-25.
Comelli?RN，Viola?IL，Gonzalez?DH(2009)Characterization?of?promoterelements?required?for?expression?and?induction?by?sucrose?of?the?ArabidopsisCOX5b-1?nuclear?gene，encoding?the?zinc-binding?subunit?of?cytochrome?c?oxidase.Plant?Mol?Biol.Jan?1.
Durieux?AC，Bonnefoy?R，Freyssenet?D(2005)Kinetic?of?transgeneexpression?after?electrotransfer?into?skeletal?muscle：importance?of?promoterorigin/strength.Biochim?Biophys?Acta.1725(3)：403-409.
Hoagland?DR，Arnon?DA(1938)The?water-culture?method?of?growing?plantswithout?soil.California?Agricultural?Experiment?Station?Circular.347：1-32.
Hoisington?D(2001)Application?of?biotechnology?to?maize?improvement：Past，Present?and?Future?Prospects.Seventh?Eastern?and?Southern?Africa?RegionalConference，pp.7-11.
Jefferson?RA(1987)Assaying?chimeric?genes?in?plants：the?GUS?gene?fusionsystem.Plant?Mol?Biol?Rep，5：387-405.
Kim?K?N，Fisher?DK(1998)Molecular?cloning?and?characterization?of?theAmylose-Extender?gene?encoding?starch?branching?enzyme?II?B?in?maize.Plant?Mol?Biol，38：945-956.
Liu?YG，Mitsukawa?N，Oosumi?T，et?al.(1995)Efficient?isolation?andmapping?of?Arabidopsis?thaliana?T-DNA?insert?junctions?by?thermal?asymmetricinterlaced?PCR.Plant?J.8(3)：457-462.
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P, Sistonen L (2001) Roles of the heat shock transcriptionfactors in regulation of the heat shock response30: the PCR product that chromosome walking is produced is connected with the plant gene expression vector that contains reporter gene;
and?beyond.FASEB?J.15(7)：1118-1131.
Wang?W，Vinocur?B，Altman?A(2003)Plant?responses?to?drought，salinity?andextreme?temperatures：towards?genetic?engineering?for?stress?tolerance.Planta218：1-14
Welchen?E，Viola?IL，Kim?HJ，Prendes?LP，Comelli?RN，Chan?Hong?J，Gonzalez?DH(2008)A?segment?containing?a?G-box?and?an?ACGT?motif?confersdifferential?expression?characteristics?and?responses?to?the?Arabidopsis?Cytc-2?gene，encoding?an?isoform?of?cytochrome?c.J?Exp?Bot.Dec?19
Wong?J，Liang?V?CT，Sachs?L?M.，and?Shi?YB(1998)Transcription?from?thethyroid?hormone-dependent?promoter?of?the?Xenopus?laevis?thyroid?hormonereceptor?bA?gene?requires?a?novel?upstream?element?and?the?initiator，but?not?aTATA?box.Journal?of?Biological?Chemistry，273：14186-14193.
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Summary of the invention
Technical problem to be solved by this invention provides a kind of promotor and application thereof of corn MADS-box gene, can respond multiple environment-stress signal, to make up the expression vector of multiple environment-stress signal induction response.
In order to solve the problems of the technologies described above, the invention provides a kind of corn MADS-box gene promoter is one of following nucleotide sequences:
(1) dna sequence dna of sequence 1 in the sequence table;
(2) dna sequence dna with said sequence 1 qualification has conforming dna sequence dna more than 85%;
The MADS-box gene promoter is predicted transcription initiation site through adopting neural network promotor predictor version 2 .2, and utilizes this promotor of method forecast analysis that provides in PlantCARE and the PLACE DB to contain the dna sequence dna element of following multiple environment-stress signal response:
3 lack of water response elements;
1 lack of water or dormin or cold response element;
1 wound response element;
1 potassium ion flows into controlling elements;
1 ABA response element;
1 enhancer element that improves genetic expression intensity;
1 copper response element;
1 pathogenic bacteria or salt response element;
2 light response elements;
1 sugared response element;
1 pathogenic bacteria response element;
1 cold or lack of water response element;
4 elements relevant with transcription rate wherein have 3 TATA boxes and 1 CAAT box.
In order to solve the problems of the technologies described above, the invention provides the application of foregoing corn MADS-box gene promoter aspect the breeding of transgenic adversity resistant plant.
Preferably, said gene promoter is used for making up and comprises arid or lack of water, dormin (ABA), potassium ion (K
+) flow into, the carrier of one or more evoked responses in cold, salt, germ, light, sugar, heavy metal and the wound, thereby be applied to the breeding of said transgenic adversity resistant plant.
0 [0048]Fig. 1 uses the method embodiment schema of chromosome walking corn clone MADS-box gene promoter for the present invention;
Description of drawings
Fig. 2 is the chromosomal dna fragment of chromosome walking pcr amplification MADS-box upstream region of gene;
Fig. 3 is the response element that contains of the dna sequence dna of the present invention clone's corn MADS-box gene promoter (Pro66) and predictability thereof;
The method that Fig. 4 makes up for the expression that detects corn MADS-box gene promoter;
Fig. 5 is startup glucuronidase (GUS, the glucuronidase) histochemical stain of the genetic expression detection of the Pro66 of amplification.
The promotor of corn MADS-box gene provided by the invention is a kind of promotor that can respond multiple environment-stress signal, is used for making up comprising arid or lack of water, dormin (ABA, ABscisic Acid), potassium ion (K
+) flow into, the carrier of cold, salt, germ, light, sugar, heavy metal and wound-induced response, thereby be applied to the breeding of transgenic adversity resistant plant.
Embodiment
Specifically, the present invention is to provide a kind of corn that comes from can be by the promotor of the MADS-box gene of lack of water abduction delivering, and this promotor contains arid or lack of water, ABA, K
+Flow into, the dna sequence dna element of cold, salt, germ, light, sugar, heavy metal and wound response, thereby may receive the expression of inducing promotor gene under the corresponding stress conditions plant.This promotor does not almost have homology with existing gene promoter of having reported for work, thereby is the promotor of the newfound a kind of plant gene of the present invention.
Below in conjunction with accompanying drawing and preferred embodiment technical scheme of the present invention is at length set forth.The embodiment that below gives an example only is used for explanation and explains the present invention, and does not constitute the restriction to technical scheme of the present invention.
Used in an embodiment of the present invention material comprises:
MADS-box expression of gene sequence label (ESTs) sequence is from this laboratory corn EST order-checking planning studies problem, and is filed in the gene pool (GenBank, call number is EC864166) of the U.S. state-run biotechnology information center (NCBI).
Intestinal bacteria (Escherichia coli) strain DH5 α preserves for this laboratory.
Restriction enzyme, T4 ligase enzyme and pcr amplified dna cloning vector pGEM-T easy Vector are available from Promega company.
X-gluc (import packing) and gel DNA reclaim test kit available from perseverance because of biotech firm.
DNA Marker, general T aq enzyme etc. are available from Fermentas company.
Glue recovery test kit and PCR product purification test kit are available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The helper plasmid pRK2073 that between thalline, shifts as helper plasmid DNA during Agrobacterium (Agrobacterium tumefacien) bacterial strain LBA4404, plant expression vector pCambia1301 and three parents combine collects preservation for this laboratory.
Chromosome walking test kit Genome Walking Kit is available from TAKARA company; The chromosomal localization of the EST of corn MADS-box gene adopts the BLASTN-HTG program in the DB of Plant Genome website (PlantGDB, its network address is http://www.plantgdb.org/).
The dna sequencing of MADS-box gene promoter entrusts Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to carry out.The prediction of genetic transcription initiation site adopts NNPP version 2.2 programs (http://www.fruitfly.org/seq_tools/promoter.html) to carry out.The take advantage of a situation controlling element prediction of plant gene promoter routine property is carried out according to the method that provides in PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE (Plant Cis-acting Regulatory DNA Elements, the http://www.dna.affrc.go.jp/PLACE/) DB.
Water culture is adopted in the cultivation of plant, and nutrient solution is Hoagland solution (Hoagland and Arnon1938).
Agrobacterium and intestinal bacteria substratum be common LA (peptone 10 grams, yeast powder 5 grams, NaCl 5 grams, agar powder 15 grams, 1000 milliliters in water, pH7.0) and LB (LA that does not add agar powder) substratum.
3 used in first round chromosome walking pcr amplification Auele Specific Primers are:
SP1：5’-CCTGAGCTGAATCTGCATGTTGTCG-3’，
SP2：5’-CCATTCGTCTTGTGCATC?CTCCAG-3’，
SP3：5’-TATCCGATC?TCCAGCTCC?CTCATG-3’。
Second takes turns the step moves 3 used in pcr amplification Auele Specific Primers and is:
Pro-SP1：5’-TGGGTCTTTGCGATCAGTTTTGCTTG-3’，
Pro-SP2：5’-AATGAGAGGGGCAATGGACAGA?CGAG-3’，
Pro-SP3：5’-TGCCGGTTGGTAGAGTTCTCGATC-3’。
The 3rd takes turns the specific PCR primer to doing
ProF：5’-GGGGTCGACTTGCCGGTTGGTAG?AG-3’，
ProR：5’-GGGCCATGGACTGCG?TTCTCCACTC-3’
The method that adopts Jefferson (1987) detects in the histological chemistry of genetic expression.
As shown in Figure 1, the method embodiment flow process for corn clone MADS-box gene promoter of the present invention comprises the steps:
10: the est sequence of corn MADS-box gene is positioned on the karyomit(e) of corn;
According to the free BLASTN-HTG programanalysis in PlantGDB (http://www.plantgdb.org/) DB, the est sequence of corn MADS-box gene is positioned on No. 8 karyomit(e)s of corn.
20: with localized chromosome walking;
The est sequence of MADS-box gene once was positioned on No. 8 karyomit(e)s of corn; Carry out the chromosome walking pcr amplification according to localized maize chromosome dna sequence dna; This chromosome walking pcr amplification adopts TAIL-PCR (Liu et al.; 1995) amplification method carries out, and the dna fragmentation that at every turn amplifies sees also Fig. 2.
Shown in Fig. 2 A; Be first round chromosome walking: the coding region sequence according to No. 8 that are positioned at corn on chromosomal is according to reverse downstream primer of 3 ' → 5 ' direction design, and with have primer that 5 ' → 3 ' direction random primer forms first round PCR to carrying out first round pcr amplification.
Shown in Fig. 2 B; Be second to take turns chromosome walking: according to the sequence of the DNA of first round PCR near 5 ' end, according to reverse downstream primer of 3 ' → 5 ' direction design and with have 5 ' → 3 ' direction random primer form second take turns PCR primer take turns pcr amplification to carrying out second.
Shown in Fig. 2 C, be the upper reaches of the target gene on karyomit(e) amplification successively as stated above, promptly taking turns the TAIL-PCR product that produces in the chromosome walking with the 2nd is template, carries out the specific PCR amplification with sequence specific primers.
M representes 1kb dna ladder type (DNA Ladder) among Fig. 2; The 1st, 2,3 corresponding amplified productions among swimming lane on the topic head 1,2, the 3 expression TAIL-PCR among A and the B among Fig. 2.The negative control of the pcr amplification that swimming lane 1 on the topic head among Fig. 2 among the C and 2 expressions are done with PCR reaction solution and sequence specific primers; Swimming lane 3 and 4 expressions be that to take turns the TAIL-PCR product that produces in the chromosome walking with the 2nd be template, the specific PCR amplified production that carries out with sequence specific primers.
M representes 1kb dna ladder type (DNA Ladder) among Fig. 2; The 1st, 2,3 corresponding amplified productions among swimming lane on the topic head 1,2, the 3 expression TAIL-PCR among A and the B among Fig. 2.The negative control of the pcr amplification that swimming lane 1 on the topic head among Fig. 2 among the C and 2 expressions are done with PCR reaction solution and sequence specific primers; Swimming lane 3 and 4 expressions be that to take turns the TAIL-PCR product that produces in the chromosome walking with the 2nd be template, the specific PCR amplified production that carries out with sequence specific primers.
30: the PCR product that chromosome walking is produced is connected with the plant gene expression vector that contains reporter gene;
40: the examining report expression of gene, and the dna fragmentation of the promotor of corn MADS-box gene identified.
Above-mentioned evaluation comprises to be predicted the dna sequence dna and the contained response element sequence thereof of clone's corn MADS-box gene promoter.
Obtained the dna fragmentation (shown in Fig. 2 C) of one section sequence-specific pcr amplification according to method flow shown in Figure 1, promptly the dna fragmentation of corn MADS-box gene promoter is confirmed its long 1061bp through order-checking.Adopt neural network promotor predictor version 2 .2 (Neural Network Promoter Prediction; NNPP version 2.2) prediction MADS-box gene transcription initiation site; Utilize the method forecast analysis that provides in PlantCARE and the PLACE DB; Find that this DNA section comprises the dna sequence dna element of following response, as shown in Figure 3:
3 lack of water response (dehydration-responsive) elements;
1 lack of water or ABA or cold response (dehydration, ABA, cold-responsive) element;
1 wound response (wounding-responsive) element;
1 K
+Flow into control (K
+Influx) element;
1 ABA response (ABA-responsive) element;
1 enhanser (enhancer) element that improves genetic expression intensity;
1 copper response (copper-responsive) element;
1 pathogenic bacteria or salt response (pathogen, salt-responsive) element;
2 photoresponses (light-responsive) element;
1 sugar response (sugar-responsive) element;
1 pathogenic bacteria response (pathogenesis-responsive) element;
1 cold or lack of water response (cold, dehydration-responsive) element;
4 elements relevant, wherein 3 TATA boxes (TATA box) and 1 CAAT box (CAAT box) with transcription rate.
The dna sequence dna of corn MADS-box gene promoter is shown in sequence 1.
The commentaries on classics beta-Glucuronidase (GUS, β-Glucuronidase) structure and the gus gene detection of expression thereof of expression vector that have corn MADS-box gene promoter (Pro66)
With the dna clone of the plan promotor (Pro66) of pcr amplification among the embodiment 1 after on the pGEM-T easy Vector plasmid vector, again through Sal I/Nco I double digestion and reclaim corresponding endonuclease bamhi.Gene expression in plants plasmid vector pCAmbia1301 removes the preceding 35S promoter DNA of gus gene (gus) in this expression vector through Sal I/Nco I double digestion.The dna fragmentation of the plan promotor Pro66 that under the ligase enzyme effect, Sal I/Nco I double digestion is also reclaimed connects and is cloned among the pCAmbia1301 of Sal I/Nco I double digestion, and constructing with gus is the new recombinant expression plasmid of reporter gene.Wherein whether the expression of gus reporter gene depends on whether the dna fragmentation that is connected this upstream region of gene has the startup effect of genetic expression.Use the same method the recombinant plasmid of the non-promoter DNA that constructs the one section 672bp that has peanut as the negative control plasmid, and the methods and strategies of structure is referring to Fig. 4.
The recombinant plasmid that constructs combines (bacillus coli DH 5 alpha that contains helper plasmid pRK2073 contains between the bacillus coli DH 5 alpha and Agrobacterium LBA4404 thalline of recombinant plasmid) to import Agrobacterium LBA4404 thalline through three parents.Utilize disposable syringe (to have 4
#Syringe needle) will be cultured to OD
600=0.6 have recombinant plasmid Agrobacterium LBA4404 thalline be injected into corn vein in tri-leaf period.26 ℃ of 1/5Hoagland nutritive mediums, 12h illumination/24h cultivates and carries out histochemical stain according to the method for Jefferson (1987) after 2 days.
The histochemical stain concrete grammar is: will be soaked in X-gluc staining fluid (100mM (pH 7.0) phosphoric acid buffer; 0.1% (V/V) Triton-X 100; 10mM EDTA; 0.5mg/ml X-Gluc; 1% (V/V) DMSO 99.8MIN.) in, 37 ℃ are spent the night, 70% ethanol decolorization.The tissue staining result sees Fig. 5.
Wherein:
A: negative control.Utilize one section non-promoter DNA of 672bp of peanut to replace the upward plasmid construction of the upper reaches CaMV35S promotor of gus gene of pCambia1301.
B: primary pCambia1301 positive control.
C: the corn MADS-box gene promoter (Pro66) that the chromosome walking pcr amplification is gone out.The Pro66 promotor (containing initiator codon, length overall 1068bp) of amplification has replaced the upward structure of the upper reaches CaMV35S promotor of gus gene of expression of plants pCambia1301.
Detect the agriculture bacillus mediated method that infects the transient expression and the conjunctive tissue chemical staining of maize leaf that adopts.The result of histochemical stain referring to Fig. 5 C, shows that Pro66 can start the expression of reporter gene gus, is a real promotor.
The purpose of present embodiment is the expression that checking corn MADS-box gene promoter Pro66 can start reporter gene gus, is a real promotor.
Claims (1)
1. a corn MADS-box gene promoter is the dna sequence dna of sequence 1 in the sequence table.
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| CN105441459A (en) * | 2015-12-29 | 2016-03-30 | 上海大学 | Corn transcription factor ZmMaDS47 gene and application thereof |
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| CN101107364A (en) * | 2004-11-25 | 2008-01-16 | 克罗普迪塞恩股份有限公司 | Plants having increased yield and a method for making the same |
| CN101200723A (en) * | 2007-10-18 | 2008-06-18 | 复旦大学 | Butterfly orchid PhPI9 gene coded sequence and uses thereof |
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| CN1769457A (en) * | 2005-09-15 | 2006-05-10 | 复旦大学 | Butterfly orchid pPI15 encoding sequence and its uses |
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