CN101665532B - Cotton disease resistance related transcription factor MEREB1 as well as coding gene and application thereof - Google Patents

Abstract

The invention discloses a protein. The provided protein is named MEREB1 and derived from sea-island cotton and is the protein shown as the following (1) or (2): (1) a protein comprising an amino acid sequence shown as a sequence 2 in a sequence table; and (2) a protein which is formed by substituting and/or lacking and/or adding one or several amino acid into the amino acid sequence of the sequence 2 in the sequence table and is related to plant disease resistance and derived from the (1). A coding gene of the protein is led into tobacco or cotton, and the expression number of disease resistance related genes in an obtained transgenic plant is improved. The MEREB1 protein can be specifically combined with GCC-box, thereby activating the expression of a target gene. The MEREB1 protein is a disease resistance related transcription factor and can be widely applied to culture a disease resistant plant.

Description

Cotton disease resistance related transcription factor MEREB1 and encoding gene thereof and application

Technical field

The present invention relates to biological technical field, particularly cotton disease resistance related transcription factor and encoding gene thereof and application.

Background technology

Cotton is one of most important cash crop of China, but verticillium is very serious to the influence of cotton planting, makes the yield and quality of cotton be subjected to considerable damage; For addressing this problem, people attempt utilizing molecular biological means to improve the disease resistance of cotton self.In recent years, discover that ERF class transcription factor is significant in plant stress-resistance mechanism and plant molecular improvement.ERFs has participated in plant to biological and abiotic stress responsing reaction, has brought into play crucial regulating and controlling effect in signal transduction pathways such as ethene.Studies show that, thereby thereby ERFs can take place to make to activate the pathogenesis-related proteins expression of gene mutually with cis-acting elements such as GCC-box improves the plant disease-resistant ability.Therefore, ERF class transcription factor can be applicable to farm crop to improvement biological and anti-(anti-) property of abiotic stress.

Summary of the invention

The object of the present invention is to provide a kind of albumen that can strengthen the plant disease-resistant related gene expression.

Albumen provided by the invention, called after MEREB1 derives from sea island cotton, is following 1) or 2) albumen:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in sequence table the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several amino acid and relevant with plant disease-resistant have 1) deutero-protein.

In order to make 1) in MEREB1 be convenient to purifying, label as shown in table 1 on proteinic N-terminal that can the aminoacid sequence shown in the sequence 2 is formed in by sequence table or C-terminal connect.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tagII	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned 2) but in the MEREB1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding gene of the MEREB1 in can be by lacking sequence in the sequence table 1 codon of one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 76-840 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The protein that sequence 2 amino-acid residues are made up of 255 amino-acid residues in the sequence table.To the 138th of carbon teminal to the 196th amino acids residue conserved domain the conservative territory of ERF function of transcription factor MEREB1 from the nitrogen end in the described sequence 2.

The above-mentioned proteic cDNA that encodes, called after MEREB1 is also in protection scope of the present invention.

Encoding gene gene provided by the invention is following 1) or 2) or 3) or 4) gene:

1) its encoding sequence be in the sequence table sequence 1 from the gene of 5 ' terminal 76-840 position;

2) its encoding sequence is the gene of sequence 1 in the sequence table;

3) under stringent condition with 1) or 2) gene recombination that limits and the nucleotide sequence of coding and plant disease resistance-related protein;

4) with 1) or 2) gene that limits has the homology more than 90%, and the coding nucleic acid molecule relevant with plant disease-resistant.

Described stringent condition is meant, with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.2,7%8DS) in, 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7%SDS, isotope-labeled nucleotide fragments), 65 ℃ of hybridization 12hr; Abandon hybridization solution, (20mmol/L sodium phosphate buffer, pH7.2 5%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid I; (20mmol/L sodium phosphate buffer, pH7.2 1%SDS), wash film 30min for 65 ℃ to add film washing liquid II.

Above-mentioned 4) the gene gene in and 1) preferably has the homology more than 95%.

MEREB1 gene cDNA total length is 1165 based compositions in the sequence table, and open reading frame is from 3 ' end the 76th to 840 bit base, and it expresses inducing of fungies such as mainly being subjected to verticillium wilt pathogen or mycotoxins.

Amplification MEREB1 full length gene or arbitrary segmental primer are to also belonging to protection scope of the present invention.

The recombinant vectors that contains said gene also belongs within protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of MEREB1 gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, pBY505 or other plant expression vector of deriving.

When using the gene constructed recombinant expression vector of MEREB1, can before its transcription initiation Nucleotide, add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin (Ubiquitin) gene promoter (pUbi), Actin promotor etc., they can use separately or be used in combination with other plant promoter.

In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as be added in the plant to express and to produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.), have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.

Above-mentioned recombinant vectors specifically is the multiple clone site with above-mentioned gene insertion vector pBI121, the recombinant vectors that obtains.

The reorganization bacterium that contains said gene also belongs within protection scope of the present invention.

The transgenic cell line that contains said gene also belongs within protection scope of the present invention.

Another object of the present invention is to provide a kind of method that disease-resistant related gene is expressed the enhanced transgenic plant of cultivating.

A kind of method that disease-resistant related gene is expressed the enhanced transgenic plant of cultivating provided by the invention is above-mentioned gene to be imported in the purpose plant obtain transgenic plant, and the expression amount of the disease-resistant related gene in the described transgenic plant is higher than the purpose plant.

Above-mentioned gene is to import in the purpose plant by above-mentioned recombinant vectors; Described plant is dicotyledons or monocotyledons; Described dicotyledons is cotton, tobacco, Arabidopis thaliana, soybean or rape; Described monocotyledons is paddy rice or wheat.Carry MEREB1 gene of the present invention plant expression vector can by Ti-plasmids, Ri plasmid, plant viral vector, as particle bombardment, pollen tube channel, microinjection, electricity lead, conventional biological method such as agriculture bacillus mediated is transformed in vegetable cell or the tissue.

Above-mentioned disease-resistant related gene is chitinase gene, β-1,3-dextranase gene or PR protein gene; Described chitinase gene be Genbank number for GHU60197 from 5 ' end the 690th to the 1302nd shown in gene, the beta-1,3-glucanase gene be Genbank number for Z68154 from the gene PR protein gene of 5 ' end shown in the 301st to the 809th be Genbank number for AF305066 from the gene shown in the 47th to the 526th at the 5 ' end.

Above-mentioned plant is dicotyledons or monocotyledons; Described dicotyledons is cotton, tobacco, Arabidopis thaliana, soybean or rape; Described monocotyledons is paddy rice or wheat.

Another purpose of the present invention is to provide a kind of method of cultivating disease-resistant transgenic plant.

A kind of method of cultivating disease-resistant transgenic plant provided by the invention is that above-mentioned gene is imported in the purpose plant, obtains transgenic plant.

Above-mentioned gene is to import in the purpose plant by above-mentioned recombinant vectors.Carry MEREB1 gene of the present invention plant expression vector can by Ti-plasmids, Ri plasmid, plant viral vector, as particle bombardment, pollen tube channel, microinjection, electricity lead, conventional biological method such as agriculture bacillus mediated is transformed in vegetable cell or the tissue.

Above-mentioned disease-resistant transgenic plant are transgenic plant of resisting verticillium

Above-mentioned plant is dicotyledons or monocotyledons; Described dicotyledons is cotton, tobacco, Arabidopis thaliana, soybean or rape; Described monocotyledons is paddy rice or wheat.

MEREB1 albumen of the present invention contains the AP2/ERF structural domain of a high conservative, a nuclear localization signal and an activation domain, belongs to the newcomer of AP2/ERF class family transcription factor.The MEREB1 gene is all expressed in Radix Gossypii, stem and leaf, and the MEREB1 expression of gene is induced by verticillium wilt pathogen simultaneously, and the mRNA accumulative speed of MEREB1 is very fast.EMSA experiment showed, that MEREB1 albumen can gel blocking can take place with the GCC-box probe mixture, and can not block with mutant GCC-box probe, and this explanation MEREB1 albumen can be specifically in conjunction with GCC-box, thus the expression of activating genes of interest.Change PR gene, chitinase gene and β-1 in the MEREB1 genetic tobacco body simultaneously, the expression amount of 3-dextranase gene obviously improves.MEREB1 albumen can strengthen the plant disease-resistant Expression of Related Genes, can be used widely in cultivating disease-resistant plants.

Description of drawings

Fig. 1 is the changing conditions of expression amount before and after MEREB1 induces in each tissue, is reference with UBQ7, and wherein a is MEREB1 expression in each tissue before inducing; B is the expression of MEREB1 in inducing each tissue of back.

Fig. 2 is MEREB1 probe and genomic dna Southern blotting figure.

Fig. 3 is the design of graphics of prokaryotic expression carrier pGEX4T-1/EREB1.

Fig. 4 is a binary expression vector pBI121/MEREB1 design of graphics.

Fig. 5 is fusion rotein GST/EREB1 and the combining of GCC-box probe.

Fig. 6 is transgenic plant, and wherein scheming A is the tobacco that changes pBI121/MEREB1; Figure B is the upland cotton that changes pBI121/MEREB1; C is that the PCR that changes the tobacco plant of pBI121/MEREB2 detects CK: wild-type plant amplification, 1-5: positive plant amplification, M:Marker DL2000.

Fig. 7 analyzes PR genetic expression in the tobacco that changes pBI121/MEREB1 under the normal growth condition for qRT-PCR, wherein Fig. 7 A is that PR1a expresses; Fig. 7 B is the expression of PR2; Fig. 7 C is the expression of PR5.

Fig. 8 changes the expression of interior each disease-resistant gene of cotton plants body of pBI121/MEREB1 down for the ordinary student long status.

Embodiment

The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.

Among the following embodiment, if no special instructions, be ordinary method.Agents useful for same all can obtain from commercial channels.

The sequential analysis of the cDNA of embodiment 1, cotton disease resistance related transcription factor MEREB1, copy number analysis and expression pattern analysis

One, the sequential analysis of the cDNA of cotton disease resistance related transcription factor MEREB1

Use Promega The total RNA extraction reagent box extracts total RNA of cotton (new extra large 20 (becoming company available from academy of agricultural sciences, Xinjiang cash crop institute the sky)) seedling in two weeks of growth, with Promaga reverse transcription test kit examination carrying out reverse transcription.

With the conservative territory design degenerated primers of ERF family transcription factor, MHF1:5-TA (C/T) AG (A/G) GGTIAG (A/G)-3; MHR1:5 '-IGG (A/G) AA (A/G) TIAG (C/T) CTIGC-3; I is time yellow sweet acid (inosine), and the PCR condition is 94 ℃, 3 minutes; 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 90 seconds-30 the circulation; 72 ℃ 10 minutes; 4 ℃ of preservations.The PCR product is connected to and is used for order-checking on the pMD18-T carrier, is that template amplification goes out est sequence with the cDNA of above-mentioned reverse transcription; Compare and utilize RACE method amplification gene 5 ' and 3 ' non-coding region by the online homology sequence of Genebank, the final full length gene cDNA that obtains, sequencing result finds that this gene cDNA clone encoded protein contains the AP2/ERF structural domain, and has complete encoder block, called after MEREB1.This full length gene cDNA sequence is 1165bp, has a single coding and reads frame (encoder block is the 76-840 position), the albumen that coding is made up of 255 amino-acid residues.In addition, on the aminoacid sequence of MEREB1 proteins encoded, found other characteristic feature of transcription factor, for example, the basic aminoacids sequence of N-stub area, the acidic region of C-end, it may be a transcriptional activation domain.Therefore, MEREB1 can encode an energy and DNA bonded ERF transcription factor, thus in cotton, play degeneration-resistant function as transcriptional control.

Two, the copy number analysis of cotton disease resistance related transcription factor MEREB1

The method that provides with reference to DIG High Primer DNA Labeling and Detection Starter Kit I (Roche) is probe with digoxin labelled probe MEREB1 gene coding region full length fragment (768bp).Take to cultivate the sea island cotton young leaflet tablet 10g in 5 weeks, extract genomic dna, after RNase A digestion, carry out purifying with the CTAB method.Genomic dna behind the purifying is cut with restriction enzyme EcoR V, HindIII, the XbaI enzyme that spends the night, use the ethanol precipitation enriched product, whole output is 1ug/ul, cumulative volume is 100ul, prepare 0.8% sepharose (every glue hole adds the DNA amount and is 33ul), through electrophoretic separation, transfer on the nylon membrane and digoxin labelled probe carries out Southern hybridization.Can observe every enzyme by final crossbreeding effect figure cuts band and has only a significantly hybridization bright band, this shows that the MEREB1 gene is a single copy gene (on behalf of restriction endonuclease EcoRI, HindIII and XbalI enzyme, Fig. 2, E, H and X cut respectively) in the cotton gene group.

Three, the expression pattern analysis of cotton disease resistance related transcription factor MEREB1

To in 28 ℃ of constant incubators, cultivate cotton (the new sea 20) seedling in two weeks, and extract seedling carefully and handle to hinder root.Adopt the verticillium wilt pathogen conidial suspension to dip in the root method material of hindering after root is handled is carried out the toxin-induced processing; The verticillium wilt pathogen of cultivating a week is diluted with sterilized water after 4 layers of filtered through gauze are added up the filtrate spore count at microscopically, 3 repetitions, being made into concentration is 1 * 10 ⁷ML ^-1Spore suspension, matching while using.Cotton seedling envrionment temperature remains on 23～28 ℃ after connecing bacterium, keeps soil humidity, is beneficial to morbidity; Get the plant leaf of inducing behind the 48h, place liquid nitrogen-80 ℃ to deposit rapidly; Gather root, stem, leaf and the complete stool of plant respectively; Extract total RNA with the CTAB method, material is put into liquid nitrogen flash freezer after coercing processing ,-80 ℃ of preservations are standby.

Extract total RNA and the different total RNA that coerce the cotton seedling under the processing that test kit has extracted the cotton seedling root of coercing after the processing, stem, leaf texture by the RNA of Promga company.Total RNA purity and quality are all higher, the A of the RNA of each sample ₂₆₀/ A ₂₈₀All more than 1.8, concentration is more than 0.8 μ g/ μ L.Detect through 1% agarose gel electrophoresis, 28S rRNA and 18S rRNA band are very clear, show that the RNA sample that is extracted can be used for the RT-PCR reaction.

With constitutive expression gene ubiquitin (UBQ-7) as interior mark, with 5 '-GAAGGCATTCCACCTGACCAAC-3 ' and 5 '-CTTGACCTTCTTCTTCTTGTGCTTG-3 ' is primer, analyze the expression of MEREB1 gene under the verticillium wilt pathogen stress conditions by RT-PCR, and the expression of MEREB1 gene in Radix Gossypii, stem and leaf.The result as shown in Figure 1.The expression of MEREB1 gene in cotton plants presented tissue-specific characteristics; The expression amount of MEREB1 in root, stem is the highest, and the expression amount in leaf minimum (Fig. 1).The expression of MEREB1 is subjected to inducing of verticillium wilt pathogen, can induce efficiently expressing of MEREB1, particularly in root and stem.

The above results explanation MEREB1 has participated in verticillium wilt pathogen and has coerced signal transduction pathway.

Acquisition of embodiment 2, disease-resistant transgenic tobacco and functional analysis thereof and Analysis on Mechanism

One, the acquisition of disease-resistant transgenic tobacco

1, the clone of the cDNA of cotton disease resistance related transcription factor MEREB1

The a pair of primer of synthetic adds Sma I and XbaI enzyme cutting site respectively at its 5 ' end:

Upstream primer: 5 '-CCCGGGATggAAATgTACCCAAgTACgA-3 ';

Downstream primer: 5 '-TCTAGA TTACgCTCCAAAACCCATATTTA-3 '.

Genome with cotton (new sea 20) is a template, carry out pcr amplification with above-mentioned upstream and downstream primer, obtain the fragment of 765bp, called after MEREB1, the PCR product that obtains is reclaimed, connect T carrier (pEASY-T3 of Beijing Quanshijin Biotechnology Co., Ltd), called after pMEREB1, heat shock changes E.coli BL21 (DE3) over to, cut screening positive clone by PCR, enzyme, and positive colony carried out sequence verification, sequencing result shows the nucleotide sequence from 5 ' end 76-840 position that fragment that pcr amplification obtains has sequence 2.

2, the structure of recombinant vectors

Building process as shown in Figure 4.

The pMEREB1 that empirical tests in the step 1 is correct obtains target gene fragment through Sma I and XbaI double digestion, be connected with the pBI121 carrier of cutting through same enzyme (purchasing company) then in Clontech, called after pBI121/MEREB1, MEREB1 promptly is inserted into the 35S promoter downstream like this.Identify that through order-checking pBI121/MEREB1 makes up correct.

3, the acquisition of disease-resistant transgenic tobacco

1) changes Agrobacterium over to

With the recombinant expression vector pBI121/MEREB1 conversion Agrobacterium LBA4404 (invitrogen) of freeze-thaw method with step 2.

Wherein the step of freeze-thaw method is as follows: take out the Agrobacterium LBA4404 competent cell of-70 ℃ of preservations, ice bath melts; Get 10-20 μ l pBI121/MEREB1 plasmid DNA (about 1-2 μ g), join in the Agrobacterium competent cell of 200ml thawing, stir evenly, left standstill several minutes with sterilization rifle head; Place liquid nitrogen 1min, 37 ℃ of water-bath 5min add 700-800 μ l LB liquid nutrient medium, 28 ℃, 200rpm, concussion 4h; 1000g 30sec removes the part supernatant, stays 100-150 μ l, inhales to beat again with the rifle head to suspend, and coats on the LB flat board that contains 50mg/L kantlex, 50mg/L Rifampin, 34mg/L paraxin, is inverted for 28 ℃ and cultivates 2d.

2) change tobacco over to

Change the reorganization Agrobacterium in the step 1) over to tobacco Nc89 (middle cigarette seed limited liability company) by the Agrobacterium infestation method, obtain changeing the tobacco of pBI121/MEREB1.

PCR identifies positive transfer-gen plant: with the transgene tobacco that obtains is template, carries out pcr amplification with following primer:

Upstream primer: 5 '-ATGGAAATGTACCCAAGTACGA-3 '

Downstream primer: 5 '-TTACGCTCCAAAACCCATATTTA-3 '

The result is shown in Fig. 6 C, and screening obtains positive plant, and following experiment, called after E1-1, E1-2, E1-4 and E1-6 are carried out in picking 4 strains (as Fig. 6 A).

Two, functional analysis

With wild-type tobacco in contrast.

1, the expression analysis of the disease-resistant related gene of disease-resistant transgenic tobacco (PR gene)

With seedling age is above-mentioned four of 2 weeks to change pBI121/MEREB1 tobacco strain system and wild-type tobacco (26 ℃ of ordinary student elongate member, illumination 18h) cultivated 14 days, with the Actin gene is internal control gene (Thangavelu, M., Belostotsky, D., Bevan, M.W., Flavell, R.B., Rogers, H.J.andLonsdale, D.M., Partial characteri--zation of the Nicotiana tabacum actin gene family:evidence for pollen-specificexpression of one of the gene family members Mol.Gen.Genet.1993.240 (2), 290-295), utilize real-time fluorescence quantitative PCR (Real Time Quantitative PCR), detect and analyzed each tobacco under the normal growth situation, disease-resistant related gene (PR gene) transcriptional level expression amount changing conditions, it is as follows wherein to detect PR gene the primer:

PR1a gene (accession number X06362):

F：5’-TTATGTTTCTCAATTGGCTGCAGAC-3’

R：5’-GGACTTTGGCCTATGACATTACC-3’

PR2 gene (accession number M60460):

F：5’-CAACATATTCAGGGATCTTAGCGAAT-3’

R：5’-TCAATAGTTGCTGCAGAGTT?TCCTT-3’

PR5 (accession number AF154636):

F：5’-ATTAGGCAAAGGCATATCAGAGTTG-3’

R：5’-CAAAACATTTCTAACACGCG?ACAG-3’

Utilize the tabulation of Excel software to sum up by qRT-PCR detection (three repetitions) situation of sea island cotton ERF gene regulating PR genetic expression and the result that will obtain, the result as shown in Figure 7, after the MEREB1 gene changes in the tobacco, under the ordinary student elongate member, just can effectively strengthen tobacco PR expression of gene, and the PR gene does not strengthen in the wild-type tobacco, this has proved that sea island cotton ERF gene brought into play vital role in the process of regulation and control disease-resistant related gene, can infer thus that ERF gene overexpression in plant can effectively strengthen the expression of disease-resistant related gene, thereby improve the disease resistance of plant, this development and utilization for the MEREB1 gene provides theoretical foundation.

Acquisition of embodiment 3, disease-resistant transgenic cotton and functional analysis thereof

One, the acquisition of disease-resistant transgenic cotton

1, the clone of the cDNA of cotton disease resistance related transcription factor MEREB1

The clone of the cDNA of MEREB1 is identical with the step 1 of embodiment 2 step 1.

2, the structure of recombinant vectors

The structure of recombinant vectors is identical with the step 2 of embodiment 2 step 1.

3, the acquisition of disease-resistant transgenic cotton

Pollen tube passage method is meant behind plant pollination, the solution that contains goal gene to the ovary injection, the pollen tube channel that utilizes plant to form, foreign gene is imported the fertilized egg cell, thereby be integrated in the genome of recipient cell the final transfer-gen plant that becomes along with the growth of zygote further.

This example utilizes pollen tube passage method to change recombinant expression vector pBI121/MEREB1 over to upland cotton (please indicate the Business Name of this cotton quality and sale), obtains changeing the cotton of pBI121/MEREB1.

PCR identifies positive transgene cotton: with the transgene cotton that obtains is template, identifies that with the PCR in the step 3 among the embodiment 2 the used primer of positive transgene tobacco carries out pcr amplification: screening obtains positive plant, and following experiment is carried out in picking 3 strains (as Fig. 6 B).

Two, functional analysis

With the wild-type cotton in contrast.

1, the expression analysis of the disease-resistant related gene of disease-resistant transgenic cotton (chitinase gene, beta-1,3-glucanase gene and PR protein gene)

That seedling age is above-mentioned 3 are changeed pBI121/MEREB1 cotton strain system and the wild-type cotton was cultivated 14 days in ordinary student elongate member (26 ℃, illumination 18h), extract total RNA of each cotton different tissues, with constitutive expression gene ubiquitin (UBQ7) as interior mark, respectively with the corresponding primer amplification transfer-gen plant cDNA template of following upland cotton disease-resistant genes involved, analyze the influence that the MEREB1 gene changes enantiopathy related gene expression behind the cotton overexpression over to by RT-PCR.Wherein, PR protein gene the primer is 5 '-ATGGGTGTTGTCACTTATGACTATG-3 ' and 5 '-CTAGTTGCAGGCTTCGGGATTAG-3 '; The chitinase gene the primer is 5 '-GGGGATACTGCTACAATAGGGAATTG-3 ' and 5 '-CTACATTGAGTCCACCGAGACT-3 '; β-1,3-dextranase gene the primer are 5 '-AGTGCAAACCTTGAATCCGTTGC-3 ' and 5 '-GCTTTCTCCA GGGCAGAGTA AAAGGTA-3 '.

Detected result shows: under the ordinary student long status, change in the upland cotton plant body of MEREB1 disease-resistant related gene (chitinase gene, β-1,3-dextranase gene, PR protein gene) over to and compare expression amount with the wild-type contrast and obviously obtained enhancing (Fig. 8).

Three, Analysis on Mechanism

The checking of MEREB1 and GCC-box combination of elements ability

It is the key factor that the ERF transcription factor is regulated and control its downstream gene expression in the environment stress signal transduction pathway that ERF class transcription factor can combine with GCC-box.The DNA of transcription factor MEREB1 is expressed in conjunction with the territory and (Electrophoreticmobility shift assay EMSA) verifies and is very important by external gel blocking.Utilization contains the probe of GCC-box element, and the design mutant:

Positive GCC-box probe:

5’-CATAAgAgCCgCCACTAAAATAAgACCgATCAAATAAgAgCCgCCAT-3’

Mutant mGCC-box probe:

5’-CATAAgATCCTCCACTAAAATAAgACCgATCAAATAAgATCCTCCAT-3’

The conservative used upstream and downstream, territory of amplification MEREB1 gene ERF primer adds SmalI and NotI restriction enzyme site and protection base respectively:

The upstream and downstream primer is respectively:

ERD1F：5’-CCCCCGGGATGAGACAGCGGTGAAAGAGGTTC-3’；

ERD1R：5’-TTGCGGCCGCATATGTTACGCTCCAAAACCCATATTTA-3’。

Genome with cotton (new sea 20) is a template, with ERD1F and ERD1R is primer, carry out pcr amplification, condition: 94 ℃ of sex change 30 seconds, annealed 40 seconds for 59 ℃, 72 ℃ were extended 1 minute, 30 circulations, product reclaims the purpose fragment through 1% agarose gel electrophoresis, obtains containing among the MEREB1 fragment of the conservative functional domain of ERF, as holding shown in the 416-813 position from 5 ' in the sequence 1, called after EREB1 is inserted into EREB1 then between the Sma I of prokaryotic expression carrier pGEX4T-1 (Amersham) and the NotI restriction enzyme site (Fig. 3), and is transformed among the efficient expression strain BL21, pass through PCR, enzyme is cut screening positive clone, and positive colony carried out sequence verification, and detect and confirm segmental insertion, finally obtained containing the conservative territory of ERF segmental pGEX4T-1 prokaryotic expression carrier positive strain.Induce GST albumen and expressing fusion protein with 0.5mMIPTG, reclaim purifying protein with supersonic method cracking inductive bacterium and MicroSpincolumns (Amersham); Finally obtained target protein.Carry out gel retardation assasy, result such as Fig. 5 (mixture of 1:GST and normal GCC-box probe; 2: fusion rotein GST/EREB1 and normal GCC-box; 3: the mixture of fusion rotein GST/EREB1 and sudden change GCC-box probe; The arrow A indication is target protein and probe mixture) shown in, the fusion rotein that contains the conservative territory of ERF among the MEREB1 is with after ERF cis-acting elements (GCC-box) mixes, in gel electrophoresis, formed clearly drag zone and the mixture of fusion rotein and mutant GCC-box (G8G11G40G43 is sported TTTT respectively) does not form drag zone, can be observed GST albumen and probe mixture simultaneously and drag zone do not occur, its no binding ability is described, having got rid of GST albumen may impact the binding characteristic of fusion rotein.These presentation of results: thus MEREB1 albumen has caused the operation in the electrophoresis process to lag behind external can the combination with ERF cis element specificity.Fusion rotein and mutant ERF cis element (mGCC) gel retardation assasy result show that MEREB1 and mGCC (G is mutated into T) all can not form tangible drag zone, have verified that sea island cotton EREBs albumen combines with the ERF cis element and has specificity.

Sequence table

＜110〉The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute

＜120〉cotton disease resistance related transcription factor MEREB1 and encoding gene thereof and application

<130>CGGNARL92544

<160>2

<210>1

<211>1165

<212>DNA

＜213〉sea island cotton (Gossypium barbadense L)

<400>1

tgaaatttca?gcttacgaac?ttgttttacc?tgaaaatttg?aatttttagc?atcggaaaag 60

ctttcaattt?tcgttatgga?aatgtaccca?agtacgattg?attccgattt?aatgatgctg 120

gactcaattc?ggagacactt?actcggcgaa?tcatccgact?tgcggttcac?ttcttcaaat 180

gactgtacca?acgtgggcgc?ggctccccct?atgttttgca?ggagctccag?ctttagccgc 240

ttgtaccctt?gtttgactga?cacctggggt?gacctcccac?tcaaagagaa?cgattctgaa 300

gacatgctag?ttttcgggta?ccttagagat?gctttaaccg?tcggttgggc?cccctccgat 360

cactcttccc?cgactttccc?gccaattaaa?ccggaacctc?aggagattcc?gacggagaca 420

gcggtgaaag?aggttcctac?ggtggcgaat?gcggcggttc?ccacggtggt?tcctgccaaa 480

gggaagcatt?acagaggagt?aaggcaaagg?ccgtggggaa?agttcgcggc?agagattagg 540

gatccggcta?aaaacggggc?tcgagtttgg?ctaggcactt?tcgagacagc?tgaggatgcg 600

gctttggctt?atgataaggc?agcttatagg?atgcgtggct?caagggcttt?gttgaatttc 660

ccattgaggg?tgaattccgg?ggaacccgac?ccagttagga?tcacgtcaaa?gagggcaaca 720

ccggagccat?ctacttcttc?gtcctcgggt?tcagaaaacg?ggtcaccaaa?aaggaggaga 780

aaagtaggta?gcgcggctcc?ggttgtagct?caagccggtt?taaatatggg?ttttggagcg 840

taaagtatgg?aagtgggaac?ttgtacatgt?ggcgaacagc?tattagtcag?tgaaatgctg 900

ctgaggtgtt?aatggtgtaa?tgtaagcaga?agtaagctta?tagttatgga?caacctccat 960

cccataggaa?ttttgtagaa?tcggaaaaaa?aaaagttgtt?tttgtcctta?aaaatactat 1020

taacagcgtg?gttaaagtaa?gcccattcta?aagaaaaatt?atacttctcc?aatggaactt 1080

gattaacata?taaaccaata?tatgataaag?aaaattatac?catccatgct?aattggtcag 1140

catagtttct?acgaaaaaaa?aaaaa 1165

<210>2

<211>255

<212>PRT

＜213〉sea island cotton (Gossypium barbadense L)

<400>2

Met?Glu?Met?Tyr?Pro?Ser?Thr?Ile?Asp?Ser?Asp?Leu?Met?Met?Leu?Asp

1 5 10 15

Ser?Ile?Arg?Arg?His?Leu?Leu?Gly?Glu?Ser?Ser?Asp?Leu?Arg?Phe?Thr

20 25 30

Ser?Ser?Asn?Asp?Cys?Thr?Asn?Val?Gly?Ala?Ala?Pro?Pro?Met?Phe?Cys

35 40 45

Arg?Ser?Ser?Ser?Phe?Ser?Arg?Leu?Tyr?Pro?Cys?Leu?Thr?Asp?Thr?Trp

50 55 60

Gly?Asp?Leu?Pro?Leu?Lys?Glu?Asn?Asp?Ser?Glu?Asp?Met?Leu?Val?Phe

65 70 75 80

Gly?Tyr?Leu?Arg?Asp?Ala?Leu?Thr?Val?Gly?Trp?Ala?Pro?Ser?Asp?His

85 90 95

Ser?Ser?Pro?Thr?Phe?Pro?Pro?Ile?Lys?Pro?Glu?Pro?Gln?Glu?Ile?Pro

100 105 110

Thr?Glu?Thr?Ala?Val?Lys?Glu?Val?Pro?Thr?Val?Ala?Asn?Ala?Ala?Val

115 120 125

Pro?Thr?Val?Val?Pro?Ala?Lys?Gly?Lys?His?Tyr?Arg?Gly?Val?Arg?Gln

130 135 140

Arg?Pro?Trp?Gly?Lys?Phe?Ala?Ala?Glu?Ile?Arg?Asp?Pro?Ala?Lys?Asn

145 150 155 160

Gly?Ala?Arg?Val?Trp?Leu?Gly?Thr?Phe?Glu?Thr?Ala?Glu?Asp?Ala?Ala

165 170 175

Leu?Ala?Tyr?Asp?Lys?Ala?Ala?Tyr?Arg?Met?Arg?Gly?Ser?Arg?Ala?Leu

180 185 190

Leu?Asn?Phe?Pro?Leu?Arg?Val?Asn?Ser?Gly?Glu?Pro?Asp?Pro?Val?Arg

195 200 205

Ile?Thr?Ser?Lys?Arg?Ala?Thr?Pro?Glu?Pro?Ser?Thr?Ser?Ser?Ser?Ser

210 215 220

Gly?Ser?Glu?Asn?Gly?Ser?Pro?Lys?Arg?Arg?Arg?Lys?Val?Gly?Ser?Ala

225 230 235 240

Ala?Pro?Val?Val?Ala?Gln?Ala?Gly?Leu?Asn?Met?Gly?Phe?Gly?Ala

245 250 255

Claims (3)

1. cultivating the method that disease-resistant related gene is expressed the enhanced transgene cotton for one kind, is that the proteic encoding gene that the aminoacid sequence shown in the sequence in the sequence table 2 is formed is imported in the cotton, obtains the transgene cotton that the disease-resistant related gene expression amount improves;

Described disease-resistant related gene is chitinase gene, β-1,3-dextranase gene or PR protein gene; Described chitinase gene be Genbank number for GHU60197 from the gene of 5 ' end shown in the 690th to the 1302nd, β-1, the 3-glucanase gene be Genbank number for Z68154 from the gene of 5 ' end shown in the 301st to the 809th, the PR protein gene be Genbank number for AF305066 from the gene shown in the 47th to the 526th at the 5 ' end.

2. method according to claim 1 is characterized in that: the encoding sequence of the proteic encoding gene that aminoacid sequence in the described sequence table shown in the sequence 2 is formed be in the sequence table sequence 1 from 5 ' terminal 76-840 position.

3. method according to claim 1 and 2, it is characterized in that: the proteic encoding gene that the aminoacid sequence in the described sequence table shown in the sequence 2 is formed is to import in the purpose plant by the recombinant vectors that contains described encoding gene, described recombinant vectors is a multiple clone site of described encoding gene being inserted carrier pBI121, the recombinant vectors that obtains.

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