CN102978218B - Cloning of apple stress-resistant related gene MdSIMYB2 and application of cloning of apple stress-resistant related gene MdSIMYB2 - Google Patents

Abstract

The invention relates to cloning of an apple stress-resistant related gene MdSIMYB2 and an application of the cloning of the apple stress-resistant related gene MdSIMYB2. A homology-based cloning and rapid-amplification of cDNA ends (RACE) technology is utilized to obtain a novel apple stress-resistant related gene MdSIMYB2 from a gala apple, the nucleotide sequence of the MdSIMYB2 is shown in SEQ.ID.NO.1, and the protein amino acid sequence is shown in SEQ.ID.NO.2. The gene is transferred into a tomato, and the stress capabilities of drought resistance, salt resistance, cold resistance and the like of a transgenic tomato strain are improved; and through the overexpressing of the gene MdSIMYB2 in the apple, the expression level of the transgenetic apple can be improved, and moreover, the stress capabilities of drought resistance, salt resistance, cold resistance and the like of a transgenic apple strain are increased.

Description

Clone and the application thereof of apple anti contravariance related gene MdSIMYB2

One, technical field

The present invention relates to clone and the application thereof of a kind of apple anti contravariance related gene MdSIMYB2, belong to molecular biology and biological technical field.

Two, background technology

Plant often suffers the invasion and attack of various poor environments and disease and pest in life, and in long-term adaptation and evolutionary process, plant has formed series of defence reaction mechanism, is used for resisting the injury of poor environment.Wherein, environment-stress mainly comprises that various biologies coerce and abiotic stress.Biology is coerced mainly from insect, herbivore, phytopathogen etc.; Abiotic stress kind is numerous, comprises that arid, salt marsh, extreme temperature, chemical pollution and oxygen injury etc. coerce.In order to adapt to numerous and diverse changeable environment, reduce the injury of adverse environment to body, in plant materials, develop and to relate to the complex process of polygene, many signal pathways, polygene product.Wherein, MdSIMYB2 gene pairs abiotic stress has resistivity.

Apple is worldwide fruit, is the Main Cultivation seeds of Temperate Region in China.Its adaptive faculty is stronger, and fruit nutritive value is higher, and storage tolerance is good, and supply cycle is long, and a lot of countries are all classified as main consumption fruit and support energetically in the world.But in the growth and development process of apple, many abiotic stress are the main limiting factor that affect apple regional distribution and output raising, especially high salt, arid and low temperature etc., world apple production state is all using the anti-salt of seed selection, drought resisting, the high quality apple new variety such as cold-resistant as main breeding objective.Because apple is perennial woody plant, genetic background is complicated, assorted and spend the problems such as high, Tong Qichang, self-compatibility are poor and brought very large obstacle to genetic breeding, and conventional breeding is made slow progress.In recent years, order-checking (the Velasco etc. of apple genome, 2010) the modern biotechnology that completes and develop rapidly has been opened up new way for addressing these problems, by genetic engineering technique, there is object to improve the anti-salt of fruit tree, drought resisting, cold tolerance, become the important research direction of fruit tree genetics improvement, the separation of a large amount of plant stress-resistance functional genes and identifying for genetically engineered genetic improvement provides goal gene.

Myb transcription factor is being played the part of very important role (Liu Lei etc., 2008) in plant defense and Stress response process.The overexpression of myb gene in plant can significantly improve the resisting abiotic stress ability of plant, in plant stress-resistance reaction process, when plant receives that external environment is coerced, plant regulates and controls the expression of genes involved in body by signal transduction pathway, and then cause a series of physiology, biochemical reaction, reduce or eliminate the injury bringing to plant.At present the research of MYB is mainly concentrated on to Arabidopis thaliana, paddy rice, on the first-class minority model plant of soybean, and research is less aspect fruit tree.Due to all many-sided reasons, can plough land area fewer and feweri, add low temperature, arid, the soil salinization and disease, insect pest causes the cultivated area of fruit tree and scope to be had a strong impact on.Therefore excavate apple myb gene and carry out functional study, for the breeding of fruit tree resistant transgenic provides potential effective candidate gene, not only there is important theory significance, and be with a wide range of applications.Apple transgenic breeding is many carries out in apple rootstock, and that apple rootstock is mainly lived in is underground, does not bloom and bears fruit, and has reduced the dispute of genetically engineered in fruit tree application.

Along with people are more and more higher to the requirement of apple quality, the improvement problem of kind becomes a vital task of researcher, so Apple breeding is constantly strengthened in the fruit tree research work of countries in the world.But apple is perennial woody plant, long by the conventional breeding seed selection resistant variety cycle.

Three, summary of the invention

In order to address the above problem, the invention provides cloning process and the nucleotide sequence of a kind of apple anti contravariance related gene MdSIMYB2, provide this gene degeneration-resistant in acquisition simultaneously, especially the application in drought resisting, anti-salt, cryophylactic transgenic Fructus Lycopersici esculenti and apple, and can be applicable to produce other food crop of improvement or cash crop.

The present invention utilizes homologous clone and RACE technology from apple loud, high-pitched sound, to obtain the novel degeneration-resistant border genes involved MdSIMYB2 of apple, and its nucleotide sequence is as shown in SEQ.ID.NO.1, and protein amino acid sequence is as shown in SEQ.ID.NO.2.Concrete grammar is as follows:

The loud, high-pitched sound apple tissue cultured seedling of processing 24h through NaCl extracts total RNA, and reverse transcription obtains cDNA.According to the conserved amino acid sequence of myb gene family in announced Arabidopis thaliana in international gene pool, design degenerate primer, carries out conventional polymerase chain reaction (Polymerase chain reaction, PCR).Gained PCR product is connected with pMD18-T carrier, transforms bacillus coli DH 5 alpha competent cell, screening recon, and carry out sequencing analysis confirmation; By 5 '-RACE and 3 '-RACE technology, obtain respectively 5 ' and 3 ' terminal sequence again, be spliced into complete cDNA sequence.According to 3 of cDNA ' and and 5 ' terminal sequence design special primer, the cDNA of loud, high-pitched sound apple tissue cultured seedling of take carries out pcr amplification as template, obtains cDNA full length sequence.

Utilize RACE method to clone this gene and obtain 1401bp, its open reading frame (open reading frame, ORF) is 1095bp.364 amino acid of this genes encoding, predicted molecular weight is about 39.10kDa, and iso-electric point is 8.17.Its aminoacid sequence is retrieved in NCBI, find and the AtMYB5(Arabidopis thaliana of having announced, NM_112200.2), AtMYB17(Arabidopis thaliana, NM_115989.3), AtMYB25(Arabidopis thaliana, NM_129546.1), PhPH4(petunia, AY973324.1) have higher homology, its amino acid identity is respectively 69.23%, 66.35%, 65.38%, 64.42%.Show thus, through above-mentioned clone's step, obtained myb gene in an apple, this gene is relevant with salt stress, participates in response abiotic stress (seeing patent 201210197919.5) again, so be MdSIMYB2 by this unnamed gene because we have identified MdSIMYB1.

This gene order is as follows:

Sequence table:

(1) information of SEQ.ID.NO.1

(a) sequence signature

Length: 1401bp

Type: nucleic acid

Topological framework: linearity

(b) analysis type: cDNA

(c) suppose: no

(d) antisense: no

(e) originate at first: apple (Malus domestica)

(f) sequence description: SEQ.ID.NO.1

TTTTTTTTTG AACACTGAAA AAGCTTTACA AGGAACC CATCGTCTTC GTCGAAAGCA 60

GCAGCAGCAG CAAGTGCTAA GATGCAAACG ACGATAACAG CGCCGTCCTC GTCGAGCAAG 120

GCGGCTGGGG TTGCTGGAGG GACCAAGACG CCGTGTTGCG CAAAGGTGGG TTTGAAGAGA 180

GGGCCTTGGA CTCCCGAAGA GGACGAGCTG CTGGCAAATT ACATCAAGAA AGAAGGGGAG 240

GGACGGTGGC GGACCCTTCC CAAGCGGGCT GGGTTGCTCC GCTGCGGTAA GAGCTGCCGC 300

CTCCGCTGGA TGAACTATCT CCGCCCTTCT GTCAAGCGCG GCCAGATCGC CCCCGATGAA 360

GAAGATCTTA TCCTTCGCCT CCATCGCCTT CTGGGCAATC GGTGGTCTTT GATAGCTGGG 420

AGGATTCCAG GCCGTACGGA CAATGAGATA AAGAACTACT GGAACACACA CCTGAGCAAG 480

AAGCTGATAA GCCAAGGCAT AGATCCCAGA ACCCACAAGC CTCTCAATTC AGATCATCAC 540

TCTGCTGCTG ATGATGCTGA CGTGGACAAC ACAAACAAAT CAACTGCTGT TGCTTCTTCT 600

TCCAAAGCCA ATGATCGGTT CTTAAACCCT AACCCTAGTC CCCCTTCTGA TCGTCTTGTC 660

CATAAAGAAG GGGATCCAAA TAACAGCCGT AATGATGGAA ACATCGCAAT TGCTGATCAT 720

GATCTGGGCA CTATAGTCCA TGGCTTTGCA AATATGATCA CGTCCATCAA CAATCCCGAT 780

GCTTCTTCTT CGGCCGCGGC AACGGGTACT TTGAGTTTGA GGAGCAACAA CAGCCACGGT 840

GGAGTACTAC TTGGGGGAGG AGGAAATGAA GAGGACGACG TCTTCTCTTC GTTTCTGAAT 900

TCGTTGATCA ATGAGGATCC ATTTCCTGGA CAACACCAAT TGCAACAAGT ACTGCAGAAT 960

GGGAATGTTA GTGCACACGC AGCTGCTGCT CGTTCCGAGA ACCTTCCTTT GATTACTATG 1020

ACTAGTACTA CGGCGCCATC AACATTTGGC TGGGAGTCTG CCGTGCTCAT GTCTTCTGCT 1080

TTCATCCAAA ATGATCACCA GAGCGTTAAT GATCAAACGG AG CAGCT AGCCACCTGT 1140

TTGATTGATC AGTCGCACTG TGCATGTTGA ACAAGCAATG CGGTTGCTTA ATTAGTTAAT 1200

TTATATGTAG AAATTAGTCC TAGCTAGGGT TTGAAATATC ATATGAAATG TGATTTTGTG 1260

TGCTATCTCT ATTCTTAATT ATTCGACTAC CCCAACATCG CTCTACGAAT GGGGGCGTAT 1320

ATCAATGTAT TGTCGATCAC GTTTGTAATA TCATCTATCT ATCTATATAT GTTACCGGTA 1380

TTGTAAAACA AACATAAAAA A 1401

Illustrate: in transparent square frame represent initiator codon, and in yellow square frame represent terminator codon.

(2) information of SEQ.ID.NO.2

(a) sequence signature

Coding region length: 364 amino acid

Type: amino acid

Topological framework: linearity

(b) analysis type: protein

(c) sequence description: SEQ.ID.NO.2

MET Arg Asn Pro Ser Ser Ser Ser Lys Ala Ala Ala Ala Ala Ser

5 10 15

Ala Lys MET Gln Thr Thr Ile Thr Ala Pro Ser Ser Ser Ser Lys

20 25 30

Ala Ala Gly Val Ala Gly Gly Thr Lys Thr Pro Cys Cys Ala Lys

35 40 45

Val Gly Leu Lys Arg Gly Pro Trp Thr Pro Glu Glu Asp Glu Leu

50 55 60

Leu Ala Asn Tyr Ile Lys Lys Glu Gly Glu Gly Arg Trp Arg Thr

65 70 75

Leu Pro Lys Arg Ala Gly Leu Leu Arg Cys Gly Lys Ser Cys Arg

80 85 90

Leu Arg Trp MET Asn Tyr Leu Arg Pro Ser Val Lys Arg Gly Gln

95 100 105

Ile Ala Pro Asp Glu Glu Asp Leu Ile Leu Arg Leu His Arg Leu

110 115 120

Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Ile Pro Gly Arg

125 130 135

Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Leu Ser Lys

140 145 150

Lys Leu Ile Ser Gln Gly Ile Asp Pro Arg Thr His Lys Pro Leu

155 160 165

Asn Ser Asp His His Ser Ala Ala Asp Asp Ala Asp Val Asp Asn

170 175 180

Thr Asn Lys Ser Thr Ala Val Ala Ser Ser Ser Lys Ala Asn Asp

185 190 195

Arg Phe Leu Asn Pro Asn Pro Ser Pro Pro Ser Asp Arg Leu Val

200 205 210

His Lys Glu Gly Asp Pro Asn Asn Ser Arg Asn Asp Gly Asn Ile

215 220 225

Ala Ile Ala Asp His Asp Leu Gly Thr Ile Val His Gly Phe Ala

230 235 240

Asn MET Ile Thr Ser Ile Asn Asn Pro Asp Ala Ser Ser Ser Ala

245 250 255

Ala Ala Thr Gly Thr Leu Ser Leu Arg Ser Asn Asn Ser His Gly

260 265 270

Gly Val Leu Leu Gly Gly Gly Gly Asn Glu Glu Asp Asp Val Phe

275 280 285

Ser Ser Phe Leu Asn Ser Leu Ile Asn Glu Asp Pro Phe Pro Gly

290 295 300

Gln His Gln Leu Gln Gln Val Leu Gln Asn Gly Asn Val Ser Ala

305 310 315

His Ala Ala Ala Ala Arg Ser Glu Asn Leu Pro Leu Ile Thr MET

320 325 330

Thr Ser Thr Thr Ala Pro Ser Thr Phe Gly Trp Glu Ser Ala Val

335 340 345

Leu MET Ser Ser Ala Phe Ile Gln Asn Asp His Gln Ser Val Asn

350 355 360

Asp Gln Thr Glu

364

The invention provides the method that apple abiotic stress genes involved MdSIMYB2 applies in tomato, apple and other plant, can improve the ability of the adverse circumstances such as transgenic plant opposing arid, low temperature and salt.Step is:

(a) utilize MdSIMYB2 gene as above, cDNA sequence is placed under Caulimovirus CaMV35S promotor, build plant Overexpression vector.

(b) by Agrobacterium (LBA4404) mediated method, the expression vector building is imported to vegetable cell, obtain transfer-gen plant.

The present invention relates to a kind of plant expression vector, comprise nucleotide sequence SEQ.ID.NO.1, for improving Genes For Plant Tolerance adverse circumstance ability, the ability of especially drought-resistant, low temperature and salt.

The present invention relates to above-mentioned plant expression vector to import in vegetable cell, introduction method is Agrobacterium well known in the art (LBA4404) mediated method, obtains the transgenic plant of drought-resistant, salt and low temperature.

Herbaceous plant tomato SN1 is one of model plant of genetically engineered research, have the advantages that life cycle is short, regenerative power is strong, genetic transformation efficiency is high, and xylophyta apple life cycle is long, genetic transformation efficiency is low, thereby the present invention is described in detail as example to take in the following embodiments tomato and apple two class plants, but in the present invention, MdSIMYB2 gene and the plant expression vector that contains this gene also can improve for the production of other transgenic plant of degeneration-resistant border ability.

The present invention adopts Isolated leaf inoculation method to carry out anti-adversity ability analysis discovery to the transgenic Fructus Lycopersici esculenti homozygote and the apple that obtain, and the overexpression strain of MdSIMYB2 in transgenic Fructus Lycopersici esculenti and apple all can significantly improve that it is drought-resistant, the ability of low temperature and salt.This gene can be proceeded on the farm crop such as the xylophytas such as peach, pear tree, willow or wheat, corn, paddy rice, thereby the degeneration-resistant border ability of raising transfer-gen plant produces great economic benefit and social value.

The present invention is separated to the MYB family gene MdSIMYB2 of a R2R3 type from apple, by transgenosis functional verification, shows: it can support high salt tolerance, low temperature, the abiotic stresses such as arid; And provide a kind of new, breeding approach fast, especially in transgenic apples stock, the excavation of MdSIMYB2 gene can not only provide new candidate gene for apple adversity gene engineering, enrich plant stress-resistance genetically engineered theoretical system, and also have important practical significance for the degeneration-resistant germplasm materials (comprising yearly plant and perennial woody plant) of cultivating other plant.

Four, accompanying drawing explanation

Fig. 1: the homology comparison of the aminoacid sequence of the MYB family plant of the conservative territory schematic diagram of MdSIMYB2 protein structure and different R2R3 types in apple.

A. the MdSIMYB2 protein structure of apple is guarded territory schematic diagram; B. the homology comparison of the aminoacid sequence of the MYB family plant of different R2R3 types.Wherein, AtMYB5 (NM_112200.2), AtMYB17 (NM_115989.3), AtMYB108(NM_111525.3), PhPH4(AY973324.1), R2, R3 represent respectively the feature structure territory of MYB family gene.

Fig. 2: the phylogenetic analysis of MdSIMYB2 albumen in apple.

Wherein, wandering jew TfMYB6 (AY456184.1), cotton GhMYB1 (L04497.1), comospore poplar PtMYB180 (XM_002327312.1), PtMYB111(XM_002302716.1), Arabidopis thaliana AtMYB5 (NM_112200.2), AtMYB17(NM_115989.3), AtMYB108 (NM_111525.3), petunia PhPH4(AY973324.1), soybean GmW2 (AB597932.1), rice Os MYB4(D88620.1), psyllium CpMYB10 (AF510112.1), Root or stem of Littleleaf Indianmulberry LjTT2a(AB300033.2), barley HvMYB2 (X70876.1), Picea koraiensis Nakai PgMYB5 (EF601068.1), soybean GmMYB54 (DQ822881.1), hop HlMYB1 (AB292244.1).

Fig. 3: the response of MdSIMYB2 gene pairs abiotic stress in loud, high-pitched sound tissue cultured seedling.

B1～B4. uses respectively arid (2%PEG), NaCl (200mM), low temperature (4 ℃), ABA (100 μ M) to process after loud, high-pitched sound apple tissue cultured seedling 0h, 6h, 12h, 24h, 48h, the relative expression quantity of MdSIMYB2 gene.

Fig. 4: the MdSIMYB2 transgenic Fructus Lycopersici esculenti seedling upgrowth situation after adverse circumstance is processed.

The sxemiquantitative RT-PCR of A, wild-type tomatoes and transgenic Fructus Lycopersici esculenti strain detects; Under B1, normal management condition, cultivate 6 weeks tomato growth situations; B2. 6 weeks consistent tomato seedlings of increment, 10d does not water and does the growing state that arid is processed continuously; B3. 6 weeks consistent tomato seedlings of increment, the growing state after 300mM NaCl processes 12d; B4. 6 weeks consistent tomato seedlings of increment, the growing state after 4 ℃ of subzero treatment 4d.Wherein, WT: contrast; OE-1, OE-3, OE-4: the transgenic line of overexpression.

Fig. 5: the MdSIMYB2 transgenic apples growth of seedling situation after adverse circumstance is processed.

The sxemiquantitative RT-PCR of A, wild-type apple and transgenic apples strain (MdSIMYB2 gene overexpression) detects; Wherein, WT: contrast; T1-T4: the transgenic line of overexpression.

B, growth 6 months and the consistent loud, high-pitched sound apple of growing way seedling and the MdSIMYB2 overexpression seedling of taking root of taking root, the growing state after different adverse circumstances are processed.

B1: 15d does not water and carries out the situation that arid is processed continuously; B2: growing state after rehydration processing 3d after arid processing; C1: process through 300mM NaCl, grow to the growing state of 14d; C2: after 300mM NaCl processes, recover to grow to the growing state of 16d; D1:4 ℃ of cold condition cultivated the growing state after 16d; D2:4 ℃ of cold condition cultivated the growing state after rearmounted home 25d.Wherein, WT: contrast; T-1, T-2, T-4: the transgenic line of overexpression.

Five, specific examples mode

Below in conjunction with specific examples, the present invention is described in detail.

Example 1: the clone of apple MdSIMYB2 gene.

One, the extraction of RNA and reverse transcription:

The 200mM NaCl salt of learning from else's experience is processed the loud, high-pitched sound apple tissue cultured seedling blade of 24h, extracts its RNA reverse transcription:

One) utilize a day root test kit RNAplant Plus Reagment to extract total RNA, concrete grammar is as follows:

1) get the Apple Leaves of the freezing grinding of 1g, add 5ml to extract reagent (4 ℃), vibration mixes.

2) room temperature is placed 5 minutes.

3) 4 ℃ 12, centrifugal 1 minute of 000rpm, supernatant proceeds to new for RNase centrifuge tube.

4) on every 10ml, reset and add 2ml5M NaCl, gentleness mixes.

5) on every 10ml, reset and add 6ml chloroform, turn upside down and mix.

6) 4 ℃ 10,000 centrifugal 15 minutes, get upper strata water and proceed to new for RNase centrifuge tube.

7) add Virahol (with gained water equal-volume), mix, room temperature is placed 10 minutes.

8) 4 ℃ 10,000 centrifugal 15 minutes.Abandon supernatant, add 5-10ml75% ethanol.

9) 4 ℃ 5, centrifugal 5 minutes of 000rpm.Abandon supernatant, room temperature is dried 3-5 minute.

10) add 100 μ l without RNase water, mix, fully dissolve RNA.Obtain RNA ,-80 ℃ save backup, or carry out immediately following reverse transcription experiment.

Two) reverse transcription cDNA the first chain is synthetic

1) in the Eppendorf tube of 0.2ml, prepare that following mixed solution (its RNA is example 1 step 1) extract to obtain; As with-80 ℃ of storage RNA, must slowly melt on ice):

2) gently mixed with rifle head, Eppendorf tube is placed on PCR instrument to (65 ℃ 5 minutes) sex change, annealing, on ice chilling;

3) in above-mentioned Eppendorf tube, continue to add following reaction solution.

4) on PCR instrument, carry out reverse transcription reaction: 30 ℃ 10 minutes, 42 ℃ 60 minutes, 70 ℃ 15 minutes, 4 ℃ of preservations.Synthetic reverse transcription product cDNA, for carrying out related experiment below.

Three) cDNA tailing

Utilize TdT terminal enzyme (DNA), at example 1) step 2) the synthetic cDNA3 ' end of reverse transcription adds Poly (C) tail, as template, carries out 5 ' RACE amplification.

In 1.5ml centrifuge tube, add successively following reagent.

1) 37 ℃ are reacted 30 minutes;

2) add 50 μ l phenol/chloroform/primary isoamyl alcohol (25:24:1), fully mix; Centrifugal, get upper strata to another new centrifuge tube;

3) add 50 μ l chloroform/primary isoamyl alcohol (24:1), fully mix; Centrifugal, get upper strata to another new centrifuge tube;

4) add successively the dehydrated alcohol of 5 μ l3M NaAC, 125 μ l precoolings, place 30-60 minute for-20 ℃;

5) 4 ℃, 12, centrifugal 15 minutes of 000rpm, 70% ethanol is washed 2 times;

6) 4 ℃, 12, centrifugal 10 minutes of 000rpm, air-dry precipitation; With 20 μ l sterilized water dissolving DNAs, 4 ℃ of preservations.The tailing cDNA obtaining is used for 5 ' RACE amplification below as template.

Two, the acquisition of cDNA full length sequence

1) amplification of MdSIMYB2 homologous sequence

The conserved amino acid sequence of myb gene family in the Arabidopis thaliana of finding according to NCBI, design degenerate primer (JN-F, JB-R), take step 1 in example 1) the synthetic cDNA of reverse transcription carries out pcr amplification as template.

Its sequence of primer 1JB-F:5 '-AGKEGEGRTGGCGATCTTCCTAA-3 ' is as shown in SEQ.ID.NO.3;

Its sequence of primer 2 JB-R:5 '-CGSVPIVLAATTTCATTATCAGT-3 ' is as shown in SEQ.ID.NO.4;

Pcr amplification system (this system is all used in the PCR reaction of carrying out below):

PCR response procedures: 94 ℃ of denaturations 5 minutes; Loop parameter is 94 ℃ of sex change 40 seconds, 40 seconds, 72 ℃ extensions of 56 ℃ of annealing 90 seconds, carries out 35 circulations; 72 ℃ are fully extended 10 minutes.

PCR reacts end, with 1.0% agarose gel electrophoresis, detect the suitably band of size, and reclaim (according to Takara company " Agarose Gel DNA PurificationKit " operation), carrier and connect and (get 5 μ lSolution I, 4 μ l PCR reclaim product and are connected with 1 μ l pMD18-T carrier, specifically by pMD18-T Vector specification sheets, undertaken), transform and (to connect product and to transform competent escherichia coli cell DH5 α, on the LB plate culture medium that contains penbritin (100mg/L), be inverted for 37 ℃ and cultivate 12-20 hour; Picking white colony, pcr amplification sieve bacterium, activation simultaneously, the positive bacterium colony that picking contains object band, overnight incubation in LB liquid nutrient medium), sequencing (bacterium shaking is got to 1ml and be put in 1.5ml centrifuge tube, sample presentation order-checking).In Beijing Liuhe Huada Genomics Technology Co., Ltd, carry out after sequencing, obtain the Partial Fragment sequence of MdSIMYB2 gene.

2) MdSIMYB25 ' RACE amplification

Factually in example 1 step 2 1) the Partial Fragment sequence that obtains, at N end design nested PCR primer (5RACE1,5RACE2) and universal primer (AAP, AUAP), carry out 5 ' RACE increase (2 take turns pcr amplification).First round PCR: template is the tailing cDNA that example 1 step 1 obtains, primer AAP and 5RACE1; Second takes turns PCR: the PCR product that template is the first round, primer AUAP and 5RACE2; The pcr amplification reaction program of two-wheeled is identical: 94 ℃ of denaturations 5 minutes; Loop parameter is 94 ℃ of sex change 40 seconds, 40 seconds, 72 ℃ extensions of 56 ℃ of annealing 90 seconds, carries out 35 circulations; 72 ℃ are fully extended 10 minutes.

Its sequence of AAP:5 '-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGG-3 ' is as shown in SEQ.ID.NO.5;

Its sequence of 5RACE1:5 '-GAAGGCGATGGAGGCGAAGGATAAG-3 ' is as shown in SEQ.ID.NO.6;

Its sequence of AUAP:5 '-GGCCACGCGTCGACTAGTAC-3 ' is as shown in SEQ.ID.NO.7;

Its sequence of 5RACE2:5 '-CTTCTTGCTCAGGTGTGTGTTCCAGT-3 ' is as shown in SEQ.ID.NO.8;

The 2nd takes turns PCR reaction finish after, carry out that 1.0% agarose gel electrophoresis, PCR product reclaim, carrier connects, transform, order-checking, concrete grammar is with in example 1 step 2 1), obtain 5 ' sequence of MdSIMYB2 gene.

3) MdSIMYB23 ' RACE amplification

Factually in example 1 step 2 1) the intermediate segment sequence that obtains, at C end design nested PCR primer (3RACE1,3RACE2) and universal primer (B26, B25), carry out 3 ' RACE increase (2 take turns pcr amplification).The first round: template is the reverse transcription cDNA that example 1 step 1 obtains, primer 3RACE1 and B26; Second takes turns: template is first round PCR product, primer 3RACE1 and B25; The PCR response procedures of two-wheeled is identical: 94 ℃ of denaturations 5 minutes; Loop parameter is 94 ℃ of sex change 30 seconds, 30 seconds, 72 ℃ extensions of 56 ℃ of annealing 90 seconds, carries out 35 circulations; 72 ℃ are fully extended 10 minutes.

Its sequence of 3RACE1:5 '-ACTATCTCCGCCCTTCTGTCAA-3 ' is as shown in SEQ.ID.NO.9;

Its sequence of B26:5 '-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTT-3 ' is as shown in SEQ.ID.NO.10;

Its sequence of 3RACE2:5 '-GATAAGCCAAGGCATAGATCCCA-3 ' is as shown in SEQ.ID.NO.11;

Its sequence of B25:5 '-GACTCGAGTCGACATCGA-3 ' is as shown in SEQ.ID.NO.12;

Obtain the 3 ' sequence (detailed process with in example 1 step 2 2) of MdSIMYB2 gene).

4) acquisition of cDNA full length sequence

Use DNAMAN software, by example 1 step 2 1), 2), 3) sequencing sequence of gained splices, risk the full-length cDNA of goal gene, splice accordingly primers MdSIMYB2-F and MdSIMYB2-R, the reverse transcription cDNA of take in example 1 step 1 is template, the cDNA total length that pcr amplification is complete.PCR response procedures: 94 ℃ of denaturations 5 minutes; Loop parameter is 94 ℃ of sex change 60 seconds, 60 seconds, 72 ℃ extensions of 56 ℃ of annealing 90 seconds, carries out 35 circulations; 72 ℃ are fully extended 10 minutes.

Its sequence of MdSIMYB2-F:5 '-ATGAGGAACCCATCGTCTTC-3 ' is as shown in SEQ.ID.NO.13;

Its sequence of MdSIMYB2-R:5 '-CTACTCCGTTTGATCATTAA-3 ' is as shown in SEQ.ID.NO.14;

After reaction finishes, carry out electrophoresis, product recovery, carrier connection, transform, check order, thereby obtain the cDNA total length of MdSIMYB2, its nucleotide sequence is as shown in SEQ.ID.NO.1; Its aminoacid sequence is as shown in SEQ.ID.NO.2.Alkaline process extracts the plasmid DNA that obtains pMD18-T-MdSIMYB2, and-20 ℃ of preservations, for follow-up function confirmatory experiment.

Example 2: apple MdSIMYB2 amino acid sequence analysis

One) MdSIMYB2 and AtMYB5, AtMYB17, AtMYB108, PhPH4 aminoacid sequence are compared

1) there is 1095bp Nucleotide MdSIMYB2 gene coding region, with software DNASTAR, carries out sequential analysis discovery, 364 amino acid of this genes encoding, and albumen size is 39.10kD, pI is 8.17.Utilize the analysis software of expasy website (http://www.expasy.org/) and the database of NCBI website (http://www.ncbi.nlm.nih.gov/), MdSIMYB2 albumen is carried out to functional domain and the comparative analysis of conservative territory, (wherein R2 comprises 50-103 amino acids to find to comprise distinctive two the conservative functional domain R2R3 of MYB family by MdSIMYB2 predicted protein, R3 comprises 108-161 amino acids), the results are shown in accompanying drawing 1A.

2) from ncbi database, search out AtMYB5 (NM_112200.2), AtMYB17 (NM_115989.3), AtMYB108(NM_111525.3), PhPH4(AY973324.1) aminoacid sequence.

3) use DNAMAN software, by MdSIMYB2(SEQ.ID.NO:2) compare with the aminoacid sequence of AtMYB5, AtMYB17, AtMYB108, PhPH4.Find MdSIMYB2 and AtMYB5(NM_112200.2), AtMYB17(NM_115989.3), AtMYB108(NM_111525.3), PhPH4(AY973324.1) there is higher homology, especially at N end, there is the R2R3 structural domain (accompanying drawing 1B) of typical MYB family, belong to MYB family, for regulating and controlling the gene of adverse circumstance in apple.

Two) with the albumen cluster analysis of different plant species ZhongMYB family

1) in ncbi database, search out the aminoacid sequence of different plant species ZhongMYB family, specifically comprise: wandering jew TfMYB6 (AY456184.1), cotton GhMYB1 (L04497.1), comospore poplar PtMYB180 (XM_002327312.1), PtMYB111(XM_002302716.1), Arabidopis thaliana AtMYB5 (NM_112200.2), AtMYB17(NM_115989.3), AtMYB108 (NM_111525.3), petunia PhPH4 (AY973324.1), soybean GmW2 (AB597932.1), rice Os MYB4(D88620.1), psyllium CpMYB10 (AF510112.1), Root or stem of Littleleaf Indianmulberry LjTT2a(AB300033.2), barley HvMYB2 (X70876.1), Picea koraiensis Nakai PgMYB5 (EF601068.1), soybean GmMYB54 (DQ822881.1), hop HlMYB1 (AB292244.1).

2) phylogenetic analysis: utilize DNAMAN5.0 software, the albumen of the different plant species ZhongMYB family that search is obtained carries out cluster analysis, constructing system evolutionary tree, the sibship of discovery apple MdSIMYB2 and petunia PhPH4 and Arabidopis thaliana AtMYB5 is (accompanying drawing 2) recently.

The response of example 3:MdSIMYB2 gene pairs abiotic stress

1) the consistent apple loud, high-pitched sound tissue cultured seedling of upgrowth situation carries out adverse circumstance processing (untreated comparing), uses respectively arid (PEG2%), NaCl(200mM), low temperature (4 ℃), ABA (100 μ M) processes 0h, 6h, 12h, 24h, 48h.The material of handling well is fixing in liquid nitrogen, and-80 ℃ of preservations, finally extract RNA together and reverse transcription obtains cDNA.

2) special primer (MdSIMYB2-BDL-R) and the apple internal reference primer (Md18S-F, Md18S-R) of design MdSIMYB2 gene.

Its sequence of Md18S-F:5 '-AAACGGCTACCACATCCA-3 ' is as shown in SEQ.ID.NO.15;

Its sequence of Md18S-R:5 '-CACCAGACTTGCCCTCCA-3 ' is as shown in SEQ.ID.NO.16;

Its sequence of MdSIMYB2-BDL-R:5 '-GATAGTTCATCCAGCGG-3 ' is as shown in SEQ.ID.NO.17;

3) take Md18S-F and the Md18S-R cDNA in primer, example 3 step 1) carries out PCR reaction as template, adjusts template cDNA, makes its concentration consistent.

Response procedures: 94 ℃ of denaturations 4 minutes; Loop parameter is 94 ℃ of sex change 25 seconds, 25 seconds, 72 ℃ extensions of 56 ℃ of annealing 30 seconds, moves 25 circulations; After 72 ℃, extend l0 minute.Amplified production carries out gel electrophoresis analysis, by instrumental analysis (LabAssistant TM Gel3000Series gel image analyser), cDNA is diluted to suitable multiple, repeatedly carries out several times, finally makes its concentration consistent.

4) take the consistent cDNA of concentration is template, MdSIMYB2-F(SEQ.ID.NO.13) with MdSIMYB2-BDL-R(SEQ.ID.NO.17) as primer carries out sxemiquantitative RT-PCR, the differential expression of MdSIMYB2 gene after detecting adverse circumstance and processing.

Response procedures: 94 ℃ of denaturations 5 minutes; Loop parameter is 94 ℃ of sex change 25 seconds, 25 seconds, 72 ℃ extensions of 58 ℃ of annealing 35 seconds, moves 32 circulations; After 72 ℃, extend 10 minutes.Amplified production carries out 1% agarose gel electrophoresis and detects analysis, and result shows MdSIMYB2 gene up-regulated after arid, salt, low temperature, ABA Stress treatment, shows that this gene is subject to arid, salt, low temperature and ABA stress-inducing (accompanying drawing 3)

The structure of example 4:MdSIMYB2 genophore

One) structure of MdSIMYB2 gene overexpression carrier

For the function of research MdSIMYB2 gene, by including MdSIMYB2 gene coding region, in interior common 1095bp fragment, correctly insert on expression vector PBI121.

1) utilize the softwares such as DNAMAN, according to the nucleotide sequence of isolated MdSIMYB2 gene (SEQ.ID.NO:1), design is with primer EMdSIMYB2-F and the EMdSIMYB2-R of restriction enzyme site, and take example 1 step 2 4) in the plasmid DNA of the pMD18-T-MdSIMYB2 that preserved be template, carry out PCR reaction.

EMdSIMYB2-F:5 '-GC tCTAGAits sequence of GCTTTACAATGAGGAACCCATCG-3 ' is as shown in SEQ.ID.NO.18;

Drawing horizontal line is partly Xba I restriction enzyme site;

EMdSIMYB2-R:5 '-GC gTCGACits sequence of CAACCGCATTGCTTGTTCAAC-3 ' is as shown in SEQ.ID.NO.19;

Drawing horizontal line is partly Sal I restriction enzyme site;

2) PCR reaction finishes, carry out electrophoresis, product recovery, carrier connection, transform, check order, choose correct positive colony, LB liquid nutrient medium is cultivated the bacterial strain of positive colony and PBI121 empty carrier, alkali test is extracted its plasmid DNA and PBI121 empty carrier plasmid, and carries out double digestion by restriction endonuclease Xba I and Sal I.

3) with T4 ligase enzyme, recovery fragment MdSIMYB2 is connected with pBI121 carrier, screening positive clone order-checking, confirms to obtain correct intestinal bacteria recon pBI121-MdSIMYB2, transforms Agrobacterium LB4404 competent cell.

4) PCR identifies, the positive bacterium colony of picking.Obtain correct Agrobacterium recon pBI121-MdSIMYB2 mono-clonal, for the conversion of tomato and apple below.

Example 5: obtain transgenic Fructus Lycopersici esculenti

1) sowing: first tomato seeds disinfection: with 70% alcohol disinfecting 1min, 0.7% clorox sterilization 23-25min, aseptic water washing 3 times, blots water respectively.The seed of handling well is laid on seed germination substratum, is secretly cultured to radicle and grows (4d), and light cultivation (5-7d) to cotyledon launches completely.

2) preculture: cut the cotyledon launching completely, go to two ends, stay middle (about 0.5cm), put (blade phototropic face upwards) on pre-culture medium, be inverted and cultivate 2d.

3) infect the preparation of bacterium liquid: picking Agrobacterium mono-clonal bacterium colony in 10mL YEP liquid nutrient medium (containing 50mg/L kantlex), 28 ℃, 200rpm, shaking culture 48h(OD600:0.6-0.8); Get lmL bacterium liquid and add in fresh 20mL YEP liquid nutrient medium, shaking culture 5h (OD600:06-0.8).Centrifugal collection thalline, suspends and dilutes 20 times with MS liquid nutrient medium, standby;

4) infect: will during pre-incubated tomato leaf immerses bacterium liquid 10min(, repeatedly rock), abandon bacterium liquid, and with aseptic filter paper exhaustion, be transferred on common substratum, sealing, 28 ℃ of dark 2d that cultivate.

5) be total to the sterilized water washing containing Pyocianil (250mg/L) 3 times for tomato leaf after cultivating, sterilizing filter paper blots, and moves under division culture medium glazing and cultivates.Every 15d, change a subculture.

6) when 1cm left and right is grown in bud differentiation, cut, part succeeding transfer culture, part moves on root media takes root.After root system development is good, after hardening, moves into and fill in the nutrition pot of matrix, greenhouse Routine Management.

Note: materials disinfection is processed, infected, inoculation etc. is all carried out on Bechtop;

Tomato Seeds Germination substratum: 1/2MS;

Tomato preculture, common culture medium: MS+IAA0.5mg/L+ZT2mg/L;

Tomato division culture medium: MS+IAA0.5mg/L+ZT2mg/L+Kana30mg/L+Cb250mg/L;

Tomato root media: MS+IAA0.1mg/L;

Culture condition: 25-28 ℃, light application time 16h/d, intensity of illumination 2000Lux.

Example 6: obtain transgenic apples

1) preculture: get the tender leaf of the loud, high-pitched sound tissue cultured seedling top 3-4 sheet expansion of cultivating about 4 weeks, in vertical vein direction 2 roads that row dry, put (face of blade upward) on pre-culture medium, secretly cultivate 2d; Be ready to infect the Agrobacterium bacterium liquid (concrete steps are with 3 in example 5) of use.

2) infect: with Agrobacterium bacterium liquid, infect pre-incubated Apple Leaves 20min (during rock several times), aseptic filter paper blots bacterium liquid, move on MS minimum medium and cultivate altogether, secretly cultivate 2d.

3) differentiation culture: the sterilized water washing containing Pyocianil (250mg/l) 3 times for the Apple Leaves after cultivating altogether, with the MS liquid nutrient medium containing Pyocianil (250mg/l), wash 1 time, sterilizing filter paper blots, and moves to dark cultivation 3 weeks on division culture medium.The division culture medium more renewing, carries out light cultivation.After resistance regeneration bud differentiates, cut budlet, on proliferated culture medium, expand numerous (within every 4 weeks, subculture is 1 time).

Note: materials disinfection is processed, infected, inoculation etc. is all carried out on Bechtop.

Loud, high-pitched sound apple tissue cultured seedling pre-culture medium: MS+6-BA0.5mg/l+NAA0.15mg/l;

Loud, high-pitched sound apple division culture medium: MS+TDZ2.0mg/l+IAA0.2mg/l+Cb250mg/L;

Loud, high-pitched sound apple tissue cultured seedling proliferated culture medium: MS+6-BA0.7mg/l+NAA0.15mg/l+Cb250mg/L.

Culture condition: 25-28 ℃, light application time 16h/d, intensity of illumination 2000Lux.

Example 7: transgenic Fructus Lycopersici esculenti is carried out to drought-resistant, low temperature and salt capability analysis

Homozygote transgenic Fructus Lycopersici esculenti strain is carried out to degeneration-resistant border capability analysis, to determine the function of transfer-gen plant.

1) MdSIMYB2 gene is at the relative expression quantity of different strain transgenic Fructus Lycopersici esculentis.

Take through the tomato conversion strain DNA of antibiotic-screening is template, take 35S and MdSIMYB2-R(SEQ.ID.NO.14) carry out PCR reaction screening transgenic strain as primer; Extract the wherein RNA of 4 transgenic lines, carry out sxemiquantitative RT-PCR(concrete grammar and see example 3, the internal reference primer of tomato is: leACTIN-F, LeACTIN-R), the expression level of MdSIMYB2 gene in detection transgenic line.The expression amount of MdSIMYB2 gene in different strains is different, and wherein OE-4 expression amount the highest (accompanying drawing 4A), chooses 3 strain OE-1 that expression amount is higher, OE-3, and OE-4, several generations individual plant sowing, obtains homozygote seed and carries out follow-up transgenosis functional verification.

Its sequence of 35S:5'-CGCACAATCCCACTATCCTT-3' is as shown in SEQ.ID.NO.20

Its sequence of LeACTIN-F:5'-CTTCAGTCCACAATCGGTGG-3' is as shown in SEQ.ID.NO.21;

Its sequence of LeACTIN-R:5'-CATTCCGAGTTGAGCTGCTG-3' is as shown in SEQ.ID.NO.21;

2) drought-resistant, the low temperature of transgenic Fructus Lycopersici esculenti and salt capability analysis:

The transgenic Fructus Lycopersici esculenti seed OE-1 that will isozygoty, OE-3, OE-4 and wild-type contrast WT, plant in the flowerpot of sterile soil is housed, and normal management is grown 6 weeks.To the transgenic Fructus Lycopersici esculenti of state consistency and contrast, carry out respectively arid, salt, the processing of cold adverse circumstance, observe upgrowth situation separately.B1: all tomato seedlings of normal growth to 6 compare; B2: 6 weeks tomato seedlings that increment is consistent, 10d does not water and is the upgrowth situation that arid is processed: B3 continuously: 6 weeks tomato seedlings that increment is consistent, with 300mM NaCl, process the upgrowth situation after 12d; B4: 6 weeks tomato seedlings that increment is consistent, the upgrowth situation after 4 ℃ of subzero treatment 4d.Result shows: after arid is processed, transfer-gen plant is grown better, and wild-type is wilted; After 300mM NaCl processes, wild-type is wilted, blade flavescence, and Lao Ye fallen leaves are serious, and the flavescence of transgenic line blade is lighter, and growth conditions is better; After 4 ℃ of deepfreezes, transgenic line is also obviously good than wild-type growth conditions, and frostbite is wilted, had to wild-type strain.These results suggest that: MdSIMYB2 gene proceeds in tomato, improved anti-salt, drought resisting and the anti-low temperature ability of Transgenic Tomato Plants.

Example 8: the Analysis of Resistance to transgenosis loud, high-pitched sound apple seedling

1) MdSIMYB2 gene is at the relative expression quantity of the different strains of transgenic apples.

Utilize screening culture medium (containing 30mg/L kantlex) to screen candidate's transgenic line of the anti-kantlex positive, obtain altogether the positive strain (T1-T4) of 4 35S:MdSIMYB2; For further identifying transgenic line, when carrying out succeeding transfer culture, the tissue cultured seedling that every strain is got 0.1g left and right, extracts corresponding RNA, reverse transcription is also carried out sxemiquantitative RT-PCR detection (concrete grammar is shown in example 3), to determine the expression level of MdSIMYB2 gene in these strains.Result shows, MdSIMYB2 gene is at T-1, T-2, overexpression in T-3 and T-4 (accompanying drawing 5A).Select T-1, T-2, T-4 partly continues to expand numerous, and part moves into carries out root culture on root media.Root system is grown (approximately 4 weeks), moves into and fills in the nutrition pot of matrix, greenhouse Routine Management after hardening.

2) adverse circumstance is processed: select consistent 3 transgenic lines (T-1, T-2, T-4) of growth conditions and contrast, carry out adverse circumstance under arid, salt and cool condition and process.B1: 15d does not water continuously, the growth conditions after arid is processed; B2: after arid is processed, the recovery upgrowth situation normally watering after management 3d; C1: process after 3 times (3d processes 1 time) upgrowth situation at 14d with 300mM NaCl; C2: salt process after to the upgrowth situation of 16d; D1:4 ℃ of cold condition cultivated the upgrowth situation after 16d; D2: after subzero treatment, be cultured to the recovery upgrowth situation of 25d 22 ℃ of normal tempss.

3) apple seedling abiotic stress is processed to rear contrast and the transgenic line growing state observed.B1～B2: arid contrasts and wilts after processing, and transgenic line is also wilted, but after rehydration, transgenic line can partially or completely recover growth, and contrast recovers to grow, because excessively arid is dead; C1～C2: after salt is processed, contrast blade is withered, almost can not normal growth, though and overexpression strain is subject to salt damage serious, substantially can grow; D1～D2: contrast spire after deepfreeze 16d and wilt seriously, blade almost reddens completely because of cold, almost can not recover growth; Although and transgenic line blade also reddens because of cold, stop growing, carry out, after the growth of normal temps managing to resume, substantially restoreing normal growth.These results have absolutely proved that MdSIMYB2 gene is a degeneration-resistant relevant gene, and the overexpression of this gene can improve the anti-adversity ability of transgenic apples.

Loud, high-pitched sound apple root media: 1/2MS or MS+IAA0.2mg/l.

According to above-mentioned technology, from apple, be separated to the myb gene MdSIMYB2 of a R2R3 type, by finding out tomato and apple transfer gene function analysis: it has obvious effect in the extraneous arid of opposing, low temperature and high salt, illustrate that MdSIMYB2 gene can participate in plant opposing abiotic stress process.Can be by annual crops such as this gene transformation wheat, corn, cottons, or the perennial woody plant such as apple, pears, improving its degeneration-resistant border ability, the ability of especially anti-salt, arid and low temperature, improves output, produces important economic benefit and social benefit.

Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Sequence table

Claims (2)

1. an apple MdSIMYB2 gene, is characterized in that its nucleotide sequence is as shown in SEQ.ID.NO.1; Its nucleotide sequence coded aminoacid sequence is as shown in SEQ.ID.NO.2.

2. the application of apple MdSIMYB2 gene as claimed in claim 1 in improving drought-resistant, the anti-salt of transgenic apples and anti-low temperature ability.