CN102464708B - Protein related to plant stress resistance and coding gene thereof as well as application thereof - Google Patents
Protein related to plant stress resistance and coding gene thereof as well as application thereof Download PDFInfo
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Abstract
The invention discloses a protein related to plant stress resistance and a coding gene thereof as well as application thereof. The protein related to plant stress resistance, provided by the invention, is as shown in (a) or (b), wherein the protein as shown in (a) is formed by an amino acid sequence as shown in a sequence 1 in a sequence table, and the protein as shown in (b) is formed by replacement and/or deletion and/or addition of one or several amino acid residue sequences to an amino acid residue sequence of the sequence 1 in the sequence table and is related to the plant stress resistance and derived from (a). The expression of the protein related to plant stress resistance, provided by the invention, is induced by adverse situations such as drought, low temperature, high salt or ABA (Abscisic Acid) and the like; the protein can participate in the response of leymus chinensis to multiple adversity stresses and increases the plant stress resistance. The protein related to the plant stress resistance and the coding gene of the protein have important theoretical and practical significance for cultivating the leymus chinensis with increased stress resistance and other new plantvarieties.
Description
Technical field
The present invention relates to albumen relevant with stress resistance of plant in the biological technical field and encoding gene thereof and application.
Background technology
Bad physical environment, act on plant as low temperature, high temperature, arid and salt marsh etc. and can cause that a series of physiological metabolism takes place in the plant materials reacts, the reversibility that shows as metabolism and growth suppresses, and when serious even cause irreversible injury, thereby causes whole plant death.In various the coercing, arid, saline and alkaline, cold (freezing) evil is particularly outstanding to the influence of plant, is the topmost environmental factor that influences plant-growth and crop yield.Utilize the low temperature patience of genetic engineering means improvement crop, the harm of avoiding as far as possible or alleviating the poor environment factor is the research focus in current biology and modern agricultural technology field, also is the key subjects that China and world agriculture are badly in need of solution.Plant is called resistance to the opposing of coercing and the ability of restraining oneself, and resistance is the adaptability to poor environment that plant forms in the long-term evolution process.For many years, people have carried out big quantity research from physiology, biochemistry, metabolism, ecology and heredity, evolution equal angles, accumulated abundant data, particularly along with development of molecular biology, people can be in genomic constitution, expression regulation and the resistance of reverse mechanism of signal conduction equimolecular level understanding plant to coercing, for the anti-performance of coercing of utilizing genetic engineering means improvement plant has been opened up new approach.Because the complicacy of plant stress-resistance proterties, adopt the resistance of traditional breeding method raising plant very difficult, along with development of molecular biology, genetic engineering means has been opened up the new way of plant stress-resistance breeding, but being separated into of efficient adversity gene limits the engineered principal element of plant stress-resistance.In the past, the clone of adversity gene and application mainly are single functional genes, and as trimethyl-glycine synthase gene, proline(Pro) synthase gene etc., though obtained certain effect, the resistance of plant is not comprehensively improved.
Stress resistance of plant is subjected to controlled by multiple genes, numerous functional genes that the plant stress-resistance proterties is exerted an influence are given full expression to and plays a role, resistance that could more effective improvement plant.Along with the continuous development of biotechnology, research emphasis has turned to the regulatory factor (as promotor and transcription factor) of each species specificity or high efficiency from general functional gene.Because a transcription factor can be regulated and control a plurality of and resistance Expression of Related Genes in the plant, strengthens the effect of a transcription factor, just can make the degeneration-resistant proterties of plant obtain comprehensive improvement.
Summary of the invention
An object of the present invention is to provide the albumen relevant with stress resistance of plant and encoding gene thereof.
The albumen relevant with stress resistance of plant provided by the present invention, name is called LcMYB13355, derives from sheep's hay (Leymus chinensis), is following (a) or protein (b):
(a) protein of being formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the amino acid residue sequence of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with resistance protein of being derived by (a).
Described and encoding gene plant adversity resistance related protein also belongs to protection scope of the present invention.
Described and encoding gene plant adversity resistance related protein are following 1)-4) in arbitrary described gene:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 133rd to the 2388th Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under stringent condition with 1) or 2) shown in dna molecule hybridize and the gene of encoding said proteins;
4) with 1) or 2) or 3) gene have homology more than 90% and the gene of encoding said proteins.
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5%SDS 65 ℃ of hybridization down, is used 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Sequence 2 in the sequence table is by 2697 based compositions, and its open reading frame (ORF) is from 5 ' terminal 133-2388 bit base, and encoding amino acid sequence is the albumen shown in the sequence 1 in the sequence table.
The expression cassette, recombinant expression vector, transgenic cell line or the reorganization bacterium that contain the encoding gene of described and plant adversity resistance related protein also belong to protection scope of the present invention.
Described recombinant expression vector is to insert the described recombinant expression vector that obtains with encoding gene plant adversity resistance related protein between the multiple clone site of p3301-121 carrier;
Described p3301-121 carrier is to obtain by the method that comprises the steps:
(1) with pCAMBIA3301 carrier process HindIII and EcoRI double digestion, reclaims the big fragment of carrier;
(2) with pBI121 carrier process HindIII and EcoRI double digestion, reclaim the fragment that comprises gus gene;
(3) the big fragment of carrier that reclaims in the step (1) is connected with the fragment that comprises gus gene of recovery in the step (2), obtains recombinant vectors p3301-121.
Described pCAMBIA3301 carrier is available from CAMBIA company; Described pBI121 carrier is available from Clontech company.
Described reorganization bacterium is recombination microzyme.
The primer of the encoding gene total length of described and plant adversity resistance related protein of increasing or its arbitrary fragment is to also belonging to protection scope of the present invention.
Described primer centering, a primer sequence is shown in sequence in the sequence table 3, and another primer sequence is shown in sequence in the sequence table 4.
Another object of the present invention provides a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant provided by the present invention is that described encoding gene with plant adversity resistance related protein is imported in the purpose plant, obtains the transgenic plant that resistance strengthens.
Described is to import in the purpose plant by described recombinant expression vector with encoding gene plant adversity resistance related protein.
Described purpose plant is dicotyledons or monocotyledons; Described dicotyledons is Arabidopis thaliana; Described resistance is the resistance to low temperature, high salt, arid, dormin and/or methyl jasmonate; Described low temperature is 4 ℃.
Available existing plant expression vector construction contains the recombinant expression vector of LcMYB13355 gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA1302, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries Leymus chinensis low-temperature induced transcription factor encoding gene LcMYB13355 of the present invention can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.Both can be monocotyledonss such as wheat, paddy rice, corn, false bromegrass by the plant transformed host, also can be dicotyledonss such as Arabidopis thaliana, tobacco, soybean, cucumber, tomato, cotton, willow, lucerne place.
Provided by the present invention and expression plant adversity resistance related protein is induced by adverse circumstances such as arid, low temperature, high salt or ABA, can participate in sheep's hay to the response of multiple environment stress, improves the resistance of plant.Have important theory and practical significance with plant adversity resistance related protein and encoding gene thereof for sheep's hay and other neies variety of plant of cultivating the resistance raising.
Description of drawings
Fig. 1 is the agarose gel electrophoresis detected result through the total RNA of chinense seedlings of 4 ℃ of subzero treatment.
Fig. 2 is the agarose gel electrophoresis detected result of the 3 ' terminal sequence of pcr amplification LcMYB13355.
Fig. 3 is the agarose gel electrophoresis detected result of the 5 ' terminal sequence of pcr amplification LcMYB13355.
Fig. 4 is the agarose gel electrophoresis detected result of pcr amplification LcMYB13355 full-length cDNA.
Fig. 5 for the LcMYB13355 gene in different low temperature, high salt, arid with abduction delivering result under the stress conditions such as cradle.
Fig. 6 is the tissue specific expression analytical results of LcMYB13355 gene.
Fig. 7 activates the activation analysis result for the LcMYB13355 gene transcription.
Fig. 8 is the building process figure of recombinant expression vector p3301-121-LcMYB13355.
Fig. 9 is the growing state that changes LcMYB13355 gene Arabidopis thaliana under the low temperature stress treatment condition.
Figure 10 is the building process of p3301-121 carrier.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
One, the albumen relevant with stress resistance of plant and the acquisition of encoding gene thereof
1, the clone of 3 ' terminal sequence of LcMYB 13355 genes
(1) extraction of vegetable material processing and total RNA
Be material with sheep's hay (lucky give birth to No., available from Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) seedling, handle after 6 hours and 12 hours for 4 ℃, mixed 6 hours and the sample of 12 hour two different treatment time, extract its total RNA, carry out 1% agarose gel electrophoresis and detect, the result as shown in Figure 1.The result shows that the RNA that extracts has two tangible electrophoretic bands, is followed successively by 28S RNA and 18S RNA from top to bottom, shows to have obtained higher, the more complete total RNA of purity.
(2) clone of LcMYB 13355 genes 3 ' terminal sequence
According to the amino acid residue sequence design primer of GS-FLX 454 (being called for short 454) order-checking sheep's hay MYB, concrete primer sequence is as follows:
LC13355racef：5′-GCCAAATCGCCACCTCAAAGTGACA-3′，
The total RNA through the chinense seedlings of subzero treatment that extracts with above-mentioned steps (1) is template, uses PrimeScript
TMThe requirement of 1st Strand cDNA Synthesized Kit test kit (Takara company) and reference reagent box specification sheets, synthetic its first chain cDNA of counter-rotating.Reaction system and reaction conditions are as follows: Oligo-dT (10pmol/ μ l) 1 μ l, Total RNA (≤1 μ g) 2 μ l, dNTP Mixture (10mmol/l each) 1.0 μ l, 5 * Buffer, 4.0 μ l, RNaseInhibitor (40U/ μ l) 0.5 μ l, PrimeScript RTase (200U/ μ l) 0.5 μ l, RNase-free distilled water11 μ l; 65 ℃ of 5min, 42 ℃ of 45min, 70 ℃ of 15min.With the first synthetic chain cDNA be stored in-20 ℃ standby.
Be template with the first chain cDNA that obtains again, pcr amplification is carried out in primer LC13355racef and primer OligodT-adaptor 5 '-GATTTCTGTCCGACGACTTTTTTTTTTTTTTTTTT-3 ' pairing; The PCR reaction system is: each 1 μ l of cDNA template, primer LC1 and primer OligodT-adaptor, 10 * Buffer, 2.5 μ l, dNTP Mixture (10mmol/l each) 2 μ l, Taq enzyme 0.25 μ l, ddH
2O 12.25 μ l; Reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of 30s then, 55 ℃ of 30s, 72 ℃ of 60s, totally 36 circulations; Last 72 ℃ are extended 10min.
After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, the result as shown in Figure 2.Wherein, swimming lane M is the dna molecular amount standard of DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is 3 ' RACE pcr amplification product, the result shows, has obtained length through pcr amplification and has been about purpose fragment about 1300bp.Reclaim and purifying 3 ' RACE product, be connected on the PMD-18T carrier, connect product transformed into escherichia coli DH5 α competent cell, screening positive clone carries out bacterium liquid PCR to be identified, the plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.The result shows that the length of this fragment is 1346bp, and 3 ' terminal sequence of known MYB genoid has higher homology in this fragments sequence and the plant, shows that this fragment may be the 3 ' terminal sequence of sheep's hay MYB class in conjunction with protein coding gene.
2, the clone of LcMYB 13355 gene 5 ' terminal sequences
Hold cDNA sequences Design primer according to LcMYB 13355 genes 3 ' that above-mentioned steps one obtains: 13355racer:5 '-TTCGTTCCAGCAGCCCCCAGTCGTCG-3 ', the total RNA through the chinense seedlings of subzero treatment that extracts with step 1 is template, adopt 5 ' RACE test kit and the reference reagent box specification sheets of Promega company, its first chain cDNA is synthesized in reverse transcription.Reaction system and condition are as follows: 1 μ l RNA, 1 μ l 5 '-CDS primerA, 1 μ l SMARTII A oligo, 1 μ l DTT (20mM), 1 μ l dNTP Mix (10mM), 1 μ l MMLV ReverseTranscriptase, 2 μ l 5X First-Strand Buffer, 2 μ l sterile H
2O; 70 ℃ of 2min, 2min on ice, 42 ℃ of 1.5h, 72 ℃ of 7min.
The first chain cDNA with acquisition is template, primer 13355racer and primer UPM (Promega company: Long (0.4 μ M): 5 '-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ', Short (2 μ M): 5 '-CTAATACGACTCACTATAGGGC-3 ') match and carry out pcr amplification; The PCR reaction system is: 1 μ l 50X Advantage 2Polymerase Mix, 34.5 μ l PCR-Grade Water, 5 μ l 10XAdvantage 2PCR Buffer, 1 μ l dNTP Mix (10mM), 1 μ l 50XAdvantage 2Polymerase Mix, 5 μ lUPM, 1 μ l primer R, 2.5 μ lcDNA templates; Reaction conditions is: first 94 ℃ of 30s, 68 ℃ of 30s then, 70 ℃ of 60s, totally 40 circulations; Last 70 ℃ are extended 10min.
After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, the result as shown in Figure 3.Wherein, swimming lane M is the dna molecular amount standard of DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is 5 ' RACE pcr amplification product, the result shows, has obtained the purpose fragment that length is about 1600bp through pcr amplification.Reclaim and purifying 5 ' RACE product, be connected on the PMD-18T carrier, connect product transformed into escherichia coli DH5 α competent cell, screening positive clone carries out bacterium liquid PCR to be identified, the plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.The result shows that the length of this fragment is 1597bp, and 5 ' terminal sequence of known MYB genoid has higher homology in this fragments sequence and the plant, shows that this fragment may be the 5 ' terminal sequence of sheep's hay MYB class in conjunction with protein coding gene.
3, the acquisition of LcMYB 13355 full length gene cDNA sequences and PCR detect
The length of utilizing above-mentioned steps 1 and step 2 to obtain is the overlap between 1346bp and the 1597bp fragment, the full length cDNA sequence of the LcMYB gene that splicing obtains by Contig software, and its deoxyribonucleotide sequence is shown in sequence in the sequence table 2.Sequence 2 is encoding sequence by 2697 based compositions from 5 ' the 133rd the-the 2388th at end in the sequence table, and coding has the protein of the amino acid residue sequence shown in the sequence 1 in the sequence table, and sequence 1 is made up of 751 amino-acid residues in the sequence table.According to the following primer of LcMYB full length gene cDNA sequences Design:
13355fullf:5 '-CTCCAAATCCCTAACTCCCCAAT-3 ' and
13355fullr：5′-GACTCCAATTGACCCAAGCTC-3′。
The total RNA through the chinense seedlings of subzero treatment that extracts with above-mentioned step 1 is template through the first synthetic chain cDNA of reverse transcription, carries out pcr amplification.Pcr amplification product is carried out 1% agarose gel electrophoresis detect, the result as shown in Figure 4.Wherein, swimming lane M is the dna molecular amount standard of DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is pcr amplification product.The result shows, obtained the fragment that length is about 2700bp through pcr amplification.Reclaim and this product of purifying, be connected on the PMD-18T carrier, connect product transformed into escherichia coli DH5 α competent cell, screening positive clone carries out bacterium liquid PCR identifies that the plasmid that extracts positive colony checks order.Sequencing result shows, the sequence that this pcr amplification product and above-mentioned splicing obtain is identical in the amplification part, pcr amplification product sequence total length is 2675bp, show that 3 ' RACE fragment and 5 ' the RACE fragment of cloning belong to same gene, be LcMYB13355 with this unnamed gene, the nucleotide sequence of this gene is shown in sequence in the sequence table 2; With the albumen called after LcMYB 13355 of this genes encoding, the aminoacid sequence of this albumen is shown in sequence in the sequence table 1.
Two, the expression pattern analysis of LcMYB 13355 genes under different abiotic stress factor conditions
With the normal growth sheep's hay in 8 weeks (lucky giving birth to No. one, available from Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) seedling carries out the low temperature (4 ℃) of different time (0,0.5,3,6,12,24 and 48 hour), high salt (400mM NaCl), arid (20% polyethylene glycol 6000), dormin (ABA respectively, 100 μ M) and methyl jasmonate (MJ, 100 μ M) coerce processing.Respectively each is handled the sample mix of different time, extract total RNA of each chinense seedlings of handling, the sxemiquantitative PCR method is analyzed the expression pattern of LcMYB13355 gene under different abiotic stress factor conditions, the result as shown in Figure 5, NaCl represents that salt stress, PEG represent that drought stress, ABA represent that dormin is coerced among Fig. 5, MJ represents that methyl jasmonate is coerced, cold represents low temperature stress.The result shows that LcMYB13355 gene transcription level obviously is subjected to inducing of low temperature and salt stress.
Three, the tissue specific expression analysis of LcMYB 13355 genes
Extract total RNA of root, rhizome, leaf sheath, leaf, stem, rhizome bud and seven kinds of tissues of young fringe of the chinense seedlings of handling through low temperature stress among the embodiment 1 respectively, utilize primer 5 '-AATCGCCACCTCAAAGTG-3 ' and 5 '-TGTATTAGCAACAAGCAGCC-3 ' carries out RT-PCR and analyzes, simultaneously with the Actin gene as interior mark.After reaction finishes, the RT-PCR product is carried out 1% agarose gel electrophoresis detect, the result as shown in Figure 6.The result shows that there is notable difference in the expression of LcMYB13355 gene in different tissues, and be minimum with expression amount in the leaf sheath, in stem, leaf and the root content secondly, expression amount is higher in rhizome bud, young fringe and the rhizome.
Four, the LcMYB13355 gene transcription activates activation analysis
The LcMYB13355 gene design primer that 1 amplification obtains according to above-described embodiment adds EcoRI and BamHI restriction enzyme site, and primer sequence is as follows:
Primer 1:G
GAATTCATGGAGAAGGAGGTGTGTGAA (underscore partly is the EcoRI restriction enzyme site),
Primer 2: CG
GGATCCCTACATGATGGGTGGAAGAA (underscore partly is the BamHI restriction enzyme site),
Be template with the LcMYB13355 gene shown in the sequence 2 in the sequence table of synthetic, carry out pcr amplification, amplified production is cut through EcoRI and BamHI enzyme, reclaim the LcMYB13355 gene fragment after enzyme is cut; Simultaneously, cut Yeast expression carrier pBridge (available from CAMBIA company) with EcoRI and BamHI enzyme, reclaim the big fragment of carrier; The big fragment of carrier that reclaims is connected with LcMYB13355 gene fragment after the enzyme of recovery is cut, obtains the purpose plasmid.The purpose plasmid is changed in the intestinal bacteria, resistance screening, the picking positive colony, positive colony is carried out liquid culture, extract the positive colony plasmid and carry out sequence verification, sequencing result shows inserted the LcMYB13355 gene fragment shown in the 133rd to 2388 of the sequence 2 in the sequence table between the EcoRI of carrier pBridge and BamHI restriction enzyme site, proves that plasmid construction is correct, with the recombinant vectors called after pBridge-BD-LcMYB13355 that obtains.It is (available from U.S. Clontech company that recombinant vectors pBridge-BD-LcMYB13355 is imported to the yeast AH109 strain that contains His3 and LacZ reporter, cat. no K1612-1) in, simultaneously Yeast expression carrier pBridge is changed over to yeast AH109 strain system as changeing the empty carrier contrast, changing pGAL4 (available from U.S. Clontech company) over to yeast AH109 strain is as over against photograph.The transgenic yeast bacterium is cultivated at the SD substratum (SD/-His-Trp) that does not contain Histidine (His) and tryptophane (Trp), and the result is shown in a among Fig. 7 and b.Among Fig. 7 a represent to change the LcMYB13355 gene yeast strain (C), change pGAL4 over to as over against the yeast strain (B) of photograph and change empty carrier contrast yeast strain (A) and lacking the selection substratum upgrowth situation of Histidine and tryptophane; B is the yeast of a among Fig. 7 situation after with the Z-buffer colour developing that contains X-gal, (betagalactosidase activity filter paper analysis) among Fig. 7.The result shows, change empty carrier contrast yeast strain (A) and do not containing on the SD substratum of His and Trp and can not grow, can and show blue and change the yeast strain (C) of LcMYB13355 gene and change pGAL4 in the SD substratum growth that does not contain His and Trp as the yeast strain (B) over against photograph.The result shows that LcMYB13355 has transcriptional activation activity.
The resistance analysis of embodiment 2, commentaries on classics LcMYB13355 gene Arabidopis thaliana plant
One, obtains to change LcMYB13355 gene Arabidopis thaliana plant
The LcMYB13355 gene design primer that 1 amplification obtains according to above-described embodiment adds BamHI and SmaI restriction enzyme site, and primer sequence is as follows:
Primer 3:CG
GGATCCATGGAGAAGGAGGTGTGTGAA (underscore is the BamHI site) (sequence 3 in the sequence table),
Primer 4:TCC
CCCGGGCTACATGATGGGTGGAAGAA (underscore is the SmaI site) (sequence 4 in the sequence table),
Be template with the LcMYB13355 gene shown in the sequence 2 in the sequence table of synthetic, carry out pcr amplification, the PCR product cloning that amplification obtains is gone into pMD18Simple-T (TaKaRa company) carrier, called after pMD18-LcMYB13355.Utilize BamHI and SmaI to carry out double digestion pMD18-LcMYB13355, reclaim the fragment of about 2.3kb, use BamHI and the big fragment of SmaI double digestion p3301-121 carrier recovery carrier simultaneously, the big fragment of carrier that reclaims is connected with the LcMYB13355 gene fragment of the 2.3kb of recovery, obtains the purpose plasmid.The purpose plasmid is changed in the intestinal bacteria, resistance screening, the picking positive colony, positive colony is carried out liquid culture, extract the positive colony plasmid and carry out sequence verification, sequencing result shows inserted the LcMYB13355 gene fragment shown in the 133rd to 2388 of the sequence 2 in the sequence table between the BamHI of carrier p3301-121 and SalI restriction enzyme site, the proof plasmid construction is correct, with the plant binary expression vector that the LcMYB13355 gene complete is read frame that contains that obtains, called after p3301-121-LcMYB13355, building process are as shown in Figure 8.The plant expression vector p3301-121-LcMYB13355 that builds changes Agrobacterium EHA105 (available from Beijing Baeyer enlightening Bioisystech Co., Ltd) over to by freeze-thaw method, carry out the Herbicid resistant screening, the picking positive colony, positive colony is carried out liquid culture, contaminate the Arabidopis thaliana inflorescence with positive colony bacterium liquid and carry out the Arabidopis thaliana conversion, transform the dark cultivation in back 2 days, carry out the second time and contaminate, the normal cultivation to gathered in the crops seed then.The seed of results carries out resistance screening containing the flat board of weedicide (1/2MS+10mg/L Glufosinate ammonium), screen positive seedling, results seed (T1), the seed of results is containing the flat board of weedicide (1/2MS+10mg/L Glufosinate ammonium) plantation, the screening offspring separates than the strain system that is 3: 1, gather in the crops its seed (T2), (T3) does degeneration-resistant experiment with its seedling.According to the method that obtains to change LcMYB13355 gene Arabidopis thaliana, with empty carrier p3301-121 arabidopsis thaliana transformation plant, obtain changeing empty carrier contrast Arabidopis thaliana plant.
Described p3301-121 carrier is to obtain by the method that comprises the steps:
(1) with pCAMBIA3301 carrier (available from CAMBIA company) process HindIII and EcoRI double digestion, reclaims the big fragment of carrier of 11246bp;
(2) (contain 35S promoter, gus reporter gene Tnos) also passes through HindIII and EcoRI double digestion, reclaims the fragment of the 2942bp that comprises gus gene with pBI121 carrier (available from Clontech company);
(3) the big fragment of carrier that reclaims in the step (1) is connected through the T4DNA enzyme with the middle fragment that comprises gus gene that reclaims of step (2), obtains recombinant vectors p3301-121 (Figure 10).
Two, change the resistance analysis of LcMYB13355 gene Arabidopis thaliana plant
The seed of the commentaries on classics LcMYB13355 gene Arabidopis thaliana plant that above-mentioned steps one is obtained, changeing seed and the wild-type Arabidopis thaliana seed of empty carrier contrast Arabidopis thaliana plant plants respectively in containing on the weedicide substratum (1/2MS+10mg/L Glufosinate ammonium), (growth conditions is: temperature is 22-24 ℃ to germination and growth, illumination-8 in the 16 hours hour dark photoperiod, atmospheric moisture is 60%-80%) after the week, be transplanted in the soil, (culturing room's culture condition: temperature is 22-24 ℃ in culturing room's cultivation, illumination-8 in the 16 hours hour dark photoperiod, atmospheric moisture is 50-60%) after the week, carry out low temperature stress at 4 ℃ and handle.Coerce to handle after 4 hours and observe phenotype, observe to coerce and handled the 7th day.Coercing processing preceding 6 days transgenic arabidopsis plant, commentaries on classics empty carrier contrast Arabidopis thaliana plant and wild-type Arabidopis thaliana plant does not all have obvious phenotypes to change, handled the 7th day from coercing, wild-type Arabidopis thaliana plant has 95% plant blade dehydration wilting symptom (A among Fig. 9) to occur; Have 85% plant not have obvious phenotypes to change (B among Fig. 9) and change LcMYB13355 gene Arabidopis thaliana seedling plant, it is consistent with the phenotype of wild-type Arabidopis thaliana plant to change empty carrier contrast Arabidopis thaliana plant.This experimental result shows that changeing LcMYB13355 gene Arabidopis thaliana compares with the wild-type Arabidopis thaliana with changeing empty carrier contrast Arabidopis thaliana, has stronger anti-low temperature ability.
Claims (8)
1. albumen, the protein of being formed by the aminoacid sequence shown in the sequence in the sequence table 1.
2. the encoding gene of the described albumen of claim 1.
3. encoding gene according to claim 2 is characterized in that: the encoding gene of described albumen for as 1) or 2) described gene:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 133rd to the 2388th Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table.
4. the expression cassette, recombinant expression vector or the reorganization bacterium that contain claim 2 or 3 described encoding genes.
5. recombinant expression vector according to claim 4 is characterized in that: described recombinant expression vector is to insert the recombinant expression vector that described encoding gene obtains between the multiple clone site of p3301-121 carrier;
Described p3301-121 carrier is to obtain by the method that comprises the steps:
(1) with pCAMBIA3301 carrier process HindIII and EcoRI double digestion, reclaims the big fragment of carrier;
(2) with pBI121 carrier process HindIII and EcoRI double digestion, reclaim the fragment that comprises gus gene;
(3) the big fragment of carrier that reclaims in the step (1) is connected with the fragment that comprises gus gene of recovery in the step (2), obtains recombinant vectors p3301-121.
6. reorganization bacterium according to claim 4, it is characterized in that: described reorganization bacterium is recombination microzyme.
7. the primer of the described encoding gene of amplification claim 2 is right, described primer centering, and a primer sequence is shown in sequence in the sequence table 3, and another primer sequence is shown in sequence in the sequence table 4.
8. a method of cultivating transgenic plant is that claim 2 or 3 described encoding genes are imported in the purpose plant, obtains the transgenic plant that resistance strengthens;
Claim 2 or 3 described encoding genes are to import in the purpose plant by claim 4 or 5 described recombinant expression vectors;
Described purpose plant is Arabidopis thaliana;
Described resistance is the resistance to low temperature; Described low temperature is 4 ℃.
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