CN101402679A - Coldproof protein, encoding gene and uses thereof - Google Patents
Coldproof protein, encoding gene and uses thereof Download PDFInfo
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Abstract
The invention discloses an antifreeze protein and coded genes thereof and an application thereof. The protein is the one with one of the following amino acid residue sequences: 1) SEQ ID No.2 in a sequence table; and 2) the protein with antifreeze activity formed by the substitution and/or deletion and/or addition of one or a plurality of amino acid residues for the amino acid residue sequence as SEQ ID No.2 in the sequence table. The antifreeze genes have antifreeze function and can be transplanted into a plant to improve the antifreeze property of the plant. The antifreeze protein and the coded genes thereof and the application thereof have great realistic significance on improving the plant, particularly the antifreeze quality of grains and commercial crops, on improving the capability thereof to withstand natural low-temperature disaster and on developing economy.
Description
Technical field
The present invention relates to a kind of Coldproof protein and encoding gene thereof and application, particularly a kind of cold-resistant albumen and encoding gene thereof that derives from South Pole fish is with they application in improving plant cold resistance.
Background technology
South Pole fish is the most cold-resistant known fish, its cell can be finished all vital movements that comprise ontogeny in-1 ℃～-2 ℃ low temperature, cold environment has been evolved down the genome encoding that the forms low temperature required all functions molecule of existence down, be important function of gene resource (Gordon, A.L. 2003.Oceanography:Thebrawniest retroflection.Nature 421:904-905).Therefore, to living in the genomic comparative analysis of South Pole fish under the differing temps environment, and clone's evaluation, functional analysis and the study on mechanism thereof of genes involved, help us to understand the mechanism that South Pole fish survive under low temperature environment extreme like this below 0 ℃.The fish that live in the South Pole have shown a series of and cold-resistant relevant physiology and biochemical trait, evolution (DeVries, A.L.1971.Glycoproteins as biological antifreeze agents in antarctic fishes.Science 172:1152-1155 as antifreeze protein; Cheng, A.L. D.a.C.-H.C.2005.Antifreeze Proteins andOrganismal Freezing Avoidance in Polar Fishes.Fish Physiology 22:155-201; De Vries, A.L.C., C.-H.C.2005.Antifreeze proteins and organismal freezing avoidance in polarfishes.fish physiology seriesThe physiology of polar fishes (eds.Farrell, A.P.﹠amp; Steffenson, J.F.) .San Diego, CA:Elsevier Academic Press 22:155-201), heat-shocked is replied (the heat-shock response, HSR) disappearance (Hofmann, G.E., Buckley, B.A., Airaksinen, S., Keen, J.E., Somero, G.N., reduce even disappear (Verde, C. 2000.2000.Heat-shock protein expression is absent in theAntarctic fish Trematomus bernacchii (Family Nototheniidae) .J.Exp.Biol.203:2331-2339), red blood corpuscle is a large amount of in the blood, E.Parisi, and G.diPrisco.2006.The evolution of thermal adaptation in polar fish.Gene 385:137-145), myofibrillar reduced number, diameter but increases or the like.What are the molecular basis of these low-temperature adaptations so? have fish produced special cold-resistant gene on which function after cold screening in several ten million years?
The activity of cold and heat air is one of fundamental cause of Changes in weather.Under specific synoptic situation, the strong cold air that accumulates in high latitude area is gone down south rapidly, invasion China, cause violent on a large scale cooling, and with phenomenons such as strong wind, sleet, freeze injuries, this class synoptic process is called cold wave or strong cold air, and the loss that is caused is called cold wave and freezing disaster thus.Cold weather can cause a large amount of underproduction of farm crop, in the U.S., cause rural economy with a toll of tens hundred million dollar owing to cold every year, for example in December, 1998, California oranges and tangerines big area death owing to freezing disaster, direct economic loss reaches 500,000,000 9 thousand ten thousand dollars.In the annual harm that on average has hundred million mu of farmlands of 6-7 to suffer cold weather of China, 20,000,000,000 kilograms of the grain reduction of income (sustainable development of China Information Network 2003.10.13).If can utilize the cold-resistant gene of having found to cultivate the novel cold-resistant transgenic plant that have, can have stronger tolerance to cold by the render transgenic farm crop, under the situation that external temperature descends suddenly, keep its physiologically active, thereby make farm crop avoid cold disaster.
Summary of the invention
The purpose of this invention is to provide a kind of Coldproof protein and encoding gene thereof and application.
Coldproof protein provided by the present invention, name is called SOD, derives from South Pole cod (Dissostichusmawsoni), is the protein with one of following amino acid residue sequences:
1) the aminoterminal residue sequence of the SEQ ID № .2 in sequence table;
2) with the aminoterminal residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and protein with antifreeze activity.
Wherein, the sequence in the sequence table 2 is made up of 154 amino-acid residues.
The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.
In order to make 1) in SOD be convenient to purifying, proteinic N end or C end that can the aminoacid sequence shown in the sequence 2 is formed in by sequence table connect label as shown in table 1.
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 |
| A Strep-tag mistake! Do not find Reference source. | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned 2) but in the SOD synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding gene of the SOD in can pass through SEQ ID № in the sequence table: the codon of one or several coded amino acid residue of disappearance in 1 the dna sequence dna, 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.
For the ease of proteic secreting, expressing, signal peptide sequence on also can adding at the N-terminal of described SOD.
The encoding gene of above-mentioned Coldproof protein (Gene2 SOD) also belongs to protection scope of the present invention.
The cDNA gene of above-mentioned Coldproof protein can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 nucleotide sequence;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Wherein, the SEQ ID № in the sequence table: 1 is made up of 465 deoxynucleotides.From SEQ ID №: the open reading frame that the 1st to 465 Nucleotide of 15 ' end is Gene2 SOD (Open Reading Frame, ORF), the nucleotide sequence of sequence 2 in the tabulation of sequence 1 code sequence in the sequence table.
The recombinant expression vector, transgenic cell line and the host bacterium that contain gene of the present invention all belong to protection scope of the present invention.
Cold-resistant gene of the present invention is a research material with these special species of South Pole cod (Dissostichus mawsoni), utilizes the scientific discovery of the structure in cDNA library and order-checking and utilizes the method for RT-PCR to obtain full length sequence.Cold-resistant gene of the present invention is changed in the tobacco, but obtained the transgene tobacco of stably express cold-resistant gene of the present invention, show through low temperature test, the tobacco that changes the cold-resistant gene of the present invention over to grows down at low temperature (4 ℃) and still kept vigorous growth conditions in 20 days, do not wilt, confirm that cold-resistant gene of the present invention has cold-resistant function, can change the winter resistance that improves plant in the plant over to.To improving plant, the cold-resistant quality of grain and cash crop particularly, the ability that improves its opposing natural low temperature disaster has important practical sense to our Economic development.
Description of drawings
Fig. 1 is the structural representation of pCAPE3.
Fig. 2 is that wild-type plant fluorescence is identified photo.
Fig. 3 identifies for changeing pCAPE3 tobacco plant fluorescence.
Fig. 4 identifies electrophorogram for transgene tobacco RT-PCR.
Fig. 5 is 4 ℃ and handles 20 days the commentaries on classics pCAPE-SOD tobacco and the phenotype of contrast.
Fig. 6 is 23 ℃ and recovers the commentaries on classics pCAPE-SOD tobacco of 15 days phenotypes of growth and the phenotype of contrast.
Fig. 7 normal cultivated 30 days and handles the Fv/Fm value of changeing pCAPE-SOD tobaccos and contrast in 20 days at 4 degree for Fig. 7.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The acquisition of embodiment 1, cold-resistant gene and proteins encoded thereof and cold-resistant application thereof
One, the acquisition of cold-resistant gene of the present invention and proteins encoded thereof
The present inventor is by having made up the cDNA library of South Pole cod (Dissostichus mawsoni), and the est sequence in this library carried out large scale sequencing, analyze by sequence assembly and BLAST at last and obtained a series of interested genes, and one of them gene that may have cold-resistant function analyzed and researched, and obtain the sequence of this gene according to following method.
Design of amplification primers, primer sequence is as follows:
Upstream primer SOD_F:5 '-CGC
GAGCTCATGGTTATGAAAGCTGTGTG-3 ' (underscore mark be the SacI enzyme recognition site);
Downstream primer SOD_R:5 '-CGC
GAATTCTTACTGGGCGATGCCGATG-3 ' (underscore mark be the EcoRI enzyme recognition site).
Utilize TRIZOL reagent to extract South Pole cod (Dissostichus mawsoni) (Karl-HermannKock1, Keith Reid, John Croxall and Stephen Nicol 2007 Fisheries in the Southern Ocean:an ecosystem approach Phil.Trans.R.Soc.B 362,2333-2349, Inst. of Genetics and Development Biology, CAS) liver rna, with this RNA is template, reverse transcription obtains the first chain cDNA, and the reverse transcription concrete grammar is as described below:
In the EP pipe, add Random Primers 1.0 μ l (100ng), the liver rna 2.0 μ l (5 μ g) of South Pole cod (Dissostichusmawsoni), dNTP mix (10mM) 1.0 μ l, water (RNA-free) 8.0 μ l; At 65 ℃ of incubation 5min, as for 5min on ice, add 5 * first-strand Buffer, 4.0 μ l then, DTT (0.1M) 2.0 μ l, Rnaseout (40U/ μ l) 1.0 μ l behind the mixing, place 2min for 25 ℃; Add 1.0 μ l SuperSeripteII RT mixing gently, place 10min down for 25 ℃.Mixture is placed 42 ℃ of reaction 50min, place 70 ℃ of heating 15min termination reactions down, chain cDNA promptly wins.
With this cDNA is template, under the guiding of primer SOD_F and primer SOD_R, with the increase cDNA sequence of cold-resistant gene of conventional PCR method.
Wherein, reaction system (50 μ l system) is the reaction solution that the contains the first chain cDNA 2 μ l of above-mentioned acquisition, 10 * buffer, 5 μ l, dNTP mixed solution (25mM) 2 μ l, SOD_F (10 μ M) 2 μ l, SOD_R (10 μ M) 2 μ l, Taq archaeal dna polymerase (2.5U/ μ l) 0.5 μ l, aqua sterilisa 36.5 μ l.
Response procedures is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s then, 56 ℃ of annealing 30s, 72 ℃ are extended 1min and carry out 35 circulations; Last 72 ℃ are extended 10min, preserve down for 4 ℃.
After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, recovery and purifying obtain the dna fragmentation of 465bp, this fragment is checked order, the result shows that the fragment that pcr amplification obtains has the nucleotide sequence of sequence 1 in the sequence table, with its called after Gene2_SOD.The nucleotide sequence of sequence 1 is made up of 465 deoxynucleotides in the sequence table, and 5 of sequence 1 ' end 1-465 position nucleotides sequence is classified encoding sequence (ORF) as in sequence table, and coding has the protein of the amino acid residue sequence of sequence 2 in the sequence table.With this albumen called after SOD.
Two, utilize cold-resistant gene of the present invention and proteins encoded thereof to improve the application of plant cold resistance
1, can in plant, express the structure of the recombinant expression vector of cold-resistant gene of the present invention
With pCAPE2 from the PEBV-VIGS carrier system (by two T-DNA plasmid (pCAPE1, pCAPE2) form), Constantin, G.D., B.N.Krath, S.A.MacFarlane, M.Nicolaisen, I.E.Johansen, and O.S.Lund.2004.Virus-induced gene silencing as a tool for functional genomics in alegume species.Plant J 40:622-631; Inst. of Genetics and Development Biology, CAS) transforms, increased MluI, EcoRI, SalI, SacI, NcoI enzyme recognition site.Concrete remodeling method is: the synthetic nucleotide fragments CACCATGGCTAGCACGCGTCCTGAGCTCATGGTGAGCAAGGGCGA that contains above-mentioned restriction enzyme site, this fragment is inserted between the NcoI and EcoRI restriction enzyme site of pCAPE2, and obtain recombinant plasmid.With the recombinant plasmid called after pCAPE3 (its structural representation as shown in Figure 1) of MluI, EcoRI, SalI, SacI, NcoI enzyme recognition site that identified correct increase.
Method amplification Gene_2SOD gene according to step 1, after using SacI and EcoRI double digestion then, enzyme cut obtain the Gene2_SOD gene fragment and be inserted between the SacI of pCAPE3 and the EcoRI restriction enzyme site and obtain recombinant vectors, this recombinant vectors is carried out the enzyme evaluation of cutting and check order, will identify the correct recombinant vectors called after pCAPE-SOD that contains the Gene2_SOD gene fragment.
2, utilize cold-resistant gene of the present invention and proteins encoded thereof to improve the tobacco winter resistance
1) acquisition of commentaries on classics pCAPE-SOD tobacco
Difference pCAPE1, pCAPE3, and behind the pCAPE-SOD commentaries on classics Agrobacterium GV3101 (available from promega company), differentiate, picking transforms successful clone, the 28 ℃ of overnight incubation of ruling are extracted plasmid and are carried out the enzyme evaluation of cutting and check order, and evaluation is shown the correct engineering strain called after GV-pCAPE1 that contains pCAPE1, evaluation is shown the correct engineering strain called after GV-pCAPE3 that contains Pcape3, will identify the correct engineering strain called after GV-pCAPE-SOD that contains pCAPE-SOD;
The above-mentioned GV-pCAPE1 of picking, GV-pCAPE3 or GV-pCAPE-SOD are inoculated into 5ml respectively and add 28 ℃ of overnight incubation in the LB substratum of 50mg/L Rifampin (Rif) and 50mg/L kantlex (Kana); Bacterium liquid with overnight incubation is transferred in the LB substratum of 50ml interpolation 50mg/L Rifampin (Rif), 50mg/L kantlex (Kana), 20 μ M Syringylethanones (Acetosyringone) and 10mM 2-(N-morpholine) ethyl sulfonic acid (MES) 28 ℃ of overnight incubation respectively then;
The above-mentioned GV-pCAPE1 of centrifugal collection, GV-pCAPE3 or GV-pCAPE-SOD bacterium liquid are resuspended in then and have added 10mM MgCl
2, 10mM 2-(N-morpholine) ethyl sulfonic acid (MES)+200 μ M Syringylethanone (Acetosyringone) the LB substratum in, transfer OD
600=2.0, at room temperature leave standstill and obtained OD in 3 hours
600Be 2.0 GV-pCAPE1, GV-pCAPE3 or GV-pCAPE-SOD bacterium liquid.
OD with above-mentioned acquisition
600Be that 2.0 GV-pCAPE1 bacterium liquid mixes according to 1: 1 volume ratio with GV-pCAPE3 bacterium liquid or GV-pCAPE-SOD bacterium liquid respectively, obtain the mixed bacteria liquid of GV-pCAPE1 and GV-pCAPE3 and the mixed bacteria liquid of GV-pCAPE1 and GV-pCAPE-SOD.
(Nicotiana benthamiana is available from Tobacco Institute, Chinese Academy of Agricultural Science) is seeded in the vermiculite with the Ben Shi cigarette, between 25 ℃ of cultivations in, illumination in 16 hours, 8 hours dark culturing long be used for Agrobacterium and inject when the 5-6 sheet launches leaf.The mixed bacteria liquid of above-mentioned GV-pCAPE1 that obtains and GV-pCAPE3 or the mixed bacteria liquid of GV-pCAPE1 and GV-pCAPE-SOD are injected the tobacco that above-mentioned 5-6 sheet launches leaf respectively, will inject the tender leaf of proximal ends during injection.In 23 degree, 40% humidity is cultivated under hour dark greenhouse, 16 hours illumination/8 and was obtained changeing pCAPE-SOD tobacco or commentaries on classics pCAPE3 tobacco in 25 days with the tobacco after the injection.
2) evaluation of commentaries on classics pCAPE-SOD tobacco
The evaluation of A, commentaries on classics pCAPE3 tobacco
The commentaries on classics pCAPE3 tobacco that step 1) is obtained carries out the evaluation of expression fluorescence, concrete grammar is: get 1 square centimeter of commentaries on classics pCAPE3 tobacco and wild-type tobacco Ben Shi cigarette (Nicotiana benthamiana) and (contrast as wild-type, WT) blade, tobacco leaf, place on the cover glass, drip one of distilled water, compressing tablet, place under the laser confocal microscope and observe, the positive plant that changes pCAPE3 and pCAPE1 over to excites at the 470nm of laser confocal microscope and shows green fluorescence down; The chloroplast(id) fluorescence that observe to change pCAPE3 tobacco and wild-type tobacco Ben Shi cigarette (Nicotiana benthamiana) with laser confocal microscope in contrast simultaneously.The result as shown in Figures 2 and 3, the result show have 6 strains change the pCAPE3 tobacco all be successfully change over to pCAPE3 and pCAPE1 and can be in tobacco the transgene tobacco of normal expression.Among Fig. 2, A is the GFP fluorescence photo of wild-type tobacco, and B is the chloroplast(id) fluorescence photo of wild-type tobacco, and C is the overlapping of fluorescence shown in A and the B.Among Fig. 3, A is for changeing the GFP fluorescence photo of pCAPE3 tobacco, and B is for changeing the chloroplast(id) fluorescence photo of pCAPE3 tobacco, and C is the overlapping of fluorescence shown in A and the B.
The evaluation of B, commentaries on classics pCAPE-SOD tobacco
With above-mentioned steps 1) evaluation by RT-PCR of the commentaries on classics pCAPE-SOD tobacco that obtains, the primer of RT-PCR is:
Upstream primer SOD-up:TGTTGAAAGGAGCTGGAGAGGC;
Downstream primer SOD-down:TACTGGGCGATGCCGATGACT.
The first chain cDNA template of the commentaries on classics pCAPE-SOD tobacco that obtains with step 1) is that template is carried out pcr amplification reaction.Simultaneously with 26S rRNA in contrast, the pcr amplification primer is that 5 ' end upstream primer is: GAAGAAGGTCCCAAGGGTTC; 3 ' end downstream primer is: TCTCCCTTTAACACCAACGG.
Wherein, reaction system (50 μ l system) is for containing the reaction solution 2 μ l of the first chain cDNA that changes the pCAPE-SOD tobacco, 10 * buffer, 5 μ l, dNTP mixed solution (25mM) 2 μ l, SOD-up (10 μ g/L) 2 μ l, SOD_down (10 μ g/L) 2 μ l, Taq archaeal dna polymerase (2.5U/ μ l) 0.5 μ l, aqua sterilisa 36.5 μ l.
Response procedures is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s then, 56 ℃ of annealing 30s, 72 ℃ are extended 1min and carry out 35 circulations; Last 72 ℃ are extended 10min, preserve down for 4 ℃.
After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, the dna fragmentation of 440bp promptly is a target fragment.
The result as shown in Figure 4, the result shows that 6 strains change that the Gene2_SOD gene all successfully changes in the tobacco by pCAPE-SOD and normal expression in changeing the pCAPE-SOD tobacco in the pCAPE-SOD tobacco.
3, the winter resistance of changeing the pCAPE-SOD tobacco is identified
Step 2 is identified that male changes pCAPE3 tobacco (contrast), change pCAPE-SOD tobacco and tobacco Ben Shi cigarette (Nicotiana benthamiana, available from Tobacco Institute, Chinese Academy of Agricultural Science) seed germination after the growth 25 days, place 4 ℃ then respectively, 16h illumination/8h dark, grew 20 days, and then 23 ℃, 16h illumination/8h dark, recover growth 15 days, be arranged on 23 ℃ simultaneously, grow under the condition of 16h illumination/8h dark, all the other conditions are in above-mentioned identical commentaries on classics pCAPE3 tobacco (contrast), change pCAPE-SOD tobacco and wild-type tobacco as positive control.In the process of cultivating plant being carried out phenotype observes.As shown in Figure 5, transgene tobacco is at 4 ℃, 16h illumination/8h dark, grow after 20 days, control group plant (change the pCAPE3 tobacco, contrast among Fig. 5) has the phenotype of tangible cold damage, the keen-witted and capable bending of plant, blade is dispirited, changes pCAPE-SOD tobacco (Gene2_SOD among Fig. 5) and then grows after 4 ℃ of subzero treatment still well, and subzero treatment does not cause it that untoward reaction is arranged.In 4 ℃ of deepfreezes of plant after 20 days, place 23 ℃, 16h illumination/8h dark, its phenotype as shown in Figure 6 after recovering to grow 15 days, can see, control group plants (contrasting among Fig. 6) places under the normal temps after deepfreeze is subjected to injury from low temperature again and cultivates, still can not return to normal growth conditions, the subzero treatment that illustrates 4 ℃ is nonvolatil to the injury of tobacco, and is expendable, and changes pCAPE-SOD tobacco (Gene2_SOD among Fig. 6), 4 ℃ of deepfreezes are after 20 days, place 23 ℃ to recover growth after 15 days, plant can continue normal growth and grow, the injury proterties that does not show any low temperature and caused.
In order further to confirm the cold tolerance of transfer-gen plant, we have further measured above-mentioned commentaries on classics pCAPE-SOD tobacco and have handled this physical signs of photosynthesizer II photochemistry efficient (Fv/Fm) (Tarantino of 20 days normal cultivation 30 days with at 4 degree, D., Vianelli, A., Carraro, L. and Soave, C. (1999) A nuclear mutantof Arabidropsis thaliana selected for enhanced sensitivity to light-chill stress is altered inPS II electron transport activity.Physiol.Plant.107,361-371.), be contrast to change the pCAPE3 tobacco.The result as shown in Figure 7, experimental data shows that the FV/Fm of control group plant descends obviously, descends very for a short time and change pCAPE-SOD tobacco plant FV/Fm, the illustrative experiment group is changeed the Gene2_SOD tobacco plant has tangible resistance to low temperature.Show that Gene2_SOD has cold-resistant function.
Sequence table
<160>2
<210>1
<211>465
<212>DNA
＜213〉South Pole cod (Dissostichus mawsoni)
<400>1
atggttatga?aagctgtgtg?tgtgttgaaa?ggagctggag?aggctagcgg?gactgtcttc 60
ttcgagcagg?agaatgattc?agcccctgtg?acgctgaccg?gagaaatcaa?aggccttact 120
cctggtgagc?atggtttcca?tgtccatgct?tttggagaca?atacaaatgg?gtgcatcagt 180
gcaggccctc?acttcaatcc?ccacaacaag?actcatgccg?gtcctactga?tgaaaatagg 240
catgttggag?acctggggaa?tgtgactgct?gcagctgata?atgttgcaaa?gctcaaaatc 300
acggacaaga?tgatcaccct?tgctggccaa?tactctatta?ttggcagaac?catggtgatc 360
catgagaagg?ccgacgacct?gggaaaagga?ggcaatgatg?agagtctaaa?gacaggcaat 420
gctggtggac?gtctggcctg?tggagtcatc?ggcatcgccc?agtaa 465
<210>2
<211>154
<212>PRT
＜213〉South Pole cod (Dissostichus mawsoni)
<400>2
Met?Val?Met?Lys?Ala?Val?Cys?Val?Leu?Lys?Gly?Ala?Gly?Glu?Ala?Ser
1 5 10 15
Gly?Thr?Val?Phe?Phe?Glu?Gln?Glu?Asn?Asp?Ser?Ala?Pro?Val?Thr?Leu
20 25 30
Thr?Gly?Glu?Ile?Lys?Gly?Leu?Thr?Pro?Gly?Glu?His?Gly?Phe?His?Val
35 40 45
His?Ala?Phe?Gly?Asp?Asn?Thr?Asn?Gly?Cys?Ile?Ser?Ala?Gly?Pro?His
50 55 60
Phe?Asn?Pro?His?Asn?Lys?Thr?His?Ala?Gly?Pro?Thr?Asp?Glu?Asn?Arg
65 70 75 80
His?Val?Gly?Asp?Leu?Gly?Asn?Val?Thr?Ala?Ala?Ala?Asp?Asn?Val?Ala
85 90 95
Lys?Leu?Lys?Ile?Thr?Asp?Lys?Met?Ile?Thr?Leu?Ala?Gly?Gln?Tyr?Ser
100 105 110
Ile?Ile?Gly?Arg?Thr?Met?Val?Ile?His?Glu?Lys?Ala?Asp?Asp?Leu?Gly
115 120 125
Lys?Gly?Gly?Asn?Asp?Glu?Ser?Leu?Lys?Thr?Gly?Asn?Ala?Gly?Gly?Arg
130 135 140
Leu?Ala?Cys?Gly?Val?Ile?Gly?Ile?Ala?Gln
145 150
Claims (9)
1, a kind of Coldproof protein is the protein with one of following amino acid residue sequences:
1) amino acid residue sequence of the SEQ ID № .2 in sequence table;
2) with the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and protein with antifreeze activity.
2, the encoding gene of the described Coldproof protein of claim 1.
3, encoding gene according to claim 2 is characterized in that: the cDNA gene of described Coldproof protein has one of following nucleotide sequence:
1) from SEQ ID №: 1 nucleotide sequence;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
4, the recombinant expression vector that contains the encoding gene of claim 2 or 3 described Coldproof proteins.
5, the transgenic cell line that contains the encoding gene of claim 2 or 3 described Coldproof proteins.
6, the host bacterium that contains the encoding gene of claim 2 or 3 described Coldproof proteins.
7, the application of the described Coldproof protein of claim 1 in the winter resistance that improves plant or animal.
8, the application of the encoding gene of claim 2 or 3 described Coldproof proteins in the winter resistance that improves plant or animal.
9, application according to claim 8 is characterized in that: described plant is a tobacco.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102633869A (en) * | 2011-02-10 | 2012-08-15 | 中国科学院遗传与发育生物学研究所 | Low temperature resistance associated protein and encoding gene and application thereof |
| CN102952816A (en) * | 2011-08-23 | 2013-03-06 | 中国科学院遗传与发育生物学研究所 | Application of ATPase proteins in stress tolerance of plants |
| CN109651494A (en) * | 2019-01-10 | 2019-04-19 | 北京大宏利辉生物科技中心 | Common nepenthes wcor413-like albumen and its application |
| CN117164674A (en) * | 2023-10-24 | 2023-12-05 | 上海水大技术转移有限公司 | Antifreeze protein, gene, yeast engineering bacteria and application thereof |
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2008
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102633869A (en) * | 2011-02-10 | 2012-08-15 | 中国科学院遗传与发育生物学研究所 | Low temperature resistance associated protein and encoding gene and application thereof |
| CN102633869B (en) * | 2011-02-10 | 2014-01-01 | 中国科学院遗传与发育生物学研究所 | Low temperature resistance associated protein and encoding gene and application thereof |
| CN102952816A (en) * | 2011-08-23 | 2013-03-06 | 中国科学院遗传与发育生物学研究所 | Application of ATPase proteins in stress tolerance of plants |
| CN109651494A (en) * | 2019-01-10 | 2019-04-19 | 北京大宏利辉生物科技中心 | Common nepenthes wcor413-like albumen and its application |
| CN109651494B (en) * | 2019-01-10 | 2022-06-03 | 济南瑞之源生物科技有限公司 | Pitcher plant wcor413-like protein and application thereof |
| CN117164674A (en) * | 2023-10-24 | 2023-12-05 | 上海水大技术转移有限公司 | Antifreeze protein, gene, yeast engineering bacteria and application thereof |
| CN117164674B (en) * | 2023-10-24 | 2024-02-02 | 上海水大技术转移有限公司 | Antifreeze protein, gene, yeast engineering bacteria and application thereof |
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