CN106119258A - Wax insect Antifreeze protein gene ep afp and the aminoacid sequence of coding thereof - Google Patents
Wax insect Antifreeze protein gene ep afp and the aminoacid sequence of coding thereof Download PDFInfo
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- CN106119258A CN106119258A CN201610546589.4A CN201610546589A CN106119258A CN 106119258 A CN106119258 A CN 106119258A CN 201610546589 A CN201610546589 A CN 201610546589A CN 106119258 A CN106119258 A CN 106119258A
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- afp
- antifreeze protein
- wax insect
- protein gene
- wax
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
Abstract
The present invention is wax insect Antifreeze protein gene ep afp and the aminoacid sequence of coding thereof, relates to molecular biology and genetic engineering field.The present invention, with the wax insect of the resistance to extreme low temperature in the Northeast of China as material, obtains purpose fragment by PCR amplification, is connected to construction recombination plasmid in pGEM carrier T, it is thus achieved that the Positive recombinant clones of ep afp gene；Utilizing homologous recombination construction prokaryotic expression carrier pET 22b/afp, recombiant plasmid proceeds to carry out in BL21 expression strain prokaryotic expression, and the heat stagnation activity utilizing differential scanning calorimetry to analyze purifying protein is 0.91 DEG C.According to Nicotiana tabacum L. codon amended wax insect Antifreeze protein gene sequence as improving the important candidate gene that plant cold resistance converts.The wax insect antifreeze protein ep afp of the present invention is the new albumen possessing freeze proof effect, has the biggest using value.
Description
Technical field
The present invention relates to molecular biology and genetic engineering field.A kind of freeze proof egg being present in wax insect
White gene ep-afp and the nucleotide sequence of coding thereof.
Background technology
Wax insect all has distribution at China's wide geographic area, within 2006, is found that wax insect first in Changchun, Jilin.So far,
The coldest monthly mean temperature-10.2 DEG C, the ice box of Annual lowest climate temperature-31.0 DEG C still with the presence of wax insect Natural Population,
Illustrate that this worm has the most uncommon adaptation ability to low temperature.Progress in Insect Cold Hardines is by number of mechanisms control, wherein antifreeze protein
The abduction delivering of antifreeze protein AFP plays the part of important function at Insect Anti excessive cold invading middle-JIAO(the spleen and stomach).AFP can by its heat stagnation activity
So that freeze point depression 3-6 DEG C, the generation of AFP not only can make insecticide body maintain metastable supercooling state for a long time,
The ice also being able to tissue body surface travels further in body fluid, additionally, AFP can not only improve the supercooling ability of hemolymph, for
The ion gradient in body is maintained also to have important function.
The major function of antifreeze protein is exactly heat stagnation activity, and the heat stagnation activity of Properties of Insect Antifreeze Proteins is compared to Fish or plant
The activity of its suppression ice-crystal growth is higher.Some Properties of Insect Antifreeze Proteins oneself be cloned and express, the specific activity of Properties of Insect Antifreeze Proteins
Fish antifreeze protein activity is high 3-4 times.The research of antifreeze protein at present is concentrated mainly on several species few in number, these things
The freezing tolerance planted is far away from wax insect.
With the Northeast, the wax insect of resistance to extreme low temperature is as research material in this research, in conjunction with high throughput sequencing technologies, excavates
There is the antifreeze protein of the stagnant activity of higher thermal, as improving the preferred genes that plant cold resistance converts.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of wax insect Antifreeze protein gene ep-with anti-freezing property
Afp and the aminoacid sequence of coding thereof.
The present invention, with the wax insect of the resistance to extreme low temperature in the Northeast of China as material, obtains purpose fragment by PCR amplification,
It is connected to construction recombination plasmid on pGEM-T carrier, it is thus achieved that the Positive recombinant clones of ep-afp gene；Utilize homologous recombination construction
Prokaryotic expression carrier pET-22b/afp, recombiant plasmid proceeds to carry out in BL21 expression strain prokaryotic expression, utilizes differential scanning amount
The heat stagnation activity of calorimetry analysis purifying protein is 0.91 DEG C.
The wax insect Antifreeze protein gene ep-afp nucleotide full length sequence of the present invention or its fragment use PCR TRAP,
The method of recombination method or synthetic obtains, and wax insect Antifreeze protein gene ep-afp is by the nucleotide shown in SEQ ID NO:1
Sequence forms, and SEQ ID NO:1 is specific as follows:
ATGACTCGTGGGGAAGCTTCTCAAATTAAGGTTCACTATAAGGGGAAGGATGAAGATTTCATTGTTCTC
GTTGATGATGAAGCTTCTTATAAGAAGTGGCTCGCTGATAAGTCTGTTCCACTCGCTCACTTCATTTCTGCTTTCAA
GATTTTCGTTACTCACAAGCAAGGGACTCAAGGGATGATGGATACTGCTTCTAAGGCTACTCTCGATAATCAATTCG
GGACTTCTGTTGATGAAGAAGTTATTAAGCAAATTCTCGAACAAGGGAACATGCAATCTTCTGAAATGCTCGATCGT
CAAGGGCCAAAGAACGATTCTATGTCTTCTATGAATGCTCGTTAG
The aminoacid sequence with wax insect Antifreeze protein gene ep-afp coding of the present invention is by shown in SEQ ID NO:2
Aminoacid sequence forms, and SEQ ID NO:2 is specific as follows:
When plant transgene, according to Nicotiana tabacum L. codon preference amendment wax insect Antifreeze protein gene ep-afp by
Nucleotide sequence composition shown in SEQ ID NO:3, SEQ ID NO:3 is specific as follows:
ATGACTCGTGGGGAAGCTTCTCAAATTAAGGTTCACTATAAGGGGAAGGATGAAGATTTCATTGTTCTC
GTTGATGATGAAGCTTCTTATAAGAAGTGGCTCGCTGATAAGTCTGTTCCACTCGCTCACTTCATTTCTGCTTTCAA
GATTTTCGTTACTCACAAGCAAGGGACTCAAGGGATGATGGATACTGCTTCTAAGGCTACTCTCGATAATCAATTCG
GGACTTCTGTTGATGAAGAAGTTATTAAGCAAATTCTCGAACAAGGGAACATGCAATCTTCTGAAATGCTCGATCGT
CAAGGGCCAAAGAACGATTCTATGTCTTCTATGAATGCTCGT
Method used in the present invention is method known to same domain technical staff.
The beneficial effects of the present invention is:
1, the major function of antifreeze protein is heating stagnation curve Thermal hysteresis activity, THA, i.e. reduces
The freezing point of solution, but do not change its fusing point, thus produce the temperature gap between freezing point and fusing point.This heat stagnation value is the biggest, anti-
The activity freezing albumen is the highest.In all antifreeze proteins, Properties of Insect Antifreeze Proteins has the highest heat stagnation activity, freeze proof for Fish
3-4 times of albumen heat stagnation activity.Therefore Properties of Insect Antifreeze Proteins has higher using value.
2, the present invention has obvious effect in an experiment, and China has numerous caste, but seldom from its internal discovery
Antifreeze protein, the wax insect antifreeze protein ep-afp of the present invention possesses the new albumen of freeze proof effect, the heat stagnation of this albumen just
Activity reaches 0.91 DEG C, has the biggest using value.
Detailed description of the invention
Method used in the present embodiment is method known to same domain technical staff.
The wax insect Antifreeze protein gene ep-afp nucleotide full length sequence of the present invention or its fragment use PCR TRAP,
The method of recombination method or synthetic obtains.Wax insect Antifreeze protein gene ep-afp is by the nucleotide shown in SEQ ID NO:1
Sequence forms, and SEQ ID NO:1 is specific as follows:
ATGACTCGTGGGGAAGCTTCTCAAATTAAGGTTCACTATAAGGGGAAGGATGAAGATTTCATTGTTCTC
GTTGATGATGAAGCTTCTTATAAGAAGTGGCTCGCTGATAAGTCTGTTCCACTCGCTCACTTCATTTCTGCTTTCAA
GATTTTCGTTACTCACAAGCAAGGGACTCAAGGGATGATGGATACTGCTTCTAAGGCTACTCTCGATAATCAATTCG
GGACTTCTGTTGATGAAGAAGTTATTAAGCAAATTCTCGAACAAGGGAACATGCAATCTTCTGAAATGCTCGATCGT
CAAGGGCCAAAGAACGATTCTATGTCTTCTATGAATGCTCGTTAG
The wax insect Antifreeze protein gene ep-afp of the present invention uses PCR TRAP, according to disclosed in this invention relevant
Nucleotide sequence, especially open reading frame sequence design special primer ATGACTCGTGGGGAAGCTTCTC and
CTAACGAGCATTCATAGAAG, and with commercially available cDNA storehouse or by the cDNA library work of routine well known by persons skilled in the art
For template, expand and obtain the nucleotide sequence of wax insect Antifreeze protein gene ep-afp.This gene is cloned into escherichia coli
Competent cell, once obtains the Positive recombinant clones of this gene, it becomes possible to obtain this gene in large quantity with recombination method.
It is typically to be cloned into prokaryotic expression carrier pET-22b, then proceeds to the competent cell of E. coli expression strains BL21, so
The prokaryotic expression of genes of interest, isolated from the host cell after propagation is carried out afterwards by the method for conventional IPTG induction
Wax insect antifreeze protein.
The aminoacid sequence with wax insect antifreeze protein Ep-AFP coding of the present invention is by the amino shown in SEQ ID NO:2
Acid sequence forms, and SEQ ID NO:2 is specific as follows:
When plant transgene, according to Nicotiana tabacum L. codon preference, wax insect Antifreeze protein gene ep-afp is repaiied
Change, obtain nucleotide sequence SEQ ID NO:3, specific as follows:
ATGACTCGTGGGGAAGCTTCTCAAATTAAGGTTCACTATAAGGGGAAGGATGAAGATTTCATTGTTCTC
GTTGATGATGAAGCTTCTTATAAGAAGTGGCTCGCTGATAAGTCTGTTCCACTCGCTCACTTCATTTCTGCTTTCAA
GATTTTCGTTACTCACAAGCAAGGGACTCAAGGGATGATGGATACTGCTTCTAAGGCTACTCTCGATAATCAATTCG
GGACTTCTGTTGATGAAGAAGTTATTAAGCAAATTCTCGAACAAGGGAACATGCAATCTTCTGAAATGCTCGATCGT
CAAGGGCCAAAGAACGATTCTATGTCTTCTATGAATGCTCGT
The nucleotide sequence of the wax insect Antifreeze protein gene ep-afp coding of the present invention, obtains in accordance with the following methods:
(1) Total RNAs extraction, library construction and high-flux sequence: gather the Northeast December-January next year and survive the winter white beeswax
Worm female adult worm, is added immediately Trizol and grinds homogenate, extract total serum IgE.
(2) cDNA synthesis: after extracting RNA, utilize chloroform, isopropanol to extract, finally the RNA of extraction is dissolved in without RNA
In the water of enzyme.With 1% agarose gel electrophoresis and ultraviolet/visible spectrophotometer (NanoDrop ND1000
Spectrophotometer) concentration and the purity of total serum IgE are measured.With 1.0 μ g total serum IgE as template, invert with Oligo (dT)
Record downstream primer, after 70 DEG C of incubation 10min rapidly more than cooled on ice 2min.Reverse transcription reaction is carried out by MLV reverse transcription,
42 DEG C of incubation 1h, cooled on ice after 70 DEG C of incubation 15min, reverse transcription product cDNA is placed in-20 DEG C of Refrigerator stores standby.
(3) clone of Antifreeze protein gene: for building Antifreeze protein gene, with cDNA as template, design primer amplification is white
The cDNA fragment of wax insect afp gene.Reaction condition: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72
℃10min.PCR primer, after 1% agarose gel electrophoresis separates, reclaims test kit recovery with cutting glue.It is then attached to pGEM-
Construction recombination plasmid pGEM-T-afp in carrier T, serves the order-checking of Hai Shenggong biotechnology Services Co., Ltd.To preparation
Recombiant plasmid solution, surveys OD value with ultraviolet spectrophotometer, calculates OD260/OD280 and analyzes plasmid purity, and records original dense
Degree.This solution is stored in-20 DEG C of refrigerators as standard substance stock solution standby.
(4) Prokaryotic expression vector construction: design with the forward primer of carrier homologous sequence and downstream primer, to obtain
Recombiant plasmid pGEM-T-afp be template, carry out PCR amplification, PCR primer after purification, and expression vector pET-22b mixing,
There is homologous recombination, recombinant products transformed clone bacterial strain DH5 α under the effect of recombinase, use the restructuring gram of the T7 primer screening positive
Grand, deliver biotech firm's order-checking.The named pET-22b/afp of recombiant plasmid.The bacterial strain correct for determining sequence, takes out after shaking bacterium
Upgrading grain, Plastid transformation BL21 expression strain, T7 primer detection positive colony, after order-checking confirms, add glycerol-70 DEG C preservation.
(5) prokaryotic expression: after IPTG (0.5M) abduction delivering, SDS-PAGE detects protein expression, finds one substantially
Band of expression, close to theoretical molecular 15kDa.
(6) heat stagnation Activity determination: utilize low pressure chromatography system to carry out the Ni post affinity purification of fusion protein, protein purification
After, use differential scanning calorimeter to measure the difference between fusing point and freezing point, with bovine serum albumin for comparison, analyze wax insect and resist
Freeze the heat stagnation activity of albumen, it is thus achieved that the wax insect Antifreeze protein gene that heat stagnation activity is higher.
Wax insect Antifreeze protein gene ep-afp, directly synthesizes in biotech firm according to above-mentioned SEQ ID NO:2 sequence；Repair
The wax insect Antifreeze protein gene ep-afp for plant transgene after changing, also can be straight according to above-mentioned SEQ ID NO:3 sequence
It is connected on biotech firm's synthesis.
SEQUENCE LISTING 1
<110>Resources Insect Inst., Chinese Academy of Forestry Sciences
<120>wax insect Antifreeze protein gene ep-afp and the aminoacid sequence of coding thereof
<130> PatentIn version 3.5
<160> 1
<170> PatentIn version 3.5
<210> 1
<400> 1
atgactcgtg gggaagcttc tcaaattaag gttcactata aggggaagga tgaagatttc 60
attgttctcg ttgatgatga agcttcttat aagaagtggc tcgctgataa gtctgttcca 120
ctcgctcact tcatttctgc tttcaagatt ttcgttactc acaagcaagg gactcaaggg 180
atgatggata ctgcttctaa ggctactctc gataatcaat tcgggacttc tgttgatgaa 240
gaagttatta agcaaattct cgaacaaggg aacatgcaat cttctgaaat gctcgatcgt 300
caagggccaa agaacgattc tatgtcttct atgaatgctc gttag 345
SEQUENCE LISTING 2
<110>Resources Insect Inst., Chinese Academy of Forestry Sciences
<120>wax insect Antifreeze protein gene ep-afp and the aminoacid sequence of coding thereof
<130> PatentIn version 3.5
<160> 1
<170> PatentIn version 3.5
<210> 1
<400> 1
Met Thr Arg Gly Glu Ala Ser Gln Ile Lys 10
Val His Tyr Lys Gly Lys Asp Glu Asp Phe 20
Ile Val Leu Val Asp Asp Glu Ala Ser Tyr 30
Lys Lys Trp Leu Ala Asp Lys Ser Val Pro 40
Leu Ala His Phe Ile Ser Ala Phe Lys Ile 50
Phe Val Thr His Lys Gln Gly Thr Gln Gly 60
Met Met Asp Thr Ala Ser Lys Ala Thr Leu 70
Asp Asn Gln Phe Gly Thr Ser Val Asp Glu 80
Glu Val Ile Lys Gln Ile Leu Glu Gln Gly 90
Asn Met Gln Ser Ser Glu Met Leu Asp Arg 100
Gln Gly Pro Lys Asn Asp Ser Met Ser Ser 110
Met Asn Ala Arg 114
SEQUENCE LISTING 3
<110>Resources Insect Inst., Chinese Academy of Forestry Sciences
<120>wax insect Antifreeze protein gene ep-afp and the aminoacid sequence of coding thereof
<130> PatentIn version 3.5
<160> 1
<170> PatentIn version 3.5
<210> 1
<400> 1
atgactcgtg gggaagcttc tcaaattaag gttcactata aggggaagga tgaagatttc 60
attgttctcg ttgatgatga agcttcttat aagaagtggc tcgctgataa gtctgttcca 120
ctcgctcact tcatttctgc tttcaagatt ttcgttactc acaagcaagg gactcaaggg 180
atgatggata ctgcttctaa ggctactctc gataatcaat tcgggacttc tgttgatgaa 240
gaagttatta agcaaattct cgaacaaggg aacatgcaat cttctgaaat gctcgatcgt 300
caagggccaa agaacgattc tatgtcttct atgaatgctc gt 342
Claims (3)
1. wax insect Antifreeze protein gene ep-afp is made up of the nucleotide sequence shown in SEQ ID NO:1.
2. the aminoacid sequence of wax insect Antifreeze protein gene ep-afp coding is by the aminoacid sequence group shown in SEQ ID NO:2
Become.
3. according to Nicotiana tabacum L. codon preference amendment wax insect Antifreeze protein gene ep-afp by the core shown in SEQ ID NO:3
Nucleotide sequence forms.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610546589.4A CN106119258A (en) | 2016-07-12 | 2016-07-12 | Wax insect Antifreeze protein gene ep afp and the aminoacid sequence of coding thereof |
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| CN201610546589.4A CN106119258A (en) | 2016-07-12 | 2016-07-12 | Wax insect Antifreeze protein gene ep afp and the aminoacid sequence of coding thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021232536A1 (en) * | 2020-05-19 | 2021-11-25 | 中国林业科学研究院资源昆虫研究所 | Construction method for system for large volume in vitro acquisition of ericerus pela dsrna |
| CN114395025A (en) * | 2021-01-27 | 2022-04-26 | 昆明学院 | Paralichthys atranus antifreeze protein gene ep-afp and application thereof |
-
2016
- 2016-07-12 CN CN201610546589.4A patent/CN106119258A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| SHU-HUI YU, ET AL.: "Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect", 《INSECT SCIENCE》 * |
| 万军 等: "昆虫抗冻蛋白研究进展", 《北方蚕业》 * |
| 于淑惠 等: "白蜡虫雄虫真蛹转录组分析", 《林业科学研究》 * |
| 杨璞 等: "白蜡虫不同虫态及不同地理种群越冬雌成虫过冷却点的研究", 《林业科学研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021232536A1 (en) * | 2020-05-19 | 2021-11-25 | 中国林业科学研究院资源昆虫研究所 | Construction method for system for large volume in vitro acquisition of ericerus pela dsrna |
| CN114395025A (en) * | 2021-01-27 | 2022-04-26 | 昆明学院 | Paralichthys atranus antifreeze protein gene ep-afp and application thereof |
| CN114395025B (en) * | 2021-01-27 | 2023-08-08 | 昆明学院 | Paraffin anti-freeze protein gene ep-afp and application thereof |
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