CN105695481A - Juicy peach fruit ICE1 gene, and cloning method and application thereof - Google Patents

Juicy peach fruit ICE1 gene, and cloning method and application thereof Download PDF

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CN105695481A
CN105695481A CN201610175621.2A CN201610175621A CN105695481A CN 105695481 A CN105695481 A CN 105695481A CN 201610175621 A CN201610175621 A CN 201610175621A CN 105695481 A CN105695481 A CN 105695481A
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ice1
peach fruit
gene
honey peach
ice1 gene
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万嗣宝
朱月莹
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University of Shanghai for Science and Technology
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University of Shanghai for Science and Technology
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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
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/65—Vectors comprising as targeting moiety peptide derived from defined protein from plants

Abstract

The invention provides a juicy peach fruit ICE1 gene, and a cloning method and application thereof. The gene has the base sequence shown by SEQ ID NO.1. After arabidopsis conversion by an agrobacterium-mediated method; PCR (polymerase chain reaction) is performed for verifying that the juicy peach fruit ICE1 gene is successfully transformed into an arabidopsis genome; the deep study can be conveniently performed on the cold-resistance mechanism of the juicy peach fruit ICE1 gene in the later stage.

Description

Honey peach fruit ICE1 gene, its cloning process and application
Technical field
The present invention relates to and relate to a kind of honey peach fruit ICE1 gene, its cloning process and application。
Background technology
ICE1(inducerofCBFexpression1) it is a transcription factor of most upstream in plant low temperature responsive genes。It binds specifically on the promoter sequence of CBF3 gene when low temperature, the expression of induction CBF3, then CBF3 is attached in the DRE sequence of genes of interest promoter downstream, the induction a series of cold acclimation protein COR in downstream and the expression of other genes worked in plant cold adaptation, thus improving the winter resistance of plant。Honey peach belongs to typical climacteric type fruit, not easily fresh-keeping, easily damages to plants caused by sudden drop in temperature in low-temperature transport, storage。Damage to plants caused by sudden drop in temperature the commodity value not only affecting fruit, and rotten, the quality deterioration after heating up can be aggravated, and then affect the shelf life of fruit。At present, yet there are no the report of honey peach ICE1 gene function。
Summary of the invention
An object of the present invention is in that to provide a kind of honey peach ICE1 gene。
The two of the purpose of the present invention are in that to provide the cloning process of this gene。
The three of the purpose of the present invention are in that the application providing this gene in agriculture bacillus mediated inflorescence dip method arabidopsis thaliana transformation。
For reaching above-mentioned purpose, the present invention adopts the following technical scheme that as follows:
A kind of honey peach fruit ICE1 gene, it is characterised in that this gene is the base sequence shown in SEQIDNO.1。
The encoding proteins of a kind of above-mentioned honey peach fruit ICE1 gene, it is characterised in that this encoding proteins is the aminoacid sequence of the base sequence shown in SEQIDNO.1。
A kind of carrier, it is characterised in that this carrier contains above-mentioned honey peach fruit ICE1 gene。
A kind of recombiant plasmid, it is characterised in that this plasmid contains above-mentioned carrier。
The cloning process of a kind of above-mentioned honey peach fruit ICE1 gene, it is characterised in that concretely comprising the following steps of the method:
A. with Fructus Mali pumilae ICE1, namely ABS50251 is probe, in NCBI, Fructus Persicae genome database is carried out blast search, retrieves a nucleotide sequence XM_007209914 with entire open reading frame, is the Fructus Persicae ICE1 gene order of prediction；
B. extracting honey peach fruit total serum IgE, then reverse transcription obtains cDNA the first chain;The forward primer S1 and downstream primer S2 of the Fructus Persicae ICE1 sequence X M_007209914 design amplification ICE1 gene according to prediction；The cDNA obtained with described reverse transcription again, for template, carries out polymerase chain PCR reaction, namely obtains honey peach fruit ICE1 gene；
Described forward primer S1 is: 5 '-TGGGCTCTCTGTCTACCT-3 '；
Described downstream primer S2:5 '-ACGACTCCCGTCTTTGGT-3 '。
The reaction system of above-mentioned PCR reaction is: 2 × High fidelity PCR Mix15 μ L, template 1 μ L, each 1 μ L of upstream and downstream primer, adds ddH2O to 30 μ L；The reaction condition of described PCR reaction is: 94 DEG C of denaturation 4min；94 DEG C of degeneration 30s, 47 DEG C of annealing 30s, 68 DEG C extend 3min, 35 circulations；68 DEG C re-extend 10min。
A kind of application according to above-mentioned honey peach fruit ICE1 gene in agriculture bacillus mediated inflorescence dip method arabidopsis thaliana transformation。
With the cDNA of reverse transcription acquisition for template, carry out amplified reaction, product and PGM-T vector construction recombiant plasmid with instant High fidelity PCR test kit, convert TOP10 competent cell and also check order。Fructus Persicae ICE1 sequence (XM_007209914) concordance of sequencing result and prediction is 99.72%。Aminoacid sequence coded by sequencing sequence is carried out domain analysis, it has been found that it has a bHLH region (containing 44 amino acid residues), and the aminoacid sequence in this region is with the bHLH domain high conservative of Fructus Mali pumilae ICE1。Visible, this sequencing sequence is honey peach fruit ICE1 gene order, and it is KT148884 that this sequence has been filed on BanKit(accession number)。
The upstream and downstream primer (introducing restriction enzyme site Kpn I and BamH I sequence respectively) of its open reading frame sequence is expanded according to the design of honey peach fruit ICE1 gene order。ICE1 gene open reading frame, gene order is: SEQIDNO.5。Amplification builds the ICE1 aligning primer of sense expression vector: forward primer ICE1-Kpn I: SEQIDNO.6；Downstream primer ICE1-BamH I: SEQIDNO.7。Amplification builds the ICE1 aligning primer of antisense expression vector: forward primer ICE1-BamH I: SEQIDNO.8；Downstream primer ICE1-Kpn I: SEQIDNO.9。
With the bacterium sample that checks order for template, expand the open reading frame sequence of honey peach fruit ICE1 gene with instant High fidelity PCR test kit。Product and PGM-T vector construction recombiant plasmid, convert TOP10 competent cell, again carry out sequence。Sequencing result is 100% with sequencing result homology first。
1. the structure of justice, Antisense construct PCAMBIAy1300-ICE1
From the bacterium solution that order-checking is correct, extract recombiant plasmid, distinguish double digestion recombiant plasmid and expression vector PCAMBIAy1300 with Kpn I and BamH I, and reclaim ICE1 gene open reading frame sequence and linearizing expression vector。T4 ligase is utilized to connect ICE1 gene open reading frame fragment and linearisation expression vector, build justice, Antisense construct respectively, then Agrobacterium competent cell is converted, PCR and the sequence verification of extracting plasmid are all correct, it was shown that justice, Antisense construct PCAMBIAy1300-ICE1 all successfully construct。
The justice of ICE1 gene and Antisense construct are used for agriculture bacillus mediated inflorescence dip method genetic transformation arabidopsis, the present invention relates to following two parts: the preparation of (1) conversional solution and arabidopsis inflorescence are contaminated and converted；(2) screening of seed and the PCR of positive plant verify。
The invention provides a kind of method expanding honey peach fruit ICE1 gene open reading frame sequence, it is provided that the protein of honey peach fruit ICE1 gene code；The honey peach fruit ICE1 gene plant expression vector reported first that the present invention builds, can be directly used for agriculture bacillus mediated Genetic Transformation in Higher Plants, provides strong foundation for plant genetic engineering breeding；The acquisition of transgenic Arabidopsis plants in the present invention, has established experiment basis for the cold resistance study mechanism of honey peach fruit ICE1 gene in follow-up test。
Accompanying drawing explanation
Fig. 1 honey peach fruit total serum IgE electrophoresis pattern；
The pcr amplification product electrophoresis pattern of Fig. 2 honey peach fruit ICE1；
(1,2 is digestion verification result for Fig. 3 cloning vehicle PGM-ICE1 digestion verification and PCR checking；1 ', 2 ' is PCR the result)；
Fig. 4 PGM-T cloning vehicle collection of illustrative plates；
Fig. 5 is PCAMBIAy1300 expression vector collection of illustrative plates；
Fig. 6 is the structure collection of illustrative plates of PCAMBIAy1300-ICE1 plant expression vector；
Fig. 7 be Fig. 6 justice, Antisense construct PCAMBIAy1300-ICE1 digestion verification (1,2 for justice plant expression vector enzyme action result；3,4 is Antisense construct enzyme action result)；
Fig. 8 turns the PCR of ICE1 gene arabidopsis and verifies that (1,2 is justice transfer-gen plant；3,4 is antisense transgene plant；5,6 is wild-type Arabidopsis plants)。
Detailed description of the invention
EXPERIMENTAL EXAMPLE 1: the clone of honey peach fruit ICE1 gene
1. the extraction of honey peach fruit total serum IgE and reverse transcription become cDNA
1.1 use the pillar plant RNA out2.0 RNA isolation kit of sky, Beijing bounties Bioisystech Co., Ltd to extract honey peach (HoneyPeach) fruit total serum IgE, and test kit description is shown in concrete operations。
1.2 to utilize the A3500ReverseTranscriptionSystem test kit of Promega Bioisystech Co., Ltd be cDNA by RNA reverse transcription, standby as pcr template。
2.PCR expands honey peach fruit ICE1 gene
Enter the online BLAST page of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), select nucleotideblast, Fructus Mali pumilae ICE1 nucleotide sequence (MdbHLH1 is inputted on EnterQuerySequence hurdle, ABS50251), OrganismOptional hurdle input Prumuspersica, click Blast, obtain the nucleotide sequence (XM_007209914) that homology with it is 86%, be the Fructus Persicae ICE1 gene order of prediction。
The PrimerPrimer5.0 software analysis design of Fructus Persicae ICE1 sequence (XM_007209914) according to prediction expands forward primer S1:5 '-TGGGCTCTCTGTCTACCT-3 ' (SEQIDNO.3) and downstream primer S2:5 '-ACGACTCCCGTCTTTGGT-3 ' (SEQIDNO.4) of honey peach fruit ICE1 gene。With honey peach fruit cDNA for template, carrying out amplified reaction with instant High fidelity PCR test kit, PCR reaction system is: 2 × High fidelity PCR Mix15 μ L, template 1 μ L, each 1 μ L of upstream and downstream primer, adds ddH2O to 30 μ L。PCR reaction condition is: 94 DEG C of denaturation 4min；94 DEG C of degeneration 30s, 47 DEG C of annealing 30s, 68 DEG C extend 3min, 35 circulations；68 DEG C re-extend 10min。PCR primer reclaims test kit with the pillar DNA glue of Shanghai Sheng Gong biological engineering company limited after 1.0% agarose gel electrophoresis and reclaims purification purpose band, with T4DNA ligase (Promega), recovery product is connected to PGM-T carrier, then converts TOP10(TIANGEN) competent cell。After PCR screens, send to Shanghai Sheng Gong biological engineering company limited and carry out sequence。
With the DNAMAN software Fructus Persicae ICE1(XM_007209914 to sequencing result and prediction) carry out sequence alignment, it has been found that and the two concordance is 99.72%。Log in HLH district referral web site (http://pfam.xfam.org/search/sequence), the aminoacid sequence corresponding to sequencing result is inputted at Proteinsequenceoptions option, click after Submit and find that it has a bHLH region (containing 44 amino acid residues), its bHLH domain high conservative with Fructus Mali pumilae ICE1。So, this sequencing sequence is honey peach fruit ICE1 gene order, and it is KT148884 that this sequence has been filed on Bankit(accession number)。
EXPERIMENTAL EXAMPLE 2: justice, Antisense construct PCAMBIAy1300-ICE1 structure
1. main agents: plasmid extraction kit is purchased from TIANGEN biochemical technology company limited；T4DNA ligase is purchased from Promega Bioisystech Co., Ltd；Restricted enzyme Kpn I and BamH I is purchased from Dalian treasured biological engineering company limited；Expression vector PCAMBIAy1300 is provided by Life Science College arabidopsis seminar of Shanghai University laboratory。
2. the structure of recombinant plant expression vector PCAMBIAy1300-ICE1
2.1 according to honey peach fruit ICE1 nucleotide sequence (KT148884) submitted in GenBank, design the 2 upstream and downstream primers to amplification ICE1 gene open reading frame sequence with PrimerPrimer5.0 software analysis, and introduce the restriction enzyme site sequence of restricted enzyme Kpn I and BamH I respectively。Amplification builds the ICE1 aligning primer of sense expression vector: forward primer ICE1-Kpn I: 5 '-GGGGTACCATGCTTGATGTTGATGATGATTG-3 ' (SEQIDNO.6) and downstream primer ICE1-BamH I: 5 '-CGGGATCCCTATATCAAGCCATGGAACCC-3 ' (SEQIDNO.7)。Amplification builds the ICE1 aligning primer of antisense expression vector: forward primer ICE1-BamH I: 5 '-CGGGATCCATGCTTGATGTTGATGATGATTG-3 ' (SEQIDNO.8) and downstream primer ICE1-Kpn I: 5 '-GGGGTACCCTATATCAAGCCATGGAACCC-3 ' (SEQIDNO.9)。
With the Positive E. coli bacterium solution after order-checking for template, carrying out amplified reaction with instant High fidelity PCR test kit, PCR reaction system is: each 1 μ L of 2 × High fidelity PCR Mix15 μ L, template 1 μ L, upstream and downstream primer, adds ddH2O to 30 μ L。PCR reaction condition is: 94 DEG C of denaturation 4min；94 DEG C of degeneration 30s, 44 DEG C of annealing 30s, 68 DEG C extend 3min, 35 circulations；68 DEG C re-extend 10min。PCR primer reclaims test kit with the pillar DNA glue of Shanghai Sheng Gong biological engineering company limited after 1.0% agarose gel electrophoresis and reclaims purification purpose band, with T4DNA ligase, recovery product is connected to PGM-T carrier, then converts TOP10 competent cell。After PCR screens, preserve bacterium solution and send to Shanghai Sheng Gong biological engineering company limited and carry out sequence。
2.2 take plasmid extraction kit method to extract the recombiant plasmid PGM-T-ICE1 in positive bacterium solution, the PGM-T-ICE1 plasmid extracted and expression vector PCAMBIAy1300 are used restricted enzyme Kpn I and BamH I double digestion respectively, after enzyme action after 1% agarose gel electrophoresis detection, cut ICE1 gene purpose band and linearizing expression vector fragment, utilize glue to reclaim RNA isolation kit and carry out reclaiming purification。Connect ICE1 genetic fragment and linearizing expression vector with T4DNA ligase, linked system is: 10 × T4Buffer1uL, T4DNALigase1uL, linearisation expression vector 3uL, ICE1 genetic fragment 2uL, ddH2O3uL, 16 DEG C overnight connect。Then, utilize freeze-thaw method respectively justice, Antisense construct to be converted TOP10 competent cell, PCR and sequence verification, filter out the escherichia coli positive bacterium solution respectively containing justice, antisense recombinant plant expression vector PCAMBIAy1300-ICE1。
2.3 extract recombiant plasmid respectively from the bacterial strain containing justice, Antisense construct PCAMBIAy1300-ICE1, with cold shock method transformed competence colibacillus Agrobacterium GV3101, method for transformation is: (1) takes-80 DEG C of centrifuge tubes equipped with 100uL Agrobacterium competent cell GV3101 preserved and treats its partial melting a moment in the palm of the hand, inserts in ice when being in frozen water admixture。(2) in centrifuge tube, add the 10uL plant expression vector extracted, dial with hands and mix at the bottom of pipe, stand 5min, liquid nitrogen 5min, 37 DEG C of water-bath 5min, ice bath 5min successively on ice。(3) the LB fluid medium of 700uL antibiotic-free is added, in 28 DEG C of shaken cultivation 2-3h。(4) the centrifugal 1min of 6000rpm receives bacterium, leaves and takes about 100uL supernatant and blows and beats on the LB flat board that resuspended truffle is coated containing 50ug/mL kanamycin gently, is inverted and is put in 28 DEG C of incubators cultivation 2d。(5) detection of picking monoclonal, chooses positive colony and shakes bacterium, for arabidopsis genetic transformation。
EXPERIMENTAL EXAMPLE 3: arabidopsis inflorescence is contaminated and positive plant PCR identifies
1. agriculture bacillus mediated inflorescence dip method arabidopsis thaliana transformation
The compound method of 1.1 osmotic medium (now with the current): dissolve 0.645gMS, 0.15gMES, 15g sucrose with sterilized water, pH value is adjusted to 5.7 and to be settled to 300mL with KOH, stand-by after being subsequently adding 6-BA and the 100uLSilwet-77 mix homogeneously of 4uL1mg/mL。
The 1.2 LB(50mg/L kanamycin+50mg/L rifampicin+50mg/L gentamycins that positive monoclonal received 150mL) in fluid medium, 28 DEG C are shaken bacterium and cultivate 24h, abandon supernatant after the centrifugal 10min of 7000rpm, then suspend to be precipitated to the osmotic medium now joined and hang completely。The alabastrum removing the Arabidopsis plant of fruit pod in advance is directly dipped in 30-60s in above-mentioned suspension, and 12h is cultivated in plant black plastic bag shading, then normally cultivates between cultivating, gathers when seed maturity。
2. the screening of seed and the PCR of positive plant identify
Wild type and transgenic arabidopsis seed are respectively put in the centrifuge tube of 1.5mL by 2.1, add 1mL75% alcohol washes three times, superclean bench uses sterile water wash 3 times, after seed is slightly dry, tiling is to sterilized screening culture medium 1/2MS(30mg/L hygromycin) on flat board, screening and culturing 7d, then by resistance transplantation of seedlings to soil, normally cultivate。
2.2 total serum IgE extracting the resistant plant through hygromycin selection, reverse transcription is cDNA, and after carrying out PCR reaction (reaction system is shown in EXPERIMENTAL EXAMPLE 1) with wildtype Arabidopsis thaliana for negative control, the agarose gel electrophoresis through 1% detects positive transgenic plant。
In sum, the present invention constructs the justice containing honey peach fruit ICE1 gene and Antisense construct PCAMBIAy1300-ICE1。Wherein honey peach fruit ICE1 gene is reported first, and justice and Antisense construct have successfully imported arabidopsis all, and the function for follow-up further investigation honey peach fruit ICE1 gene has established solid experiment basis。The foregoing is only presently preferred embodiments of the present invention, so the equivalent variations done with the present patent application the scope of the claims, still fall within the scope that the present invention contains。
<110>Shanghai University
<120>honey peach fruit ICE1 gene, its cloning process and application
<160>9
<210>1
<211>1278
<212>DNA
<213>gene order
<400>1
GTTGGGCTCTCTGTCTACCTTCAAATCCATGCTTGATGTTGATGATGATTGGTATCTGCC60
CAATAATAGCATTCAGAGCCATTCAGATGTCCGAGACATAGCCTTCTCGCCGAGCTTTGC120
TGACCCAGAAAGCTTGCTGCTCAACCCAGTGGACTCTTCCTCTTCTTGCTCACCGTCTTC180
CTCGGTTTTCAACAATCTTGACCCAAATCAGGTTCATTACTATTTGCCTCAAAATTCCAC240
CTTGTCTTCGCTCCTTAATGTTGTTCCTAATAACCCTTTGGAGCATGGCTTCGATTTGGG300
CTGTGATATTGGGTTTCTTGACACTCAAGCATCGAGTGGTTCAAGTTTAATGAACAGGGG360
AGGTGGGGTTTTAACTGGGTTCAGTGGTTTGAGCTCAAATAACCCGATGAATGCTCAGGA420
TTTGTGTTCAAATTTGCAATTTTCAACCACCCGTCTGCCTCAATTGCTTGAAAATAGTTC480
AAATTTTTCGGGCTTTCGGGGTTTTGAGGATACTTCAGCGAATGCTTTGTTTCCCAATAG540
GCCTAAATTATTGAGACCCCTTGACTCCGTCCCTGATGTGGGAGCACCGCCCACTCTTTT600
TCAGAAAAGAGTTCAGAAGAACTTAGCTGATAATGGAGGAAATTTGGGTGCTTTAGGCTC660
AGAAGGGGGCCTGGCTTTGAATCATATGGGTGAGGAGAATGAGAGGAAGAGGAAATTGAG720
CGGGGGAGATGATTTTGAAGATGTGAGTATCGATGGTTCGGGTTTGAACTATGATTCAGA780
TGACTTTACTGAAAACACTAAGATGGATGACGGTGCTAAGAATGGTGGTAATAGCTCGAA840
TGCAAATAGTACTGTTACTGGCGGAGGGGATCATAAGGGGAAGAAGAAAGGGTTGCCTGC900
TAAGAATTTGATGGCTGAGAGGCGCCGCCGGAAGAAGCTCAATGATAGGCTGTATATGCT960
TAGATCCGTTGTTCCAAAGATCAGCAAAATGGACAGGGCATCAATCCTTGGGGATGCAATT1020
GAGTACTTGAAGGAACTTCTGCAAAAGATCAACAACCTCCATAATGAATTGGAGTCAATC1080
CCTCCGGGGTCTTCATTGACGCCTACCGGAAGTACATTCCACCCTTTGACACCCACTCCG1140
GCTACCCTGCCAAGCCGTATCAAGGAAGAACTTTGCCCCAGCTCATTACCTAGCCCAAAC1200
GGCCAACCCGCAAGGGTTGAAGTTAGGCTACGAGAAGGAAGAGCAGTGAATATCCACATG1260
TTTTGTGGGCGCAGACCTGGTCTCTTGCTCTCGACCATGAGAACCTTAGATAGCCTTGGG1320
CTAGACATCCAGCAAGCTGTCATTAGCTGTTTCAATGGTTTTGCTATGGATGTCTTCCGT1380
GCTGGGCAATGCAAGGAAGGTCAAGACGTCCACCCGGATCAGATAAAAGCAGTACTTTTG1420
GAGTCAGTCGGGTTCCATGGCTTGATATAGATTGCAGCAAAACAACAAACTGAAGTTGCT1480
TTAACCAAAGACGGGAGTCGTCATGCAATTTGTATATGTTTTGCTTTGCCATAGCTCAAT1540
TGCGTCGTGCATCAAATTTGAATTAGCAGCTTTTGAA1577
<210>2
<211>1278
<212>RPT
<213>aminoacid sequence
<400>1
MLDVDDDWYLPNNSIQSHSDVRDIAFSPSFADPESLLLNPVDSSSSCSPSSSVFNNLDPN60
QVHYYLPQNSTLSSLLNVVPNNPLEHGFDLGCDIGFLDTQASSGSSLMNRGGGVLTGFSG120
LSSNNPMNAQDLCSNLQFSTTRLPQLLENSSNFSGFRGFEDTSANALFPNRPKLLRPLDS180
VPDVGAPPTLFQKRVQKNLADNGGNLGALGSEGGLALNHMGEENERKRKLSGGDDFEDVS240
IDGSGLNYDSDDFTENTKMDDGAKNGGNSSNANSTVTGGGDHKGKKKGLPAKNLMAERRR300
RKKLNDRLYMLRSVVPKISKMDRASILGDAIEYLKELLQKINNLHNELESIPPGSSLTPT360
GSTFHPLTPTPATLPSRIKEELCPSSLPSPNGQPARVEVRLREGRAVNIHMFCGRRPGLL420
LSTMRTLDSLGLDIQQAVISCFNGFAMDVFRAGQCKEGQDVHPDQIKAVLLESVGFHGLI480
<210>3
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TGGGCTCTCTGTCTACCT18
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ACGACTCCCGTCTTTGGT18
<210>5
<211>1278
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<400>1
ATGCTTGATGTTGATGATGATTGGTATCTGCCCAATAATAGCATTCAGAGCCATTCAGAT60
GTCCGAGACATAGCCTTCTCGCCGAGCTTTGCTGACCCAGAAAGCTTGCTGCTCAACCCA120
GTGGACTCTTCCTCTTCTTGCTCACCGTCTTCCTCGGTTTTCAACAATCTTGACCCAAAT180
CAGGTTCATTACTATTTGCCTCAAAATTCCACCTTGTCTTCGCTCCTTAATGTTGTTCCT240
AATAACCCTTTGGAGCATGGCTTCGATTTGGGCTGTGATATTGGGTTTCTTGACACTCAA300
GCATCGAGTGGTTCAAGTTTAATGAACAGGGGAGGTGGGGTTTTAACTGGGTTCAGTGGT360
TTGAGCTCAAATAACCCGATGAATGCTCAGGATTTGTGTTCAAATTTGCAATTTTCAACC420
ACCCGTCTGCCTCAATTGCTTGAAAATAGTTCAAATTTTTCGGGCTTTCGGGGTTTTGAG480
GATACTTCAGCGAATGCTTTGTTTCCCAATAGGCCTAAATTATTGAGACCCCTTGACTCC540
GTCCCTGATGTGGGAGCACCGCCCACTCTTTTTCAGAAAAGAGTTCAGAAGAACTTAGCT600
GATAATGGAGGAAATTTGGGTGCTTTAGGCTCAGAAGGGGGCCTGGCTTTGAATCATATG660
GGTGAGGAGAATGAGAGGAAGAGGAAATTGAGCGGGGGAGATGATTTTGAAGATGTGAGT720
ATCGATGGTTCGGGTTTGAACTATGATTCAGATGACTTTACTGAAAACACTAAGATGGAT780
GACGGTGCTAAGAATGGTGGTAATAGCTCGAATGCAAATAGTACTGTTACTGGCGGAGGG840
GATCATAAGGGGAAGAAGAAAGGGTTGCCTGCTAAGAATTTGATGGCTGAGAGGCGCCGC900
CGGAAGAAGCTCAATGATAGGCTGTATATGCTTAGATCCGTTGTTCCAAAGATCAGCAAA960
ATGGACAGGGCATCAATCCTTGGGGATGCAATTGAGTACTTGAAGGAACTTCTGCAAAAG1020
ATCAACAACCTCCATAATGAATTGGAGTCAATCCCTCCGGGGTCTTCATTGACGCCTACC1080
GGAAGTACATTCCACCCTTTGACACCCACTCCGGCTACCCTGCCAAGCCGTATCAAGGAA1140
GAACTTTGCCCCAGCTCATTACCTAGCCCAAACGGCCAACCCGCAAGGGTTGAAGTTAGG1200
CTACGAGAAGGAAGAGCAGTGAATATCCACATGTTTTGTGGGCGCAGACCTGGTCTCTTG1260
CTCTCGACCATGAGAACCTTAGATAGCCTTGGGCTAGACATCCAGCAAGCTGTCATTAGC1320
TGTTTCAATGGTTTTGCTATGGATGTCTTCCGTGCTGGGCAATGCAAGGAAGGTCAAGAC1380
GTCCACCCGGATCAGATAAAAGCAGTACTTTTGGAGTCAGTCGGGTTCCATGGCTTGATA1440
TAG1447
<210>6
<211>31
<212>DNA
<213>artificial primer
<400>1
GGGGTACCATGCTTGATGTTGATGATGATTG32
<210>7
<211>29
<212>DNA
<213>artificial primer
<400>1
CGGGATCCCTATATCAAGCCATGGAACCC29
<210>8
<211>31
<212>DNA
<213>artificial primer
<400>1
CGGGATCCATGCTTGATGTTGATGATGATTG31
<210>9
<211>29
<212>DNA
<213>artificial primer
<400>1
GGGGTACCCTATATCAAGCCATGGAACCC29

Claims (7)

1. a honey peach fruit ICE1 gene, it is characterised in that this gene is the base sequence shown in SEQIDNO.1。
2. the encoding proteins of a honey peach fruit ICE1 gene according to claim 1, it is characterised in that this encoding proteins is the aminoacid sequence shown in SEQIDNO.2。
3. a carrier, it is characterised in that this carrier contains honey peach fruit ICE1 gene according to claim 1。
4. a recombiant plasmid, it is characterised in that this plasmid contains carrier according to claim 3。
5. the cloning process of a honey peach fruit ICE1 gene according to claim 1, it is characterised in that concretely comprising the following steps of the method:
A. with Fructus Mali pumilae ICE1, namely ABS50251 is probe, in NCBI, Fructus Persicae genome database is carried out blast search, retrieves a nucleotide sequence XM_007209914 with entire open reading frame, is the Fructus Persicae ICE1 gene order of prediction；
B. extracting honey peach fruit total serum IgE, then reverse transcription obtains cDNA the first chain;The forward primer S1 and downstream primer S2 of the Fructus Persicae ICE1 sequence X M_007209914 design amplification ICE1 gene according to prediction；The cDNA obtained with described reverse transcription again, for template, carries out polymerase chain PCR reaction, namely obtains honey peach fruit ICE1 gene；
Described forward primer S1 is: 5 '-TGGGCTCTCTGTCTACCT-3 '；
Described downstream primer S2:5 '-ACGACTCCCGTCTTTGGT-3 '。
6. method according to claim 5, it is characterised in that the reaction system of described PCR reaction is: 2 × High fidelity PCR Mix15 μ L, template 1 μ L, each 1 μ L of upstream and downstream primer, adds ddH2O to 30 μ L；The reaction condition of described PCR reaction is: 94 DEG C of denaturation 4min；94 DEG C of degeneration 30s, 47 DEG C of annealing 30s, 68 DEG C extend 3min, 35 circulations；68 DEG C re-extend 10min。
7. a honey peach fruit ICE1 gene according to claim 1 application in agriculture bacillus mediated inflorescence dip method arabidopsis thaliana transformation。
CN201610175621.2A 2016-03-26 2016-03-26 Juicy peach fruit ICE1 gene, and cloning method and application thereof Pending CN105695481A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018161921A1 (en) * 2017-03-08 2018-09-13 南京农业大学 Method for epigenetically manipulating plant phenotypic plasticity

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CN1649483A (en) * 2002-05-01 2005-08-03 美国亚利桑那大学董事会 ICE1, a regulator of cold induced transcriptome and freezing tolerance in plants

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CN1649483A (en) * 2002-05-01 2005-08-03 美国亚利桑那大学董事会 ICE1, a regulator of cold induced transcriptome and freezing tolerance in plants

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018161921A1 (en) * 2017-03-08 2018-09-13 南京农业大学 Method for epigenetically manipulating plant phenotypic plasticity

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