CN102277359A - Phosphate transporter gene and preparation method thereof - Google Patents

Phosphate transporter gene and preparation method thereof Download PDF

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CN102277359A
CN102277359A CN2011102204550A CN201110220455A CN102277359A CN 102277359 A CN102277359 A CN 102277359A CN 2011102204550 A CN2011102204550 A CN 2011102204550A CN 201110220455 A CN201110220455 A CN 201110220455A CN 102277359 A CN102277359 A CN 102277359A
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sequence
gene
primer
cdna
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李慧
丛郁
常有宏
蔺经
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Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention relates to a phosphate transporter gene, protein encoded by the phosphate transporter gene and a method for preparing the gene. The phosphate transporter gene is derived from rosaceous plants, a nucleotide sequence of the phosphate transporter gene is shown as SEQ ID NO:1, and an amino acid sequence of the protein encoded by the phosphate transporter gene is shown as SEQ ID NO:2; the phosphate transporter gene can be cloned by designed primers; and the phosphate transporter gene can be used for expression research on phosphate transporter genes of the rosaceous plants, and the phosphate transporter genes meet the requirements of the plants on phosphate transport by regulating own expression levels under different phosphorus supply conditions.

Description

Phosphorus transporter protein gene and preparation method thereof
Technical field
The invention belongs to biological technical field, relate to the phosphorus transporter protein gene and the clone thereof that in rosaceous plant, express specifically.
Background technology
Phosphorus is the important nutritive element in the plant life process, constitute 5% of vegetable cell dry weight, the material that it shifts as energy, at activation body internal protein, the whole metabolic process of regulation and control plant materials, harmonic growth and aspect such as degeneration-resistant play a significant role (Bucher M. Functional biology of plant phosphate uptake at root and mycorrhiza interfaces [J]. New Phytologist, 2007,173 (1): 11-26).Plant mainly absorbs phosphorus in the soil with inorganic phosphorus (Pi) form, and soil makes in the soil solution the absorbable solubility content of inorganic phosphorus of plant very low to the strong adsorption of phosphorus, usually less than 10 μ mol L -1(Raghothama K G. and Karthikeyan A S. Phosphate acquisition [J]. Plant and Soil, 2005,274 (1-2): 37-49).Therefore, can plant have fundamental influence to plant-growth by efficient absorption phosphorus.Plant is the active transport process of a contrary concentration to the absorption of phosphorus, phosphorus transporter protein family on the main dependence root system cytolemma is finished (Liu J Y, Versaw W K, Pumplin N, Gomez S K, Blaylock L A, Harrison M J. Closely related members of the Medicago truncatulaPHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities [J]. Journal of Biological Chemistry, 2008,283 (36): 24673-24681), the proteic quantity of phosphorus transporter and its are to phosphate radical (H on the cytolemma 2PO 4 -) ionic affinity decision plant phosphorus assimilated efficiency.Plant phosphorus transporter albumen is divided into Pht1, Pht2, Pht3, Pho1 and Pho2 five extended familys, wherein Pht1Gene family belongs to H 2PO 4 -/ nH +Transport body altogether, phosphorus transporter albumen for high-affinity, be positioned on the root system cytolemma, drive absorption (Catarecha P, the Segura M D of plant titanium pigment in the soil solution by utilizing the hydrogen ion concentration gradient on the plasma membrane, Franco-Zorrilla J M, Garc í a-Ponce B, Lanza M, Solano R, Paz-Ares J, Leyva A. A mutant of the ArabidopsisPhosphate transporter PHT1; 1 displays enhanced arsenic accumulation [J]. Plant Cell, 2007,19 (3): 1123-1133; Miao J, Sun J H, Liu D, Li B, Zhang A, Li Z, Tong Y. Characterization of the promoter of phosphate transporter TaPHT1.2 differentially expressed in wheat varieties [J]. Journal of Genetics and Genomics, 2009,36 (8): 455-466; Preuss C P, Huang C Y, Gilliham M, Tyerman S D. Channel-like characteristics of the low-affinity barley phosphate transporter PHT1; 6 when expressed in Xenopus oocytes [J]. Plant Physiology, 2010,152 (3): 1431-1441).The Pht1 family member is at paddy rice (Yu Futong, Zhang Aimin, Chen Shouyi at present, Zhang Fusuo. a high-affinity rice root phosphorus transporter segmental clone of albumen candidate gene [J]. Acta Genetica Sinica, 2001,28 (2): 144-151), wheat (ever victorious closing, Shu Haiyan, Tong Yiping, Qin Guangyong, Li Bin, Li Zhensheng. separation, Function Identification and the expression study [J] of two wheat phosphorus transporter protein genes. the northwest Botany Gazette, 2004,24 (10): 1779-1785; Miao J, Sun J H, Liu D, Li B, Zhang A, Li Z, Tong Y. Characterization of the promoter of phosphate transporter TaPHT1.2 differentially expressed in wheat varieties [J]. Journal of Genetics and Genomics, 2009,36 (8): 455-466), barley (Rae A L, Cybinski D H, Jarmey J M, Smith F W. Characterization of two phosphate transporters from barley; Evidence for diverse function and kinetic properties among members of the Pht1 family [J]. Plant Molecular Biology, 2003,53 (1-2): 27-36; Preuss C P, Huang C Y, Gilliham M, Tyerman S D. Channel-like characteristics of the low-affinity barley phosphate transporter PHT1; 6 when expressed in XenopusOocytes [J]. Plant Physiology, 2010,152 (3): 1431-1441), tomato (Liu C, Muchhal U S, Uthappa M, Kononowicz A K, Raghothama K G. Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus [J]. Plant Physiology, 1998,116 (1): 91-99), corn (Nagy R, Vasconcelos M J, Zhao S, McElver J, Bruce W, Amrhein N, Raghothama K G, Bucher M. Differential regulation of five Pht1 phosphate transporters from maize ( Zea maysL.) [J]. Plant Biol (Stuttg), 2006,8 (2): 186-197) and Para rubber tree (Tang Yinhui, He Peng. the clone of Para rubber tree phosphorus transporter protein gene and bioinformatic analysis [J]. the tropical crops journal, 2010,31 (5): 758-766) obtained the clone in the plant such as grade.Arabidopis thaliana Pht1Family Pht1; 1The disappearance utmost point of this gene of mutant analysis revealed weakened plant significantly and absorbed phosphatic ability (Shin H, Shin H S, Dewbre G R, Harrison M J. Phosphate transport in Arabidopsis: Pht1; 1 and Pht1; 4 play a major role in phosphate acquisition from both low-and high-phosphate environments [J]. Plant Journal, 2004,39 (4): 629-642), and overexpression Pht1The gene family member can significantly improve receptivity (Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, Harashima S, the Shibata D. Overexpression of an of recipient plant to inorganic phosphorus Arabidopsis thalianaHigh-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions [J]. Proceedings of the National Academy of Sciences of the United States of America, 1997,94 (13): 7098-7102; Catarecha P, Segura M D, Franco-Zorrilla J M, Garc í a-Ponce B, Lanza M, Solano R, Paz-Ares J, Leyva A. A mutant of the ArabidopsisPhosphate transporter PHT1; 1 displays enhanced arsenic accumulation [J]. Plant Cell, 2007,19 (3): 1123-1133; Park M R, Baek S H, de los Reyes B G, Yun S J. Overexpression of a high-affinity phosphate transporter gene from tobacco ( NtPT1) enhances phosphate uptake and accumulation in transgenic rice plants [J]. Plant and Soil, 2007,292 (1-2): 259-269).Show research thus Pht1Gene expression characteristics and control methods help to improve by the biotechnology means phosphorus assimilated efficiency of crop.
The Rosaceae mainly comprises the plant of Ramulus et Folium Spiraeae Salicifolia subfamily, apple subfamily, Rosoideae and four subfamilies of Li Yake.This section comprises that famous fruit is (as apple; Chinese pear-leaved crabapple; Malus spectabilis; pears; peach; Lee; apricot; plum; cherry; loquat Quinces Quince; hawthorn; strawberry and raspberry etc.); medicinal plant is (as garden burnet; Agrimony; Herba Potentillae Discoloris; Semen Pruni; Jin Yingzi and pawpaw etc.); ornamental plant is (as Ramulus et Folium Spiraeae Salicifolia; embroider the line plum; Cortex Sorbariae Arboreae; rose; Chinese rose; Malus spectabilis; plum blossom; oriental cherry; flowering peach; Sorbus alnifloria; kerria and pearlbush etc.) and industrial cut stock (as immature fruit of Juteleaf Raspberry; the root micromicro of Flos rosae multiflorae and garden burnet is to extract tannin, peach kernel; almond and almond benevolence can be extracted oil and rose; Flos rosae multiflorae; the petal of tea rose etc. can extract perfume oil).In view of the extensive use of rosaceous plant, the phosphorus transporter molecular biology research of this section plant is had important practical significance.
Yet find there be not the description and the report of discovery and rosaceous plant phosphorus transporter protein gene so far through literature search to prior art.
Summary of the invention
The objective of the invention is to,, provide a kind of rosaceous plant phosphorus transporter protein gene cDNA sequence and its coded protein in view of the blank that the phosphorus transporter molecular biology research of rosaceous plant exists, and the cloning process of gene.
The object of the present invention is achieved like this: a kind of phosphorus transporter protein gene is characterized in that: it comes from the phosphorus transporter protein gene of rosaceous plant, and the nucleotides sequence of described phosphorus transporter protein gene is classified the nucleotide sequence of SEQ ID NO:1 in the sequence table as.
In the present invention: the proteins encoded of phosphorus transporter protein gene has the aminoacid sequence of sequence table SEQ ID NO:2 protein active polypeptide, and the coded protein of nucleotide sequence of described aminoacid sequence and SEQ ID NO:1 the 13rd ~ 1629 bp position has 70% homology at least.
In the present invention: the aminoacid sequence SEQ ID NO:2 of coded protein utilizes BioXM software that the nucleotide sequence coded district part of SEQ ID NO:1 is translated the back to obtain; Described SEQ ID NO:2 is the albumen that contains 538 amino-acid residues.
A kind of preparation method of above-mentioned phosphorus transporter protein gene is characterized in that: utilize RACE technology clone unknown gene, concrete steps are:
Utilize reverse transcription primer oligo (dT) 15After synthetic conserved regions fragment amplification is used cDNA first chain,, carry out the conserved regions fragment that the RT-PCR amplification obtains SEQ ID NO:1 sequence by upstream primer SEQ ID NO:3 and downstream primer SEQ ID NO:4 pairing;
According to the conserved regions fragments sequence information of SEQ ID NO:1 sequence, design gene specific upstream primer SEQ ID NO:5, SEQ ID NO:6,5 ' RACE reverse transcription primer SEQ ID NO:7 and gene specific downstream primer SEQ ID NO:8, SEQ ID NO:9;
After utilizing in the test kit the synthetic 3' end sequence amplification of 3 ' RACE reverse transcription primer AP with cDNA first chain, by gene specific upstream primer SEQ ID NO:5 and SEQ ID NO:6 respectively with test kit in the 3' end sequence of universal primer 3 ' AUAP pairing carrying out nido RT-PCR amplification SEQ ID NO:1 sequence;
After utilizing the synthetic 5' end sequence amplification of 5 ' RACE reverse transcription primer SEQ ID NO:7 with cDNA first chain, by universal primer 5 ' AAP pairing in gene specific downstream primer SEQ ID NO:8 and the test kit, and the increase 5' end sequence of SEQ ID NO:1 sequence of universal primer 5 ' AUAP pairing carrying out RT-PCR in gene specific downstream primer SEQ ID NO:9 and the test kit;
The conserved regions fragment of the SEQ ID NO:1 sequence that obtains, the 3' end sequence and the 5' end sequence of SEQ ID NO:1 sequence are spliced, obtain PcPht1The cDNA total length;
According to the splicing result, design full length gene upstream primer SEQ ID NO:10 and full length gene downstream primer SEQ ID NO:11 utilize reverse transcription primer oligo (dT) 15The amplification of synthetic gene total length uses cDNA first chain as template, and is right PcPht1The cDNA total length carry out RT-PCR amplification checking;
The System for Rapid Amplification of cDNA Ends that described 3 ' RACE test kit is an Invitrogen company, catalog number (Cat.No.) is 18373-019; 5 ' RACE test kit is the System for Rapid Amplification of cDNA Ends of Invitrogen company, and catalog number (Cat.No.) is 18374-058.
The invention has the advantages that: phosphorus transporter protein gene ubiquity in rosaceous plant, because this coded by said gene albumen has conservative aminoacid sequence, can from rosaceous plant, clone acquisition phosphorus transporter protein gene by method of the present invention, and in the phosphorus transporter process of rosaceous plant, under different phosphorus supply conditions, this gene adapts to the needs of plant to phosphorus transporter by regulating the expression level of self, shows application prospects.
Description of drawings
Fig. 1 beans pears PcPht1The pcr amplification of gene is electrophorogram as a result.
Among the figure:
A is the beans pears PcPht1The conserved regions fragment; B is the beans pears PcPht13' end sequence nido RT-PCR second takes turns amplified production; C is the beans pears PcPht15' end sequence nido RT-PCR second takes turns amplified production; D is the beans pears PcPht1CDNA total length amplified production; M1 among A ~ C is the DL2 of TaKaRa company, 000 DNA Marker; M2 among the D is 250 bp DNA Ladder Marker of TaKaRa company.
The different plant phosphorus transporter of Fig. 2 albumen Pht1The amino acid identity analysis of genes encoding.
Among the figure:
PcPht1 be the beans pears ( Pyrus calleryana) Pht1Dna encoding the protein; GmPht1:7 be soybean ( Glycine max) Pht1:7Dna encoding the protein, GenBank sequence number are FJ797407; HbPht1 be rubber ( Hevea brasiliensis) Pht1Dna encoding the protein, GenBank sequence number are HM015901; LePT1 be tomato ( Lycopersicon esculentum) Pht1; 1Dna encoding the protein, GenBank sequence number are AF022873; LePT2 be tomato ( Lycopersicon esculentum) Pht1; 2Dna encoding the protein, GenBank sequence number are AF022874; PhPht1; 1 be petunia ( Petunia x hybrida) Pht1; 1Dna encoding the protein, GenBank sequence number are EF564180; PtrPht1; 7 be willow ( Populus trichocarpa) Pht1; 7Dna encoding the protein, GenBank sequence number are XM_002306809; StPT1 be potato ( Solanum tuberosum) Pht1; 1Dna encoding the protein, GenBank sequence number are X98890; StPT2 be potato ( Solanum tuberosum) Pht1; 2Dna encoding the protein, GenBank sequence number are X98891.
Beans pears under the different phosphorus supply conditions of Fig. 3 PcPht1Expression of gene is analyzed.
Among the figure:
PcPht1Be beans pears phosphorus transporter protein gene; ActinFor beans pears actin gene, as the internal reference gene; The A ~ C swimming lane is respectively and contains NH 4H 2PO 4Be 0.023,0.230 or 2.30 g L -1The Hoagland nutritive medium handle 24 h beans pears root cDNA amplifications; The D ~ F swimming lane is respectively and contains NH 4H 2PO 4Be 0.023,0.230 or 2.30 g L -1The Hoagland nutritive medium handle 24 h beans pears leaf cDNA amplifications.
Embodiment
Not marked concrete experimental technique in the following example all can carry out according to ordinary method.Condition described in J. Sa nurse Brooker (Sambrook J.) and D.W. Russell (Russell D W.) work " molecular cloning experiment guide " (Science Press, 2005), or according to the operation instruction of biological reagent production firm.
The clone of embodiment 1 beans pears phosphorus transporter protein gene cDNA
The design primer is right:
Upstream primer: SEQ ID NO:3
5'-GTGCTNAATGCACTNGATGT-3'
Downstream primer: SEQ ID NO:4
5'-CCTDGCHGGGAAAATCTCNGC-3'
With 0.230 g L -1NH 4H 2PO 4The beans pears root of handling 24 h is a vegetable material, extracts total RNA with RNAplant Plus Kit (available from TIANGEN company), and operating process is in strict accordance with the extraction flow process of specification sheets.Through DNase I (RNase Free, TIANGEN company) gets the total RNA of 2 μ g after the digestion and be used for synthetic (the TIANScript cDNA first chain synthetic agent box of conserved regions fragment amplification with cDNA first chain, TIANGEN company), the reverse transcription primer is oligo (dT) 15, with RNase A(DNAase and Protease-free, GenStar company) and digestion conserved regions fragment amplification cDNA first chain, method is carried out according to the reagent specification sheets.The conserved regions fragment amplification that obtains with reverse transcription is a template with cDNA first chain, utilize primer SEQ ID NO:3 and SEQ ID NO:4 pairing, with Taq Plus DNA Polymerase(TIANGEN company) carry out RT-PCR amplification, the specification sheets of application of sample system reference enzyme, its PCR response procedures is: 94 ℃ of sex change 5min, carry out 30 circulating reactions (94 ℃ of 30s subsequently, 50 ℃ of 30s, 72 ℃ of 2min), 72 ℃ are extended 10min, and amplification obtains conserved regions fragment (A among Fig. 1).
The design gene specific primer:
Gene specific upstream primer: SEQ ID NO:5
5'-TTGAAGCAGAGCCGCAGAAG-3'
Gene specific upstream primer: SEQ ID NO:6
5'-TGGACTCACAAGGACAACCG-3'
5 ' RACE reverse transcription primer: SEQ ID NO:7
5'-CCAAGCAAGTGAAGTCCATG-3'
Gene specific downstream primer: SEQ ID NO:8
5'-TCTGCGGCTCTGCTTCAATT-3'
Gene specific downstream primer: SEQ ID NO:9
5'-AAGACAGCGGCAATGAAAGC-3'。
Carry out reverse transcription and pcr amplification in conjunction with primer in the RACE test kit:
Primer in 3 ' RACE test kit
3 ' RACE reverse transcription primer AP:SEQ ID NO:14
5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′
Universal primer 3 ' AUAP:SEQ ID NO:15
5′-GGCCACGCGTCGACTAGTAC-3′
Utilize 3 ' RACE reverse transcription primer AP(SEQ ID NO:14 in the test kit) synthetic 3' end sequence amplification with cDNA first chain after, by SEQ ID NO:15 pairing in 3 ' RACE System for Rapid Amplification of cDNA Ends test kit (catalog number is 18373-019) of gene specific upstream primer SEQ ID NO:5 and Invitrogen company, right PcPht1The 3' end sequence of gene cDNA sequence carries out the 1st and takes turns nido RT-PCR amplification, and the 1st takes turns the PCR product dilutes 10 times as template, with gene specific upstream primer SEQ ID NO:6 and SEQ ID NO:15 pairing, carries out the 2nd and takes turns nido RT-PCR amplification, obtains PcPht1The amplified production (B among Fig. 1) of gene 3' end unknown nucleotide sequence.
Primer in 5 ' RACE test kit:
Universal primer 5 ' AAP:SEQ ID NO:16
5′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′
Universal primer 5 ' AUAP:SEQ ID NO:17
5′-GGCCACGCGTCGACTAGTAC-3′
After utilizing the synthetic 5' end sequence amplification of 5 ' RACE reverse transcription primer SEQ ID NO:7 with cDNA first chain, by universal primer 5 ' AAP(SEQ ID NO:16 in 5 ' RACE System for Rapid Amplification of cDNA Ends test kit (catalog number is 18374-058) of gene specific downstream primer SEQ ID NO:8 and Invitrogen company) pairing, right PcPht1The 5' end sequence of gene cDNA sequence carries out the 1st and takes turns nido RT-PCR amplification, the 1st takes turns the PCR product dilutes 10 times as template, with universal primer 5 ' AUAP(SEQ ID NO:17 in 5 ' RACE System for Rapid Amplification of cDNA Ends test kit (catalog number is 18374-058) of gene specific downstream primer SEQ ID NO:9 and Invitrogen company) pairing, carry out the 2nd and take turns nido RT-PCR amplification, obtain PcPht1The amplified production (C among Fig. 1) of gene 5' end unknown nucleotide sequence.
Described amplification program and application of sample system are all carried out according to the 3 ' RACE System for Rapid Amplification of cDNA Ends test kit and the middle specification sheets of 5 ' RACE System for Rapid Amplification of cDNA Ends test kit (catalog number is respectively 18373-019 and 18374-058) of Invitrogen company.
The 3' end that nido RT-PCR amplification is obtained and the amplified production of 5' end unknown nucleotide sequence check order, last and PcPht1The conserved regions fragment assembly of gene cDNA sequence obtains full length cDNA sequence (D among Fig. 1).
Design full length gene primer:
Full length gene upstream primer: SEQ ID NO:10
3'-GCTAGCTTAGCCATGGCTAG-5
Full length gene downstream primer: SEQ ID NO:11
3'-GACTGAAGGACAAGATTACTCG-5'
With 0.230 g L -1NH 4H 2PO 4The beans pears root of handling 24 h is a vegetable material, with RNAplant Plus Kit(available from TIANGEN company) extract total RNA, operating process is in strict accordance with the extraction flow process of specification sheets.Through DNase I (RNase Free, TIANGEN company) gets the total RNA of 2 μ g after the digestion and be used for synthetic (the TIANScript cDNA first chain synthetic agent box of full length gene amplification with cDNA first chain, TIANGEN company), the reverse transcription primer is oligo (dT) 15, utilize primer SEQ ID NO:10 and SEQ ID NO:11 pairing, with high-fidelity hot resistant DNA polymerase (Pfu DNA Polymerase, TIANGEN company) splicing is obtained PcPht1The gene cDNA full length sequence carries out RT-PCR amplification checking, the specification sheets of application of sample system reference enzyme, and its PCR response procedures is: 94 ℃ of 5min; 94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 3min, 30cycles; 72 ℃ of 10min.
The beans pears PcPht1The cDNA full length sequence of gene is:
GCTAGCTTAG CCATGGCTAG AGAGCAATTA CAGGTTCTTA ATGCGCTTGA TGTAGCAAAA 60
ACACAATGGT ACCATTTCAC TGCAATTATC ATTGCTGGAA TGGGATTCTT CACAGATGCA 120
TATGATCTCT TCTGCATATC TTTGGTGACC AAGTTGCTCG GCCGCATATA CTACCACGTT 180
GAAGGCGCAG CGAAGCCTGG GATATTGCCT CCGAATGTGT CAGCTGCCGT AAATGGTGTG 240
GCATTTTGTG GAACCCTGGC AGGCCAGCTC TTCTTTGGCT GGCTTGGTGA CAAGATGGGA 300
AGGAAGAAAG TTTATGGAAT GACTCTTATG CTTATGGTCA TATGTTCTGT TGCTTCAGGT 360
CTCTCATTTG GGCATACCCC AAAATCCGTT ATGTCAACGC TTTGTTTCTT CCGGTTCTGG 420
CTAGGGTTTG GTATTGGCGG TGACTACCCT CTCTCTGCCA CAATCATGTC TGAGTATGCT 480
AACAAGAAGA CTCGAGGCGC TTTCATTGCC GCTGTCTTTG CCATGCAGGG GTTTGGAATT 540
TTAGCTGGTG GAATATTTGC TATCATCTCT GCTGCTGCAT TTAAAGCGTT GTTTGATGCT 600
CCAACATATG AGGTCAATGC ACTTGCCTCA ACCGTTCCAC AAGCGGACTA TCTGTGGAGG 660
ATTATTGTGA TGGTAGGAGC AATTCCAGTC GCAATGACTT ACTACTGGCG CATGAAGATG 720
CCTGAAACTG CCCGTTACAC CGCCCTGGTT GCAAAGAATG CGCAACAGGC TGCATCCGAC 780
ATGTCAAAGG TTCTGCAGGT TGAAATTGAA GCAGAGCCGC AGAAGGCTGA GCCGACTGCT 840
AATACATTTG CTTTGTTCTC CAAGGAGTTT ATGCGCCGCC ATGGACTTCA CTTGCTTGGA 900
ACAACAAGCA CTTGGTTCTT GCTTGACATT GCATTCTACA GCCAAAATCT GTTCCAAAAG 960
GATATTTTCA GTGCGATCGG ATGGATTCCT CCTGCAAAGA CTATGAATGC TATTGAAGAA 1020
GTTTACAGAA TCGCAAGGGC GCAAACTCTT ATTGCATTGT GCAGTACCGT CCCGGGCTAC 1080
TGGTTTACGG TAGCTCTTAT TGACAGGATT GGAAGATTTG CAATTCAGTT GATGGGATTC 1140
TTCTTCATGA CGGTGTTCAT GTTTGCACTG GCTATTCCCT ATGATCACTG GACTCACAAG 1200
GACAACCGAA TTGGGTTCGT GGCGATCTAC TCATTGACCT TCTTTTTTGC AAACTTTGGT 1260
CCTAATGCAA CAACATTTGT TGTGCCGGCT GAGATCTTCC CAGCTAGGTT CCGGTCTACT 1320
TGTCATGGAA TCTCAGCTGC ATCCGGGAAG CTTGGCGCCA TAGTTGGTGC ATTCGGTTTC 1380
TTGTACTTGG CTCAGAACCA AGATAAGGCC AAGGCAGATG CAGGGTACCC TGCAGGCATC 1440
GGGGTTAAAA ACTCGCTCAT CGCGCTGGGT GTGGTCAACT TCTTGGGGAT ATTGTTCACT 1500
TTCTTGGTGC CCGAATCGAA TGGGAGGTCG TTGGAGGAGA TGTCGGGTGA GAACGAAGAA 1560
GAAAGCGAAA GCGTGGCAGT GGAGTTGGAG CACTCAGACT ATAACAACAG GACAGTTCCA 1620
GTTGTGTAGG TTGGATTTAG CAGTGTGCAG GAAATAATCT TGTAATGTAA GCCTGTTGTT 1680
AATTTTGGGG AGGGAAAAAG CCTACTTTTC GACTTCCTAC TTTGTTGTAT TCACTTGAGT 1740
CTAGGTCCTG GCTTTTACAA AGCCAGACAA AGGGTACGTT TTTCATGTAT AACATGTACG 1800
TATCAATATC AACAGGCAAG ACCATCGAGT AATCTTGTCC TTCAGTCAAA AAAAAAAAAA 1860
AAAAA 1865
This sequence length is 1865 bp, its coding region be SEQ.ID.NO.1 hold the 13rd to 1629 bit bases from 5', totally 1617 bp.This sequence called after PcPht1Gene.
The sequence information and the homology analysis of embodiment 2 beans pears phosphorus transporter protein genes
PcPht1BioXM software is adopted in the gene nucleotide translation, PcPht1Gene coded protein is striden film and is distinguished TMHMM Server v. 2.0 programs (http://www.cbs.dtu.dk/services/TMHMM) that adopt of analysing.Secondary structure prediction and domain analyses are respectively by PBIL LYON-GERLAND(http: //npsa-pbil.ibcp.fr/cgi-bin/npsa_ automat. pl/page=/NPSA/npsa_hnn.html) and InterPro(http: //www.ebi.ac.uk/InterProScan) finish.With online software PSORT(http: //psort.ims.utokyo.ac.jp/form.html) carry out Subcellular Localization.The aminoacid sequence comparison adopts DNAMAN software to finish, and the Pht1 protein sequence of used other plant is from http://www.ncbi.nlm.nih.gov.
PcPht1Gene coded protein is an albumen that contains 538 amino-acid residues:
MAREQLQVLN ALDVAKTQWY HFTAIIIAGM GFFTDAYDLF CISLVTKLLG RIYYHVEGAA 60
KPGILPPNVS AAVNGVAFCG TLAGQLFFGW LGDKMGRKKV YGMTLMLMVI CSVASGLSFG 120
HTPKSVMSTL CFFRFWLGFG IGGDYPLSAT IMSEYANKKT RGAFIAAVFA MQGFGILAGG 180
IFAIISAAAF KALFDAPTYE VNALASTVPQ ADYLWRIIVM VGAIPVAMTY YWRMKMPETA 240
RYTALVAKNA QQAASDMSKV LQVEIEAEPQ KAEPTANTFA LFSKEFMRRH GLHLLGTTST 300
WFLLDIAFYS QNLFQKDIFS AIGWIPPAKT MNAIEEVYRI ARAQTLIALC STVPGYWFTV 360
ALIDRIGRFA IQLMGFFFMT VFMFALAIPY DHWTHKDNRI GFVAIYSLTF FFANFGPNAT 420
TFVVPAEIFP ARFRSTCHGI SAASGKLGAI VGAFGFLYLA QNQDKAKADA GYPAGIGVKN 480
SLIALGVVNF LGILFTFLVP ESNGRSLEEM SGENEEESES VAVELEHSDY NNRTVPVV 538
This sequence is SEQ.ID.NO.2.
PcPht1The iso-electric point (pI) of gene coded protein prediction is 8.08, and the protein relative molecular mass is 59.08 KDa. PcPht1Gene coded protein is made up of 12 hydrophobic diaphragm areas (TM) of striding, each membrane spaning domain is formed spiral by 17 ~ 25 amino-acid residues, secondary structure is " 6-ring-6 " structure, promptly comprise 6 N end stride film district and 6 C ends stride the film district, separate by a central hydrophilic loop (Hydrophilicloop) between the middle part (TM6 and TM7), and PcPht1The N end of gene coded protein, C end and the big ring of intermediary all are distributed in tenuigenin. PcPht1The secondary structure of gene coded protein is made up of a large amount of alpha-helixs, random coil and a spot of extended chain structure, and wherein α-Luo Xuanjiegou has 316, account for 58.74%, and extended chain and random coil accounts for 7.25% and 34.01% respectively. PcPht1Gene coded protein comprises H 2PO 4 -/ nH +Transport body, sugar transport body and 3 structural domains of MFS universal substrate transporter altogether.Carrying out Subcellular Localization by online software PSORT finds PcPht1Gene coded protein is positioned on the cytoplasmic membrane.The Pht1 albumen homology analytical results of different plant origins shows: the beans pears PcPht1The aminoacid sequence of cDNA sequence encoding compare homology relative higher (Fig. 2) with other species.With soybean ( Glycine max) homology of GmCBL1-7 albumen (the GenBank sequence number is ACY74618) is 88%; With rubber ( Hevea brasiliensis) homology of HbPht1 albumen (the GenBank sequence number is ADL27918) is 86%; With tomato ( Lycopersicon esculentum) Pht1; The homology of 1 albumen (the GenBank sequence number is AAB82146) is 82%; With tomato ( Lycopersicon esculentum) Pht1; The homology of 2 albumen (the GenBank sequence number is AAB82147) is 74%.
Generally speaking, the clone of this institute PcPht1The aminoacid sequence mark sheet of gene understands that this gene belongs to plant high-affinity phosphorus transporter albumen Pht1Family.
The expression study of embodiment 3 beans pears phosphorus transporter protein genes
Treat that beans pears seedling grows to 4 true leaves when big, select the plant of growing way unanimity in deionized water, behind hungry 24 h, to place NH respectively 4H 2PO 4Be 0.023,0.230 or 2.30 g L -1The Hoagland nutritive medium in handle 24 h, the beans pears blade and the root of results different treatment, it is stand-by to be stored in-70 ℃ of refrigerators.Hoagland nutrient solution prescription: 1.02 g L -1KNO 3, 0.492 g L -1Ca (NO 3) 2, 0.023,0.230 or 2.30 g L -1NH 4H 2PO 4, 0.49 g L -1MgSO 47H 2O, 24.5 mg L -1Ironic citrate, 2.230 mg L -1MnSO 4, 0.240 mg L -1CuSO 45H 2O, 0.290 mg L -1ZnSO 47H 2O, 1.860 mg L -1H 3BO 3, 0.035 mg L -1(NH 4) 6Mo 7O 244H 2O, 0.028 mg L -1CoSO 4H 2O.
Extract total RNA with RNAplant plus plant total RNA extraction reagent (TIANGEN company), operating process is in strict accordance with the extraction flow process of specification sheets.Through DNase I(RNase Free, TIANGEN company) after the digestion, get the total RNA reverse transcription of 2 μ g synthetic gene expression research cDNA first chain (the TIANScript cDNA first chain synthetic agent box, TIANGEN company) of respectively handling sample, the reverse transcription primer is oligo (dT) 15, with RNase A(DNAase and Protease-free, GenStar company) and digestion gene expression research cDNA first chain, operating process is carried out in strict accordance with specification sheets.Gene expression research is stored in-20 ℃ with cDNA first chain and is ready for use on gene expression research.
The design primer is right:
Specificity upstream primer: SEQ ID NO:12
5'-TTGAAGCAGAGCCGCAGAAG-3'；
Specificity downstream primer: SEQ ID NO:13
5'-AGAAGTTGACCACACCCAGC-3'。
Upstream primer B-1:SEQ ID NO:18
5'-GGAATGGTCAAGGCTGGGTT-3'；
Upstream primer B-1 referring to [Li Hui, Cong Yu, Chang Youhong, Lin's warp is contained Baolong. the clone of beans pears cystatin gene and coerce expression [J]. northwest Botany Gazette, 2011,31 (5): 861-867]
Downstream primer B-2:SEQ ID NO:19
5'-CAAAGCATCTGTGAGGTCACG-3'
Downstream primer B-2 referring to [Li Hui, Cong Yu, Chang Youhong, Lin's warp is contained Baolong. the clone of beans pears cystatin gene and coerce expression [J]. northwest Botany Gazette, 2011,31 (5): 861-867].
Is template with gene expression research with cDNA first chain 2 μ L, utilizes PcPht1Gene-specific primer SEQ ID NO:12 and SEQ ID NO:13 pairing are with TaKaRa Ex Taq(TaKaRa company) to different concns NH 4H 2PO 4(0.023,0.230 or 2.30 g L -1) the beans pears root and the leaf cNDA that handle 24h carry out sxemiquantitative RT-PCR amplification, the specification sheets of application of sample system reference enzyme.Reaction parameter: 94 ℃ of 5min; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 2min, 30 circulations; 72 ℃ of 5min.Select beans pears Actin muscle for use ActinGene is the internal reference gene, adopts upstream primer SEQ ID NO:18 and downstream primer SEQ ID NO:19 to increase comparative studies under the same conditions PcPht1The expression of gene situation.
Normal phosphorus supply (NH 4H 2PO 40.230 g L -1) under the condition, PcPht1Expression is all arranged in beans pears blade and root, and expression amount is higher than blade in the root, only can detect faint expression signal in the blade; Low-phosphorous (NH 4H 2PO 40.0230 g L -1) can induced strong PcPht1Expression in root increases PcPht1Expression in blade, and in the root expression amount much larger than blade; Improve phosphorus supply concentration (NH 4H 2PO 42.30 g L -1), directly cause PcPht1Expression by inhibitation system in root does not have in blade and expresses (Fig. 3).Show in the phosphorus transporter process of beans pears, PcPht1Gene is by regulating the expression level of self, adapts to that plant shows application prospects to the needs of phosphorus transporter under the different phosphorus supply conditions.
In view of the beans pears are a kind of pear tree, belong to the apple subfamily fruit tree in the Rosaceae, it is one of China's widely used stock in pears producing region, south, above embodiment is a test materials with the beans pears only, but this is not a limitation of the present invention, no matter use rosaceous other plant to repeat above-mentioned experimentation and also can obtain identical technique effect, be to adopt which kind of plant of the Rosaceae to test, and the step of its gene clone and the research of gene expression characteristics all are provided with according to the standard of claim.
SEQUENCE?LISTING
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉phosphorus transporter protein gene and preparation method thereof
＜130〉specification sheets
<160>?19
<170>?PatentIn?version?3.3
<210>?1
<211>?1865
<212>?cDNA
＜213〉the beans pears ( Pyrus calleryanaDcne)
＜221〉beans pears phosphorus transporter protein gene ( PcPht1Gene) cDNA sequence
<222>?(1)..(1865)
<400>?1
GCTAGCTTAG CCATGGCTAG AGAGCAATTA CAGGTTCTTA ATGCGCTTGA TGTAGCAAAA 60
ACACAATGGT ACCATTTCAC TGCAATTATC ATTGCTGGAA TGGGATTCTT CACAGATGCA 120
TATGATCTCT TCTGCATATC TTTGGTGACC AAGTTGCTCG GCCGCATATA CTACCACGTT 180
GAAGGCGCAG CGAAGCCTGG GATATTGCCT CCGAATGTGT CAGCTGCCGT AAATGGTGTG 240
GCATTTTGTG GAACCCTGGC AGGCCAGCTC TTCTTTGGCT GGCTTGGTGA CAAGATGGGA 300
AGGAAGAAAG TTTATGGAAT GACTCTTATG CTTATGGTCA TATGTTCTGT TGCTTCAGGT 360
CTCTCATTTG GGCATACCCC AAAATCCGTT ATGTCAACGC TTTGTTTCTT CCGGTTCTGG 420
CTAGGGTTTG GTATTGGCGG TGACTACCCT CTCTCTGCCA CAATCATGTC TGAGTATGCT 480
AACAAGAAGA CTCGAGGCGC TTTCATTGCC GCTGTCTTTG CCATGCAGGG GTTTGGAATT 540
TTAGCTGGTG GAATATTTGC TATCATCTCT GCTGCTGCAT TTAAAGCGTT GTTTGATGCT 600
CCAACATATG AGGTCAATGC ACTTGCCTCA ACCGTTCCAC AAGCGGACTA TCTGTGGAGG 660
ATTATTGTGA TGGTAGGAGC AATTCCAGTC GCAATGACTT ACTACTGGCG CATGAAGATG 720
CCTGAAACTG CCCGTTACAC CGCCCTGGTT GCAAAGAATG CGCAACAGGC TGCATCCGAC 780
ATGTCAAAGG TTCTGCAGGT TGAAATTGAA GCAGAGCCGC AGAAGGCTGA GCCGACTGCT 840
AATACATTTG CTTTGTTCTC CAAGGAGTTT ATGCGCCGCC ATGGACTTCA CTTGCTTGGA 900
ACAACAAGCA CTTGGTTCTT GCTTGACATT GCATTCTACA GCCAAAATCT GTTCCAAAAG 960
GATATTTTCA GTGCGATCGG ATGGATTCCT CCTGCAAAGA CTATGAATGC TATTGAAGAA 1020
GTTTACAGAA TCGCAAGGGC GCAAACTCTT ATTGCATTGT GCAGTACCGT CCCGGGCTAC 1080
TGGTTTACGG TAGCTCTTAT TGACAGGATT GGAAGATTTG CAATTCAGTT GATGGGATTC 1140
TTCTTCATGA CGGTGTTCAT GTTTGCACTG GCTATTCCCT ATGATCACTG GACTCACAAG 1200
GACAACCGAA TTGGGTTCGT GGCGATCTAC TCATTGACCT TCTTTTTTGC AAACTTTGGT 1260
CCTAATGCAA CAACATTTGT TGTGCCGGCT GAGATCTTCC CAGCTAGGTT CCGGTCTACT 1320
TGTCATGGAA TCTCAGCTGC ATCCGGGAAG CTTGGCGCCA TAGTTGGTGC ATTCGGTTTC 1380
TTGTACTTGG CTCAGAACCA AGATAAGGCC AAGGCAGATG CAGGGTACCC TGCAGGCATC 1440
GGGGTTAAAA ACTCGCTCAT CGCGCTGGGT GTGGTCAACT TCTTGGGGAT ATTGTTCACT 1500
TTCTTGGTGC CCGAATCGAA TGGGAGGTCG TTGGAGGAGA TGTCGGGTGA GAACGAAGAA 1560
GAAAGCGAAA GCGTGGCAGT GGAGTTGGAG CACTCAGACT ATAACAACAG GACAGTTCCA 1620
GTTGTGTAGG TTGGATTTAG CAGTGTGCAG GAAATAATCT TGTAATGTAA GCCTGTTGTT 1680
AATTTTGGGG AGGGAAAAAG CCTACTTTTC GACTTCCTAC TTTGTTGTAT TCACTTGAGT 1740
CTAGGTCCTG GCTTTTACAA AGCCAGACAA AGGGTACGTT TTTCATGTAT AACATGTACG 1800
TATCAATATC AACAGGCAAG ACCATCGAGT AATCTTGTCC TTCAGTCAAA AAAAAAAAAA 1860
AAAAA 1865
<160>?19
<170>?PatentIn?version?3.3
<210>?2
<211>?538
<212>?PRO
＜213〉the beans pears ( Pyrus calleryanaDcne)
＜221〉beans pears phosphorus transporter protein gene ( PcPht1Gene) aminoacid sequence
<222>?(1)..(538)
<400>?2
MAREQLQVLN ALDVAKTQWY HFTAIIIAGM GFFTDAYDLF CISLVTKLLG RIYYHVEGAA 60
KPGILPPNVS AAVNGVAFCG TLAGQLFFGW LGDKMGRKKV YGMTLMLMVI CSVASGLSFG 120
HTPKSVMSTL CFFRFWLGFG IGGDYPLSAT IMSEYANKKT RGAFIAAVFA MQGFGILAGG 180
IFAIISAAAF KALFDAPTYE VNALASTVPQ ADYLWRIIVM VGAIPVAMTY YWRMKMPETA 240
RYTALVAKNA QQAASDMSKV LQVEIEAEPQ KAEPTANTFA LFSKEFMRRH GLHLLGTTST 300
WFLLDIAFYS QNLFQKDIFS AIGWIPPAKT MNAIEEVYRI ARAQTLIALC STVPGYWFTV 360
ALIDRIGRFA IQLMGFFFMT VFMFALAIPY DHWTHKDNRI GFVAIYSLTF FFANFGPNAT 420
TFVVPAEIFP ARFRSTCHGI SAASGKLGAI VGAFGFLYLA QNQDKAKADA GYPAGIGVKN 480
SLIALGVVNF LGILFTFLVP ESNGRSLEEM SGENEEESES VAVELEHSDY NNRTVPVV 538
<160>?19
<170>?PatentIn?version?3.3
<210>?3
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉upstream primer of beans pears phosphorus transporter protein gene cDNA sequence conserved regions fragment amplification
<222>?(1)..(20)
<400>?3
GTGCTNAATG CACTNGATGT 20
<160>?19
<170>?PatentIn?version?3.3
<210>?4
<211>?21
<212>?DNA
＜213〉artificial sequence
＜221〉downstream primer of beans pears phosphorus transporter protein gene cDNA sequence conserved regions fragment amplification
<222>?(1)..(21)
<400>?4
CCTDGCHGGG AAAATCTCNG C 21
<160>?19
<170>?PatentIn?version?3.3
<210>?5
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉upstream primer of beans pears phosphorus transporter protein gene cDNA sequence 3' end sequence amplification
<222>?(1)..(20)
<400>?5
TTGAAGCAGA GCCGCAGAAG 20
<160>?19
<170>?PatentIn?version?3.3
<210>?6
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉upstream primer of beans pears phosphorus transporter protein gene cDNA sequence 3' end sequence amplification
<222>?(1)..(20)
<400>?6
TGGACTCACA AGGACAACCG 20
<160>?19
<170>?PatentIn?version?3.3
<210>?7
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉beans pears phosphorus transporter protein gene 5 ' RACE reverse transcription primer
<222>?(1)..(20)
<400>?7
CCAAGCAAGT GAAGTCCATG 20
 
<160>?19
<170>?PatentIn?version?3.3
<210>?8
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉downstream primer of beans pears phosphorus transporter protein gene cDNA sequence 5' end sequence amplification
<222>?(1)..(20)
<400>?8
TCTGCGGCTC TGCTTCAATT 20
<160>?19
<170>?PatentIn?version?3.3
<210>?9
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉downstream primer of beans pears phosphorus transporter protein gene cDNA sequence 5' end sequence amplification
<222>?(1)..(20)
<400>?9
AAGACAGCGG CAATGAAAGC 20
<160>?19
<170>?PatentIn?version?3.3
<210>?10
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉upstream primer of beans pears phosphorus transporter protein gene cDNA full length sequence amplification
<222>?(1)..(20)
<400>?10
GCTAGCTTAG CCATGGCTAG 20
<160>?19
<170>?PatentIn?version?3.3
<210>?11
<211>?22
<212>?DNA
＜213〉artificial sequence
＜221〉downstream primer of beans pears phosphorus transporter protein gene cDNA full length sequence amplification
<222>?(1)..(22)
<400>?11
GACTGAAGGA CAAGATTACT CG 22
<160>?19
<170>?PatentIn?version?3.3
<210>?12
<211>?20
<212>?DNA
＜213〉artificial sequence
The upstream primer of RT-PCR amplification when＜221〉studying beans pears phosphorus transporter protein gene expression level
<222>?(1)..(20)
<400>?12
TTGAAGCAGA GCCGCAGAAG 20
<160>?19
<170>?PatentIn?version?3.3
<210>?13
<211>?20
<212>?DNA
＜213〉artificial sequence
The downstream primer of RT-PCR amplification when＜221〉studying beans pears phosphorus transporter protein gene expression level
<222>?(1)..(20)
<400>?13
AGAAGTTGAC CACACCCAGC 20
<160>?19
<170>?PatentIn?version?3.3
<210>?14
<211>?37
<212>?DNA
＜213〉artificial sequence
＜221〉beans pears phosphorus transporter protein gene 3 ' RACE reverse transcription primer AP
<222>?(1)..(37)
<400>?14
GGCCACGCGT CGACTAGTAC TTTTTTTTTT TTTTTTT 37
<160>?19
<170>?PatentIn?version?3.3
<210>?15
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉universal primer 3 ' AUAP in 3 ' RACE System for Rapid Amplification of cDNA Ends test kit (catalog number is 18373-019)
<222>?(1)..(20)
<400>?15
GGCCACGCGT CGACTAGTAC 20
<160>?19
<170>?PatentIn?version?3.3
<210>?16
<211>?36
<212>?DNA
＜213〉artificial sequence
＜221〉universal primer 5 ' AAP in 5 ' RACE System for Rapid Amplification of cDNA Ends test kit (catalog number is 18374-058)
<222>?(1)..(36)
<400>?16
GGCCACGCGT CGACTAGTAC GGGIIGGGII GGGIIG 36
<160>?19
<170>?PatentIn?version?3.3
<210>?17
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉universal primer 5 ' AUAP in 5 ' RACE System for Rapid Amplification of cDNA Ends test kit (catalog number is 18374-058)
<222>?(1)..(20)
<400>?17
GGCCACGCGT CGACTAGTAC 20
<160>?19
<170>?PatentIn?version?3.3
<210>?18
<211>?20
<212>?DNA
＜213〉artificial sequence
＜221〉beans pears Actin muscle ActinThe upstream primer of gene amplification
<222>?(1)..(20)
<400>?18
GGAATGGTCA AGGCTGGGTT 20
<160>?19
<170>?PatentIn?version?3.3
<210>?19
<211>?21
<212>?DNA
＜213〉artificial sequence
＜221〉beans pears Actin muscle ActinThe downstream primer of gene amplification
<222>?(1)..(21)
<400>?19
CAAAGCATCT GTGAGGTCAC G 21
 

Claims (4)

1. phosphorus transporter protein gene, it is characterized in that: it comes from the phosphorus transporter protein gene of rosaceous plant, and the nucleotides sequence of described phosphorus transporter protein gene is classified the nucleotide sequence of SEQ ID NO:1 in the sequence table as.
2. phosphorus transporter protein gene according to claim 1, it is characterized in that: the proteins encoded of phosphorus transporter protein gene has the aminoacid sequence of sequence table SEQ ID NO:2 protein active polypeptide, and the coded protein of nucleotide sequence of described aminoacid sequence and SEQ ID NO:1 the 13rd ~ 1629 bp position has 70% homology at least.
3. phosphorus transporter protein gene according to claim 2 is characterized in that: the aminoacid sequence SEQ ID NO:2 of coded protein utilizes BioXM software that the nucleotide sequence coded district part of SEQ ID NO:1 is translated the back to obtain; Described SEQ ID NO:2 is the albumen that contains 538 amino-acid residues.
4. the preparation method of a phosphorus transporter protein gene as claimed in claim 1 is characterized in that: utilize RACE technology clone unknown gene, concrete steps are:
Utilize reverse transcription primer oligo (dT) 15After synthetic conserved regions fragment amplification is used cDNA first chain,, carry out the conserved regions fragment that the RT-PCR amplification obtains SEQ ID NO:1 sequence by upstream primer SEQ ID NO:3 and downstream primer SEQ ID NO:4 pairing;
According to the conserved regions fragments sequence information of SEQ ID NO:1 sequence, design gene specific upstream primer SEQ ID NO:5, SEQ ID NO:6,5 ' RACE reverse transcription primer SEQ ID NO:7 and gene specific downstream primer SEQ ID NO:8, SEQ ID NO:9;
After utilizing in the test kit the synthetic 3' end sequence amplification of 3 ' RACE reverse transcription primer AP with cDNA first chain, by gene specific upstream primer SEQ ID NO:5 and SEQ ID NO:6 respectively with test kit in the 3' end sequence of universal primer 3 ' AUAP pairing carrying out nido RT-PCR amplification SEQ ID NO:1 sequence;
After utilizing the synthetic 5' end sequence amplification of 5 ' RACE reverse transcription primer SEQ ID NO:7 with cDNA first chain, by universal primer 5 ' AAP pairing in gene specific downstream primer SEQ ID NO:8 and the test kit, and the increase 5' end sequence of SEQ ID NO:1 sequence of universal primer 5 ' AUAP pairing carrying out nido RT-PCR in gene specific downstream primer SEQ ID NO:9 and the test kit;
The conserved regions fragment of the SEQ ID NO:1 sequence that obtains, the 3' end sequence and the 5' end sequence of SEQ ID NO:1 sequence are spliced, obtain PcPht1The cDNA total length;
According to the splicing result, design full length gene upstream primer SEQ ID NO:10 and full length gene downstream primer SEQ ID NO:11 utilize reverse transcription primer oligo (dT) 15The amplification of synthetic gene total length uses cDNA first chain as template, and is right PcPht1The cDNA total length carry out RT-PCR amplification checking.
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CN113444732A (en) * 2021-07-20 2021-09-28 周口师范学院 Application of gene TaPT16 in improving resistance of plants to powdery mildew
CN113969293A (en) * 2020-07-07 2022-01-25 中国科学院分子植物科学卓越创新中心 Crop phosphorus high-efficiency and high-yield gene and application thereof

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110714012A (en) * 2019-07-13 2020-01-21 周口师范学院 Application of gene TaPT13 in improving resistance of plant to variety of gaeumannomyces graminis
CN110714012B (en) * 2019-07-13 2022-07-08 周口师范学院 Application of gene TaPT13 in improving resistance of plant to variety of gaeumannomyces graminis
CN113969293A (en) * 2020-07-07 2022-01-25 中国科学院分子植物科学卓越创新中心 Crop phosphorus high-efficiency and high-yield gene and application thereof
CN113969293B (en) * 2020-07-07 2024-02-09 中国科学院分子植物科学卓越创新中心 Crop phosphorus high-efficiency and high-yield gene and application thereof
CN113444732A (en) * 2021-07-20 2021-09-28 周口师范学院 Application of gene TaPT16 in improving resistance of plants to powdery mildew
CN113444732B (en) * 2021-07-20 2022-07-08 周口师范学院 Application of gene TaPT16 in improving resistance of plants to powdery mildew

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Application publication date: 20111214