CN112794891B - ABC transport protein for transporting lycorine and encoding gene and application thereof - Google Patents
ABC transport protein for transporting lycorine and encoding gene and application thereof Download PDFInfo
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Abstract
The invention relates to ABC transport protein and a coding gene and application thereof. The invention discloses ABC transporter protein of lycoris radiata, which has the functions of combining lycorine and transporting the lycorine across cell membranes for the first time. The invention also discloses a polynucleotide for encoding the ABC transporter, a vector and a host cell for expressing the ABC transporter, and a method for realizing the inward transport of lycorine across a biological membrane.
Description
Technical Field
The present technology relates to the fields of biotechnology and plant biology; more specifically, the invention relates to an ABC transporter derived from lycoris plants, and a coding gene and application thereof.
Background
The transport of biomolecules across cell membranes is one of the important events in plant physiology, and is an important step in the communication transport and accumulation of substances in plants. ABC transporter (ATP binding cassette transporter) comprises metabolites such as polysaccharide, polypeptide, alkaloid, hormone and the like, and realizes transmembrane transport of specific substrates by hydrolyzing energy generated by ATP, so that the ABC transporter has important physiological functions in plants. ABC transporters are not only involved in the substrate transport process but are also in close relationship with their substrate yields. Transport proteins CjABCB1 and CjABCB2, which transport berberine located on the cell membrane, are found for example in coptis chinensis. Wherein the expression level of CjABCB1 has close relation with the berberine content in rhizoma Coptidis. cjABCB2 is involved in the long distance transport of berberine from root to rhizome.
Product toxicity is one of the bottlenecks in microbial production of compounds, and transporter mediated transport of molecules across membranes provides an effective way to solve this problem. For example, induction of ABC transporter-Snq p expression in beta-carotene producing yeast cells can enhance the efflux of beta-carotene and increase the intracellular beta-carotene content by about 1.7 times.
The Lycoris aurea (Lycoris aurea) is a perennial herb of Lycoris genus of Lycoris family, and contains rich Lycoris alkaloids in the body, such as galanthamine, lycorine, doxofylline, crine and the like, and other alkaloids and other natural products. The natural products have certain biological activity and application value. For example, galantamine is clinically useful as an acetylcholinesterase inhibitor in the treatment of moderate to mild alzheimer's disease. The dihydrolycorine generated by hydrogenation of lycorine has stronger function of resisting amoebic dysentery and smaller toxicity, so that the dihydrolycorine is used for clinic. The lactam salt prepared from lycorine has certain antitumor effect. These lycoris alkaloids have strong application and market demands. The biosynthesis thereof has great market potential. However, the influence and the effect of the transport protein on biosynthesis are considered while solving the synthesis path of lycoris alkaloids. At present, transport proteins transporting lycoris alkaloids have not been isolated and cloned. Therefore, there is a need in the art to develop transport proteins for lycoris alkaloids. The protein and the coding gene thereof can carry out the biotransformation and biosynthesis of lycoris alkaloids by transgenic plants and heterologous expression.
Disclosure of Invention
The object of the present invention is to provide an ABC transporter of lycoris plants, said ABC transporter being selected from the group consisting of:
(a) The amino acid sequence is shown as SEQ ID NO; 1: or (b)
(b) A protein which is formed by substitution, deletion or addition of one or more amino acid residues on the amino acid sequence of SEQ ID NO. 1 and has lycorine transport activity and is derived from (a); or (b)
(c) A derivatized protein having at least 85% sequence identity to the amino acid sequence of SEQ ID No. 1 and having lycorine transport activity.
The protein shown in SEQ ID NO. 1 is a novel ABC transporter isolated from Lycoris aurea. For convenience of description, the protein shown in SEQ ID NO. 1 is designated LaABCB11.
In a preferred embodiment, the transport function refers to a function of transporting lycorine from the outside to the inside of the biological membrane.
In another preferred embodiment, the sequence (c) further comprises: a fusion protein formed by adding a sequence tag, a signal sequence or a secretion signal sequence to (a) or (b).
It is another object of the present invention to provide an isolated polynucleotide which is a polynucleotide encoding said ABC transporter.
In a preferred embodiment, the nucleotide sequence is shown in SEQ ID NO. 2.
It will be appreciated that, given the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons appropriate for expression of a particular species, as desired. Thus, the polynucleotide of the ABC transporter also comprises a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 2 and is coded to have the activity of the ABC transporter.
It is another object of the present invention to provide a vector comprising said polynucleotide. The vector is a recombinant expression vector capable of expressing the ABC transporter or a gene silencing or gene editing vector for inhibiting the expression of the ABC transporter-encoding polynucleotide.
In a preferred embodiment, the vector is a recombinant expression vector pYES2-LaABCB11 comprising the sequence shown in SEQ ID NO. 2 encoding said ABC transporter.
It is a further object of the invention to provide an expression construct. The expression constructs comprise genes encoding the following proteins and/or enzymes and/or expression gene cassettes;
said ABC transporter; and/or
Related synthetases in the lycorine synthesis pathway.
The gene expression cassette is a biological element required for the expression and regulation of the protein in a host cell, and comprises a promoter, an enhancer, an attenuator, a ribosome binding site, a kazak sequence, an intron, a transcription terminator and the like; in addition, tag coding sequences and/or signal (peptide) coding sequences and the like may be included.
It is a further object of the present invention to provide a host cell comprising said vector or expression construct or genome having said polynucleotide integrated therein. The host cell is a prokaryotic cell or a eukaryotic cell, and commonly used prokaryotic host cells comprise escherichia coli, bacillus subtilis, pseudomonas mobilis, lactobacillus and the like; common eukaryotic host cells include fungal cells, plant cells, insect cells, mammalian cells, and the like. The fungal cells include yeast cells. And introducing the recombinant expression vector or the gene silencing vector or the expression construct into the proper host cell to obtain the genetically engineered strain, the transgenic cell line, the transgenic callus, the transgenic tissue, the transgenic plant or the genetically engineered plant for expressing the ABC transporter. More preferably, the host cell is a cell in which lycoris alkaloids or precursors thereof are endogenously present.
It is a further object of the present invention to provide the use of said ABC transporter for the transport of lycorine across cell membranes.
In a preferred embodiment, the ABC transporter transports lycorine across a cell membrane into a yeast cell.
It is a further object of the present invention to provide the use of said expression construct for transporting lycorine from the outer tank across the cell membrane.
In a preferred embodiment, the method comprises: transforming a host cell with said expression construct, and using the transformed host cell to transport lycorine from extracellular to intracellular; the host cell is a prokaryotic cell or a eukaryotic cell, and commonly used prokaryotic host cells comprise escherichia coli, bacillus subtilis, pseudomonas mobilis, lactobacillus and the like; common eukaryotic host cells include fungal cells, plant cells, insect cells, mammalian cells, and the like. The fungal cells include yeast cells.
The invention discloses an ABC transporter LaABCB11 from lycoris radiata of lycoris radiata for the first time, which has a good function of transporting lycorine across cell membranes inwards. Polynucleotides encoding the ABC transporters, expression vectors and host cells expressing the ABC transporters are also disclosed. The application of the ABC transporter from lycoris plants can realize the inward transport of lycorine across cell membranes.
Drawings
FIG. 1 is a DNA electrophoresis diagram of the gene encoding ABC transporter LaABCB11
FIG. 2 shows the expression level of the ABC transporter LaABCB11 encoding gene in tissues of the Indian smile
FIG. 3 is a phenotypic map of the ABC transporter LaABCB11 for the transport of lycorine
FIG. 4 is the intracellular content of the ABC transporter LaABCB11 transporting lycorine.
Detailed Description
The invention will be further elucidated with reference to specific embodiments and with reference to the accompanying drawings.
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 cloning of the gene encoding the ABC transporter gene LaABCB11
The synthesized two primers have the nucleic acid sequences of SEQ ID NO. 3 and SEQ ID NO. 4 in the sequence table respectively.
PCR was performed using cDNA obtained by reverse transcription of RNA extracted from Oryza Glutinosa as a template, and the above two primers SEQ ID NO. 3 and SEQ ID NO. 4. The DNA polymerase is Phanta of biological technology Co.Ltd TM Super Fidelity DNA polymerase. The amplification system was 50. Mu.L, and the procedure was: 98 ℃ for 2min;98 ℃ for 10s,56 ℃ for 10s and 72 ℃ for 2min, and 32 cycles are performed; the temperature is 72 ℃ for 10min, and the temperature is reduced to 10 ℃. The PCR products were detected by agarose gel electrophoresis.
Under ultraviolet lamp irradiation, the target DNA band was excised. And then recovering DNA from the agarose gel by using a DNA purification kit (Nanjinouzan FastPure Gel DNA Extraction Mini Kit), namely the amplified DNA fragment of the ABC transporter encoding gene. The recovered DNA fragment was ligated to pMD19-T vector using pMD19-T cloning kit from Takara Bio-engineering (Dalian) limited, and the constructed vector was named pMDT-LaABCB11.
The ABC transporter LaABCB11 encoding gene has a nucleotide sequence of SEQ ID NO. 2 in a sequence table. The open reading frame (Open Reading Frame, ORF) of the ABC transporter LaABCB11 encoding gene from nucleotide numbers 1-3873 of the 5' -end of SEQ ID NO. 2, the 1-3 nucleotide numbers 1-3 of the 5' -end of the SEQ ID NO. 2 being the start codon ATG of the ABC transporter LaABCB11 encoding gene, and the 3871-3873 nucleotide numbers 3871 of the 5' -end of the SEQ ID NO. 2 being the stop codon TAG of the ABC transporter LaABCB11 encoding gene. The ABC transporter LaABCB11 encoding gene encodes a protein LaABCB11 containing 1290 amino acids, has the amino acid sequence of SEQ ID NO. 1, predicts that the theoretical molecular weight of the protein is 139 Da and the isoelectric point pI is 7.58 by software.
Example 2 detection of expression level of the ABC transporter Gene LaABCB11 in Oriental laugh
Two primers of SEQ ID NO. 5 and SEQ ID NO. 6 of the sequence list are synthesized. Quantitative PCR amplification was performed using cDNA from each tissue of Setaria elegans as a template using a qTOWER 2.2 fluorescent quantitative PCR instrument. The expression level of the LaABCB11 gene in the leaf, the scape and the stamens of the laABCB11 gene is relatively high through calculation and analysis.
EXAMPLE 3 construction of ABC transporter LaABCB11 recombinant expression vector
Two primers having the nucleotide sequences of SEQ ID No. 7 and SEQ ID No. 8 in the sequence Listing are synthesized. Two enzyme cutting sites of BamHI and EcoRI and protecting base sequences thereof are respectively arranged at the 5' -end of the synthesized primers SEQ ID NO. 7 and SEQ ID NO. 8, and PCR amplification is carried out by taking the cDNA of the laugh as a template. PCR amplification procedure the PCR amplification product was detected by agarose gel electrophoresis, separated, excised and recovered, and then digested with BamHI and EcoRI, and ligated with the same BamHI and EcoRI digested pYES2 vector (Invitrogen) using T4DNA ligase from Takara Shuzo Co., ltd. The ligation product was transformed into competent cells of E.coli DH 5. Alpha. From Nanjing department of Biotechnology, inc., and plated onto LB plates with 50. Mu.g/mL kanamycin. Positive transformants were obtained by colony PCR verification. The recombinant plasmid pYES 2-EcoRI-LaABCB 11-BamHI was further verified by sequencing to be successful in construction and contained between the BamHI and EcoRI cleavage sites the sequence of SEQ ID NO:2, and a full-length polynucleotide sequence of seq id no. The recombinant plasmid obtained was designated pYES2-LaABCB11.
Example 4 expression of ABC Transporter LaABCB11
And (3) transforming the recombinant plasmid pYES2-LaABCB11 into the saccharomycete INVSc1 competent cells by using a PEG chemical transformation method to obtain the recombinant saccharomycete INVSc1/pYES2-LaABCB11. And (3) selecting the monoclonal antibody, culturing the monoclonal antibody in YNB-Ura liquid culture medium containing 2% glucose until bacterial liquid grows to have absorbance of 0.4 at a wavelength of 600nm, collecting bacterial cells, and then performing induction culture on the bacterial liquid by using YNB-Ura culture medium containing 2% raffinose.
Example 5 ABC transporter transport of lycorine
The 12h induction yeast culture broth was collected and centrifuged at 8000rpm at 4℃for 5min, and the supernatant was discarded and the cells were washed with 2% glycerol solution 1 time. The cells were resuspended in fresh YNB-Ura medium containing 2% raffinose and transferred to 250mL sterile Erlenmeyer flasks, and lycorine (purchased from Shanghai Seiyaku BioCo., ltd.) stock solution was added to a final concentration of 0.3mM, mixed well and cultured with shaking at 100rpm at 30℃for 6-12h.
Yeast cells were collected and centrifuged at 8000rpm at 4℃for 5min, the supernatant was discarded, the cells were washed 3 times with a 2% glycerol solution, resuspended in methanol, and 1/5 volume of acid glass beads (diameter 4-5 μm, sigma) were added and disrupted by shaking in a mill at 30Hz for 15 min. Centrifuge at 12000rpm for 10min at 4 ℃. The supernatant was filtered through a 0.45 μm pore size filter, and the filtrate was analyzed by High Performance Liquid Chromatography (HPLC). The analysis conditions were: the method comprises the steps of adopting an LC-20A high performance liquid chromatograph (Shimadzu, japan), wherein a chromatographic column is a reverse chromatographic column InertSustatin C18 (5 mu m,4.6 mm-250 mm), a column temperature is 35 ℃, a diode array detector is at a wavelength of 290nm, a sample injection amount is 20 mu L, a mobile phase A is 0.1% n-dibutylamine aqueous solution, a mobile phase B is acetonitrile, a flow rate is 0.8mL/min, and gradient elution is carried out. The analysis results are shown in fig. 4, and the results indicate that: the ABC transporter LaABCB11 can transport lycorine into cells through a membrane, so that the content of lycorine in the cells reaches 10-15 nmol/(g FW). It is understood that the ABC transporter LaABCB11 can specifically transport lycorine across biological membranes and has transport activity for its transport into cells.
Sequence listing
<110> institute of plant Material of national academy of sciences of China, jiangsu province
<120> ABC transporter for transporting lycorine, and coding gene and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1290
<212> PRT
<213> smile (Lycoris aurea)
<400> 1
Met Gly Ile Glu Glu Glu Lys Asp Lys Ile Ser Pro Ser Val Asn Gly
1 5 10 15
Ser Asn Glu Pro Ser Ser Ser Asn Ser Thr Val Lys Glu Ala Asn Gly
20 25 30
Glu Lys Ser Asn Glu Lys Arg Asp Glu Ala Lys Tyr Thr Val Pro Phe
35 40 45
Tyr Lys Leu Phe Ala Phe Ala Asp Ser Thr Asp Val Leu Leu Met Thr
50 55 60
Ala Gly Ser Ile Gly Ala Val Ala Asn Gly Leu Ala Leu Pro Leu Met
65 70 75 80
Thr Val Leu Phe Gly Asn Leu Ile Gln Ser Phe Gly Gly Ala Thr Asn
85 90 95
Ile His Asp Val Val His Arg Val Ser Lys Val Cys Leu Glu Phe Val
100 105 110
Tyr Leu Ala Val Gly Ser Gly Ile Ala Ser Phe Phe Gln Val Ser Cys
115 120 125
Trp Met Ala Thr Gly Glu Arg Gln Ala Ala Arg Ile Arg Asn Leu Tyr
130 135 140
Leu Lys Thr Ile Leu Arg Gln Glu Ile Ala Phe Phe Asp Lys Glu Thr
145 150 155 160
Asn Thr Gly Glu Val Val Gly Arg Met Ser Gly Asp Thr Val Leu Ile
165 170 175
Gln Asp Ala Met Gly Glu Lys Val Gly Lys Phe Ile Gln Leu Val Ser
180 185 190
Thr Phe Phe Gly Gly Phe Val Val Ala Phe Val Gln Gly Trp Leu Leu
195 200 205
Thr Leu Val Met Leu Thr Thr Ile Pro Leu Leu Val Ile Ala Gly Ala
210 215 220
Ala Met Gly Ala Ala Ile Ser Lys Met Ala Ser Met Gly Gln Thr Ala
225 230 235 240
Tyr Ala Glu Ala Ala Val Val Val Glu Gln Thr Ile Gly Ser Ile Arg
245 250 255
Thr Val Ala Ser Phe Thr Gly Glu Lys Gln Ser Val Ile Lys Tyr Lys
260 265 270
Lys Ser Leu Lys Ser Ala Tyr Glu Ser Ser Val Arg Glu Ser Leu Ala
275 280 285
Ser Gly Leu Gly Leu Gly Thr Val Met Leu Ile Met Phe Cys Gly Tyr
290 295 300
Gly Leu Gly Ile Trp Tyr Gly Ser Lys Leu Ile Leu Asp Lys Gly Tyr
305 310 315 320
Thr Gly Ala Asp Val Ile Asn Val Ile Phe Ala Val Leu Thr Gly Ser
325 330 335
Phe Ser Leu Gly Gln Ala Ser Pro Cys Leu Thr Ala Phe Ala Ala Gly
340 345 350
Gln Ala Ala Ala Phe Lys Met Phe Glu Thr Ile Asn Arg Lys Pro Glu
355 360 365
Ile Asp Ala Thr Asp Pro Thr Gly Lys Lys Leu Asp Asp Ile Val Gly
370 375 380
Asp Val Glu Phe Lys Asp Val Tyr Phe Ser Tyr Pro Thr Arg Gln Asp
385 390 395 400
Glu Gln Ile Phe Arg Gly Phe Ser Leu Phe Ile Gln His Gly Thr Thr
405 410 415
Val Ala Leu Val Gly Glu Ser Gly Ser Gly Lys Ser Thr Val Ile Ser
420 425 430
Leu Ile Glu Arg Phe Tyr Asp Ala Gln Ala Gly Glu Val Leu Ile Asp
435 440 445
Gly Ile Asn Leu Lys Glu Phe Gln Leu Lys Trp Ile Arg Gly Lys Ile
450 455 460
Gly Leu Val Ser Gln Glu Pro Val Leu Phe Ala Ser Ser Ile Arg Asp
465 470 475 480
Asn Ile Ala Tyr Gly Lys Glu Gly Ala Thr Thr Glu Glu Ile Arg Ala
485 490 495
Ala Thr Glu Leu Ala Asn Ala Ser Lys Phe Ile Asp Lys Met Pro Gln
500 505 510
Gly Leu Asp Thr Met Val Gly Glu His Gly Thr Gln Leu Ser Gly Gly
515 520 525
Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala Ile Leu Lys Asp Pro Arg
530 535 540
Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Ala Glu Ser Glu
545 550 555 560
Arg Ile Val Gln Glu Ala Leu Asp Arg Ile Met Ala Asn Arg Thr Thr
565 570 575
Val Ile Val Ala His Arg Leu Ser Thr Val Arg Asn Ala Asp Thr Ile
580 585 590
Ala Val Ile His Arg Gly Ser Ile Val Glu Lys Gly Ser His Ser Glu
595 600 605
Leu Leu Lys Asp Ser Asn Gly Ala Tyr Cys Gln Leu Ile Arg Leu Gln
610 615 620
Glu Met Asn Gln Asn Ser Asp His Pro Ser Gln Ser Ser Lys Ala Lys
625 630 635 640
Leu Asn Leu Ser Ser Asp Ala Gly Arg Arg Ser Ser Arg His Met Ser
645 650 655
Leu Asn Arg Ser Ile Ser Arg Glu Ser Ser Ser Ile Gly Asn Ser Ser
660 665 670
Arg Ser Phe Thr Leu Pro Leu Gly Leu Pro Pro Gly Ile Asp Leu Gln
675 680 685
Glu Thr Lys Ser Glu Gly Ala Thr Asn Thr Glu Val Pro Pro Ala Gln
690 695 700
Val Lys Glu Val Ser Leu Arg Arg Leu Ala Tyr Leu Asn Lys Pro Glu
705 710 715 720
Ile Pro Val Leu Ile Ile Gly Val Ile Ser Ala Val Val Asn Gly Leu
725 730 735
Val Phe Pro Ile Phe Gly Leu Leu Leu Ser Ser Val Ile Asn Thr Phe
740 745 750
Tyr Gln Pro Pro Asp Lys Leu Lys Lys Asp Ser Lys Phe Trp Ser Leu
755 760 765
Leu Phe Ala Ile Phe Gly Val Ile Ser Phe Leu Ala Leu Pro Ala Arg
770 775 780
Thr Tyr Phe Phe Gly Val Ala Gly Ala Lys Leu Ile Gln Arg Ile Arg
785 790 795 800
Ser Met Thr Phe Glu Lys Val Val His Met Glu Val Ala Trp Phe Asp
805 810 815
Glu Pro Glu Asn Ser Ser Gly Ala Ile Gly Ala Arg Leu Ser Ala Asp
820 825 830
Ala Ala Thr Val Arg Gly Leu Val Gly Asp Ala Leu Ala Leu Val Val
835 840 845
Gln Asn Ile Thr Thr Leu Ile Ala Gly Leu Val Ile Ala Phe Val Ala
850 855 860
Asn Trp Gln Leu Ser Phe Ile Ile Leu Ala Leu Ile Pro Leu Ile Gly
865 870 875 880
Ile Asn Gly Tyr Ile Gln Val Lys Phe Met Thr Gly Phe Ser Ala Asp
885 890 895
Ala Lys Met Met Tyr Glu Glu Ala Ser Gln Val Ala Asn Asp Ala Val
900 905 910
Gly Ser Ile Arg Thr Val Ala Ser Phe Ser Ala Glu Asp Lys Val Met
915 920 925
Glu Leu Tyr Lys Lys Lys Cys Glu Gly Pro Met Arg Thr Gly Ile Arg
930 935 940
Gln Gly Leu Ile Ser Gly Ile Gly Phe Gly Ala Ser Phe Phe Leu Leu
945 950 955 960
Phe Cys Val Tyr Ala Thr Ser Phe Tyr Ala Gly Ala Arg Leu Val Glu
965 970 975
Asp Gly Lys Thr Thr Phe Gly Lys Val Phe Lys Val Phe Phe Ala Leu
980 985 990
Ser Met Ala Ala Ile Gly Ile Ser Gln Ser Ser Ser Leu Ala Pro Asp
995 1000 1005
Ser Ser Lys Ala Lys Ser Ala Thr Ala Ser Val Phe Ala Val Leu Asp
1010 1015 1020
Arg Lys Ser Lys Ile Asp Pro Ser Asp Glu Ser Gly Met Thr Leu Glu
1025 1030 1035 1040
Arg Leu Lys Gly Asn Ile Glu Phe Arg His Val Ser Phe Lys Tyr Pro
1045 1050 1055
Thr Arg Pro His Val Gln Ile Phe Gln Asp Leu Cys Leu Ser Ile His
1060 1065 1070
Ala Gly Lys Thr Val Ala Leu Val Gly Glu Ser Gly Ser Gly Lys Ser
1075 1080 1085
Thr Ala Ile Ala Leu Leu Gln Arg Phe Tyr Asp Pro Asp Ser Gly His
1090 1095 1100
Ile Leu Ile Asp Gly Met Glu Ile Glu Lys Phe Gln Val Arg Trp Leu
1105 1110 1115 1120
Arg Gln Gln Met Gly Leu Val Ser Gln Glu Pro Ala Leu Phe Asn Asp
1125 1130 1135
Thr Ile Arg Ala Asn Ile Ala Tyr Gly Lys Glu Gly Asp Ala Thr Glu
1140 1145 1150
Ala Glu Val Val Ala Ala Ala Glu Ser Ala Asn Ala His Lys Phe Ile
1155 1160 1165
Cys Ser Leu Gln Gln Gly Tyr Asp Thr Val Val Gly Glu Arg Gly Ile
1170 1175 1180
Gln Leu Ser Gly Gly Gln Lys Gln Arg Val Ala Ile Ala Arg Ala Ile
1185 1190 1195 1200
Val Lys Glu Pro Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala Leu
1205 1210 1215
Asp Ala Glu Ser Glu Arg Val Val Gln Asp Ala Leu Asp Arg Val Met
1220 1225 1230
Val Asn Arg Thr Thr Ile Val Ile Ala His Arg Leu Ser Thr Ile Arg
1235 1240 1245
Gly Ala Asp Val Ile Ala Val Val Lys Asn Gly Met Ile Ile Glu Lys
1250 1255 1260
Gly Asn His Asp Ser Leu Ile Asn Ile Lys Asp Gly Ala Tyr Ala Ser
1265 1270 1275 1280
Leu Val Ala Leu His Thr Asn Ser Ser Thr
1285 1290
<210> 2
<211> 3873
<212> DNA
<213> smile (Lycoris aurea)
<400> 2
atgggtattg aagaagagaa ggacaaaatt agtccttcag tcaatggatc aaatgagcca 60
tcttctagta attcaacggt gaaagaagca aacggcgaga agagcaatga gaaaagggac 120
gaggcgaagt acaccgttcc gttctacaag ctgttcgctt ttgcagactc gacagatgtg 180
cttctcatga ccgcaggctc gattggagct gtggctaatg ggcttgcatt gcctctcatg 240
acggtgctct ttggaaattt gatccaatct ttcggaggag ctactaatat tcatgatgtg 300
gttcataggg tttccaaggt ctgtcttgag tttgtctact tggctgtggg atcagggata 360
gcgtcatttt ttcaggtgtc ttgttggatg gcgacggggg agaggcaggc tgcaagaata 420
aggaatttgt atttgaaaac catattacgg caagaaatcg cattctttga caaggaaaca 480
aatactggag aggttgtggg gagaatgtca ggtgatactg ttctcattca agatgccatg 540
ggtgaaaagg ttggcaagtt catccagcta gtatcaacat tctttggggg ttttgtagtt 600
gcatttgttc aaggatggct tctcactctt gtgatgctaa ccacaatccc gctcctcgtg 660
atagcaggcg cggcaatggg agccgcaata tcaaaaatgg catcgatggg gcaaacagct 720
tatgcggagg cagctgttgt cgttgagcaa acaatcggtt caattagaac agttgcatct 780
tttactggcg aaaagcaatc ggtcataaag tacaagaaat ctctcaagag tgcttatgag 840
tctagtgttc gagagagtct cgcttcagga ttaggtcttg gtactgtaat gctaatcatg 900
ttttgcggct acgggttggg aatttggtat gggtcaaaat tgatattgga taaaggctac 960
actggtgctg atgtaatcaa cgtgatattt gcagtcttga caggctcatt ttccttggga 1020
caggcatctc catgcttgac ggcatttgca gcagggcaag ccgcggcatt caagatgttt 1080
gaaacgatca acagaaagcc agagatagat gcaaccgatc caacagggaa aaaactagat 1140
gacatagttg gagatgtaga atttaaagat gtttacttca gctatccaac aagacaagat 1200
gagcaaatat ttagaggatt ttctctattc atacagcatg gcacgactgt agctttggtc 1260
ggagagagtg gaagcgggaa gtctacagtt ataagtctga ttgagagatt ttatgacgca 1320
caagcaggtg aagttcttat agatggcata aaccttaagg aatttcaact caaatggatc 1380
agaggtaaaa ttgggctagt gagtcaagag cccgtactct ttgcatcaag cattagagat 1440
aacatagcct atggaaagga aggggcaacc actgaagaaa ttagagccgc aactgagctt 1500
gctaatgctt caaagtttat agacaagatg ccacagggac ttgacaccat ggttggtgag 1560
catgggaccc agctatccgg gggccaaaaa caaaggattg cgattgcaag agctattctg 1620
aaagacccac gaattctgct tcttgatgaa gccaccagtg ctttggatgc agaatctgaa 1680
aggatagtgc aagaggcact tgataggata atggcaaacc gtacgactgt cattgttgct 1740
catcgcttga gtacagtgag aaatgcagat actattgctg ttatacacag aggttcaatt 1800
gtggaaaaag gttcgcattc cgagctacta aaggattcaa atggtgctta ttgccaactc 1860
atacgcttgc aagaaatgaa tcaaaattct gaccatccat ctcagtccag caaggctaag 1920
ctcaatcttt catctgacgc tgggaggcgc tctagcagac atatgtctct aaatcgttca 1980
ataagccgtg aatcatcttc tataggcaac agtagtcgct ctttcacatt acccttagga 2040
ttgcccccag gaattgatct tcaagagacc aaatcagagg gagcaacaaa cactgaggtt 2100
cctccagcac aagtaaagga agtatcactc cgacgccttg cctacctcaa taaacctgaa 2160
attccagttc ttataattgg tgtgatctct gctgttgtta atgggcttgt gtttcccata 2220
tttggactac tcttatcgag cgtgataaat acattctacc aaccacctga taaactcaaa 2280
aaggattcaa aattttggtc gttgttgttt gctatatttg gtgtgatttc tttcctagca 2340
cttcccgcta gaacttactt ttttggagtg gccggagcca aattaataca aaggataaga 2400
tcaatgacct tcgagaaggt ggtacatatg gaggttgctt ggtttgatga acctgaaaat 2460
tctagtggag caattggcgc aagattatca gctgatgcag cgacggttcg gggcctcgtg 2520
ggtgatgcac ttgctttagt tgttcagaac attacaactt taattgctgg tttggtaatt 2580
gcttttgttg caaactggca attgtctttt attatcttgg ctctgatacc actcataggc 2640
atcaacggat atatccaagt gaagttcatg actggattta gtgctgatgc aaagatgatg 2700
tatgaggagg ctagtcaggt tgccaatgat gccgtgggaa gtataagaac cgttgcttct 2760
ttctcagctg aagataaagt gatggaactt tacaagaaga aatgtgaagg acctatgagg 2820
actggaattc ggcaaggatt gattagcggt attggatttg gagcctcctt cttcttgcta 2880
ttttgtgttt atgcaaccag tttttatgcg ggagctcgcc ttgtggagga tggaaagact 2940
acatttggaa aagtttttaa ggttttcttt gctctttcta tggcagctat tggaatttct 3000
caatcaagct ccctagcgcc agactctagc aaagccaaat ccgctacagc ttctgtgttt 3060
gctgttcttg atcgtaagtc aaagattgat ccaagcgatg aatccgggat gacactagaa 3120
agattaaagg gaaacattga gttccggcat gtcagtttca agtatccaac aaggccacat 3180
gtgcagattt tccaagactt gtgcttgtcg attcatgctg gaaagactgt tgcattggtt 3240
ggcgagagcg gaagtgggaa atcaactgcg atagcattgt tacaaagatt ttatgatccc 3300
gattccggtc atatactgat agatggaatg gagattgaga aatttcaggt gcggtggcta 3360
aggcagcaaa tgggtctggt tagtcaagaa ccagctttgt ttaatgacac gattcgagcc 3420
aacattgcct acgggaaaga aggagatgcc accgaggctg aagttgtagc tgcggccgag 3480
tcagcaaatg cccacaagtt catatgcagt ttacaacagg gttatgacac ggtggttgga 3540
gagcgaggga tccagctatc aggtggtcaa aagcaacggg tggcgattgc acgtgccatt 3600
gtgaaagagc caaaaattct acttctagac gaagcaacaa gcgcgctcga tgctgaatct 3660
gaacgagtgg ttcaagatgc tttagatcga gtcatggtca accgtacaac aatagtgatc 3720
gcacatcggt tgtctacgat cagaggtgcc gatgtgattg cagtggttaa gaatggaatg 3780
attattgaga aaggaaacca tgattcattg atcaatatca aggatggtgc ttatgcatca 3840
ttagttgcac ttcacacaaa ttcttctacg tag 3873
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgggtattg aagaagagaa gg 22
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctacgtagaa gaatttgtgt g 21
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ccactcgttc agattcagca 20
<210> 7
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ctcggatcca tgggtattga agaagagaag g 31
<210> 8
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gcagaattcc tacgtagaag aatttgtgtg 30
Claims (6)
1. An ABC transporter, wherein said ABC transporter is selected from the group consisting of: the amino acid sequence is shown in SEQ ID NO:1, and a protein shown in the formula 1.
2. A polynucleotide encoding the ABC transporter of claim 1; the polynucleotide comprises an isolated polynucleotide, and is characterized in that the nucleotide sequence of the polynucleotide is shown as SEQ ID NO. 2.
3. A vector comprising the polynucleotide of claim 2.
4. Use of the vector of claim 3, wherein the ABC transporter of claim 1 is expressed.
5. A host cell comprising the vector of claim 3 or the polynucleotide of claim 2 integrated into the genome.
6. Use of an ABC transporter according to claim 1 for the inward transport of lycorine across a cell membrane.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11276172A (en) * | 1998-03-27 | 1999-10-12 | Tanabe Seiyaku Co Ltd | Abc transporter gene |
| WO2015105273A1 (en) * | 2014-01-07 | 2015-07-16 | 포항공과대학교 산학협력단 | Composition for promoting cytokinin transport, comprising abcg14 protein of plant |
| CN109652427A (en) * | 2019-01-24 | 2019-04-19 | 安徽农业大学 | A kind of strawberry FaABCC1 transporter gene and its application |
-
2021
- 2021-03-31 CN CN202110354954.2A patent/CN112794891B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11276172A (en) * | 1998-03-27 | 1999-10-12 | Tanabe Seiyaku Co Ltd | Abc transporter gene |
| WO2015105273A1 (en) * | 2014-01-07 | 2015-07-16 | 포항공과대학교 산학협력단 | Composition for promoting cytokinin transport, comprising abcg14 protein of plant |
| CN109652427A (en) * | 2019-01-24 | 2019-04-19 | 安徽农业大学 | A kind of strawberry FaABCC1 transporter gene and its application |
Non-Patent Citations (2)
| Title |
|---|
| 忽地笑ＡＢＣＧ５ 转运蛋白基因(ＬａＡＢＣＧ５ )的 克隆和表达分析;刘彦彤等;植物资源与环境学报;第第29卷卷(第第2期期);全文 * |
| 忽地笑LaABCG3转运蛋白基因的克隆与表达分析;韩小康等;植物生理学报;第第54卷卷(第第4期期);全文 * |
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