CN102220351A - Function identification and application of delta-5 desaturase gene PinD5 of Phytophthora infestans in potato - Google Patents
Function identification and application of delta-5 desaturase gene PinD5 of Phytophthora infestans in potato Download PDFInfo
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Abstract
The invention relates to function identification and an application of delta-5 desaturase gene PinD5 of Phytophthora infestans in a potato. The delta-5 desaturase gene PinD5 relating to fish oil synthesis is separated form the Phytophthora infestans in the potato by an RT-PCR (reverse transcription polymerase chain reaction) method, and the invention belongs to the field of molecular biology and biological technologies. TtThe new delta-5 desaturase gene is determined through yeast expression identification, and coded delta 5 desaturase which allows dohono-gamma-linolenic acid to be converted into arachidonic acid can be used for fish oil production in plants especially oil crops.
Description
(1) technical field
The present invention relates to Function Identification and the application of Δ 5 delta 8 desaturase genes PinD5 in potato phytophthora infestans (Phytophthora infestans) overlength chain polyunsaturated fatty acid metabolic pathway of synthesizing, belong to molecular biology and biological technical field.
(2) background technology
Overlength chain polyunsaturated fatty acid generally contains 20-22 carbon, a 4-6 cis-double bonds, for example arachidonic acid (AA, 20:4 Δ
5,8,11,14), EPA (20:5 Δ
8,14,14,17) and DHA (22:6 Δ
4,7,10,13,16,19) human nutrition and health are had important effect.Often mend food EPA and this class lipid acid of DHA and not only tire, infant's pallium and neuronic growth are had vital role (Crawford, 2000), and sickness rate (Thies et al., 2003 that can reduce diseases such as cardiovascular and cerebrovascular diseases, hypertension and inflammation; Kinsella et al., 1990; Yamazaki et al., 1992).At present, this class lipid acid is mainly derived from fish oil.Because overfishing causes ocean wild fish resource exhausted day by day, fish oil output can not satisfy the needs (Pauly et al., 2003) that people are just increasing rapidly.In addition, because environmental pollution etc. cause the content of toxic substance such as heavy metal ion and organochlorine in the fish oil to raise, have a strong impact on edible safety (Yokoo et al., 2003 of fish oil; Drexler et al., 2003).Therefore, press for lasting, stable, a safe fish oil and substitute the production approach.
Higher plant can be synthesized linolic acid (LA, 18:2 Δ in a large number
9,12) and linolenic acid (ALA, 18:3 Δ
9,12,15), but can not synthesize overlength chain polyunsaturated fatty acid.Some fungies and little algae etc. can transform generation EPA and DHA by a series of chain extension enzymes in " Δ 6 desaturation approach " and desaturase katalysis with LA and the ALA of self.LA and ALA generate gamma-linolenic acid (GLA, 18:3 Δ under the katalysis of Δ 6 desaturases
6,9,12) and therapic acid (SDA, 18:4 Δ
6,9,12,15), and then under Δ 6 chain extension enzyme catalysis, generate dihomo-gamma-linolenic acid (DGLA, 20:3 Δ
8,11,14) and eicosatetraenoic acid (ETA, 20:4 Δ
8,11,14,17).At last, under Δ 5 desaturase katalysis, generate AA and EPA.EPA generates DHA through Δ 5 chain extensions and Δ 4 desaturations.
By the metabolic gene engineering overlength chain polyunsaturated fatty acid metabolic pathway of synthesizing in marine microalgae or the fungi is transplanted in the plant, thereby particularly producing this type of lipid acid in the oil crop seeds by using plant, will be that an ideal fish oil substitutes the production approach.At present, the genes involved in this metabolic pathway of synthesizing is cloned into (Yadav et al., 1993 higher plant, marine alga, fungi, nematode, liver moss and animal etc. successively; Wallis and Browse, 1999; Qi et al., 2002; Michaelson et al., 1998; Sayanova et al., 2006; Kajikawa et al., 2006; Cho et al., 1999; Sperling et al., 2000; Leonard et al., 2000; Watts and Browse, 1999), and all patent applied for protections.Excavate this genoid resource of clone as early as possible, the gene that acquisition has independent intellectual property right lays the foundation for China produces this type of health care lipid acid by transgenic technology in oil crops, is the free-revving engine that this invention is created.
(3) summary of the invention
The present invention at first extracts the total RNA of potato phytophthora infestans, after reverse transcription becomes cDNA, be PCR with primer 5 '-ATGACCACGGATGACATCAC-3 ' and 5 '-CTCGAGTTAGCCCATGTGGACGGT-3 ', after the fragment glue recovery with amplification, be connected among the cloning vector pMD18-T.Bioinformatic analysis, the cDNA fragment open reading frame that obtains partly is 1551bp, 516 the amino acid whose polypeptide of encoding, possess " front-end " N terminal cell pigment b5 structural domain (HPGG) that desaturase had, and three the Histidine mount structure territories (HxxxH, HxxHH and LNxQxxHHLFP) relevant with electron transport.This gene cDNA sequence is as follows:
Sequence table
(1) information of SEQ ID NO.1
(a) sequence signature
* length: 1551 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: potato phytophthora infestans (Phytophthora infestans)
(f) sequence description: SEQ IN NO.1
1?ATGACCACGG?ATGACATCAC?ACTCCAATTC?GCACTCACGG?CCGGAAAGAC?CATCGGAACC
61?TCTGCTTCCC?CTGCATTGTC?ACCACTAATT?GACGAAGTAG?AAGCTGCCAT?GGCCCCTATC
121?GAGACCGAGA?AGACCATCGT?TAACGAGGGT?CTTTACCAGC?GCAAGACTGC?TGCTGATGCA
181?GCTACTAACA?AGTCTGCCGC?CACCTACACG?TGGCAGGACG?TGGCCAAGCA?CAACACAGAC
241?AACAGCGCTT?GGGTCATCAT?CCGCGGAATC?GTATACGACG?TAACTGAATG?GGCGGATCGC
301?CACCCTGGAG?GCCGTGAACT?CGTGCTGCTG?CACTCTGGTC?GCGAGTGCAC?CGACACATTC
361?GACTCGTACC?ACCCATTCTC?TAACCGCGCC?GAGAAGATCC?TGGCCAAATA?CGCCATTGGC
421?AAGCTTGTCG?GAGGCTCCGA?GTTCCCCACC?TACAAGCCTG?ACACAGGTTT?CTACAGGGAA
481?TGCTGCGACC?GTGTGCACCA?GTACTTCAAG?GACAACAACC?TGGACCCTCG?CAGTCCGTAC
541?TCGGGTCTGT?TGCGCATGCT?TTTCGTGGCT?ATCCTTGGTG?CGGTGTCCTA?CGTGGGTATG
601?AACCAGCTGC?TGTCCGACAA?CATCTACGCA?CACTACGCAT?GGGGCGCCCT?CTTCGGTGCG
661?TGCCAAGCTC?TACCTTTGCT?GCACGTGATG?CACGATGCCT?CACACGCGGC?CATTACCAGC
721?AGCCCTACAG?GCTGGAGACT?TATCGGGCGT?CTTGCAATGG?ACTGGGTGGC?CGGCGCCAAC
781?ATGGTGTCTT?GGCTCAACCA?GCACGTTGTG?GGCCACCACA?TCTACACGAA?CGTTGCGGGA
841?GCTGACCCTG?ACCTTCCCGT?GGACTTCAAG?AGCGACGTGC?GTCGCATAGT?GTACCGCCAG
901?GTGTTGCTTC?CAATCTACAA?GTTCCAACAC?ATCTACCTAC?CTCCGCTCTA?CGGCGTGCTC
961?GGCCTCAAGT?TCCGTGTTCA?GGACATCTTT?GAGACGTTCA?TCTCCCTCAC?CAACGGACCG
1021?CTACGTGTGA?ACCCCCTCTC?TATCGGTGAC?TGGGCTGAAA?TGATCATCTC?CAAGGCCTTC
1081?TGGGCGTTCT?ACCGCATCTA?CATCCCGCTG?GTTGTGCTGC?AGGTTAACCC?CGCTCGCTTC
1141?TGGGGTGTTT?TCCTTCTGGC?CGAGTTTGTG?ACGGGCTGGT?ACTTGGCCTT?CAACTTCCAA
1201?GTTAGTCACG?TGTCCACGGC?ATGCGACTAC?CCTGGTGGTG?ATGAGGAGGT?ATCGGCTATC
1261?GAAGACGAGT?GGGCTATCTC?GCAGATCAAG?TCATCGGTAG?ACTACGCGCA?CGGCTCGTTC
1321?CTCACGGCGT?TCCTCACGGG?TGCACTGAAC?TACCAGGTGA?CCCACCACCT?CTTCCCCGGC
1381?GTCTCGCAGT?ATCATTACCC?TGCTATCGCC?CCGATCATCA?TGGACGTGTG?CAACAAGTAC
1441?AAGATCAAGT?ACACGGTGCT?GCCTACGTTC?TCAGCGGCTC?TGGCTGGACA?CTTTGAGCAT
1501?CTCGTTGTCA?TGGGAAAGAT?GGGCAAGCGT?GTGACCGTCC?ACATGGGCTA?A
(2) information of SEQ IN NO.2
(a) sequence signature
* length: 516 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
Sequence description
MET?Thr?Thr?Asp?Asp?Ile?Thr?Leu?Gln?Phe?Ala?Leu?Thr?Ala?Gly
1 5 10 15
Lys?Thr?Ile?Gly?Thr?Ser?Ala?Ser?Pro?Ala?Leu?Ser?Pro?Leu?Ile
20 25 30
Asp?Glu?Val?Glu?Ala?Ala?MET?Ala?Pro?Ile?Glu?Thr?Glu?Lys?Thr
35 40 45
Ile?Val?Asn?Glu?Gly?Leu?Tyr?Gln?Arg?Lys?Thr?Ala?Ala?Asp?Ala
50 55 60
Ala?Thr?Asn?Lys?Ser?Ala?Ala?Thr?Tyr?Thr?Trp?Gln?Asp?Val?Ala
65 70 75
Lys?His?Asn?Thr?Asp?Asn?Ser?Ala?Trp?Val?Ile?Ile?Arg?Gly?Ile
80 85 90
Val?Tyr?Asp?Val?Thr?Glu?Trp?Ala?Asp?Arg?His?Pro?Gly?Gly?Arg
95 100 105
Glu?Leu?Val?Leu?Leu?His?Ser?Gly?Arg?Glu?Cys?Thr?Asp?Thr?Phe
110 115 120
Asp?Ser?Tyr?His?Pro?Phe?Ser?Asn?Arg?Ala?Glu?Lys?Ile?Leu?Ala
125 130 135
Lys?Tyr?Ala?Ile?Gly?Lys?Leu?Val?Gly?Gly?Ser?Glu?Phe?Pro?Thr
140 145 150
Tyr?Lys?Pro?Asp?Thr?Gly?Phe?Tyr?Arg?Glu?Cys?Cys?Asp?Arg?Val
155 160 165
His?Gln?Tyr?Phe?Lys?Asp?Asn?Asn?Leu?Asp?Pro?Arg?Ser?Pro?Tyr
170 175 180
Ser?Gly?Leu?Leu?Arg?MET?Leu?Phe?Val?Ala?Ile?Leu?Gly?Ala?Val
185 190 195
Ser?Tyr?Val?Gly?MET?Asn?Gln?Leu?Leu?Ser?Asp?Asn?Ile?Tyr?Ala
200 205 210
His?Tyr?Ala?Trp?Gly?Ala?Leu?Phe?Gly?Ala?Cys?Gln?Ala?Leu?Pro
215 220 225
Leu?Leu?His?Val?MET?His?Asp?Ala?Ser?His?Ala?Ala?Ile?Thr?Ser
230 235 240
Ser?pro?Thr?Gly?Trp?Arg?Leu?Ile?Gly?Arg?Leu?Ala?MET?Asp?Trp
245 250 255
Val?Ala?Gly?Ala?Asn?MET?Val?Ser?Trp?Leu?Asn?Gln?His?Val?Val
260 265 270
Gly?His?His?Ile?Tyr?Thr?Asn?Val?Ala?Gly?Ala?Asp?Pro?Asp?Leu
275 80 285
Pro?Val?Asp?Phe?Lys?Ser?Asp?Val?Arg?Arg?Ile?Val?Tyr?Arg?Gln
290 295 300
Val?Leu?Leu?Pro?Ile?Tyr?Lys?Phe?Gln?His?Ile?Tyr?Leu?Pro?Pro
305 310 315
Leu?Tyr?Gly?Val?Leu?Gly?Leu?Lys?Phe?Arg?Val?Gln?Asp?Ile?Phe
320 25 330
Glu?Thr?Phe?Ile?Ser?Leu?Thr?Asn?Gly?Pro?Leu?Arg?Val?Asn?Pro
335 40 345
Leu?Ser?Ile?Gly?Asp?Trp?Ala?Glu?MET?Ile?Ile?Ser?Lys?Ala?Phe
350 355 360
Trp?Ala?Phe?Tyr?Arg?Ile?Tyr?Ile?Pro?Leu?Val?Val?Leu?Gln?Val
365 370 375
Asn?Pro?Ala?Arg?Phe?Trp?Gly?Val?Phe?Leu?Leu?Ala?Glu?Phe?Val
380 385 390
Thr?Gly?Trp?Tyr?Leu?Ala?Phe?Asn?Phe?Gln?Val?Ser?His?Val?Ser
395 400 405
Thr?Ala?Cys?Asp?Tyr?Pro?Gly?Gly?Asp?Glu?Glu?Val?Ser?Ala?Ile
410 15 420
Glu?Asp?Glu?Trp?Ala?Ile?Ser?Gln?Ile?Lys?Ser?Ser?Val?Asp?Tyr
425 430 435
Ala?His?Gly?Ser?Phe?Leu?Thr?Ala?Phe?Leu?Thr?Gly?Ala?Leu?Asn
440 445 450
Tyr?Gln?Val?Thr?His?His?Lau?Phe?Pro?Gly?Val?Ser?Gln?Tyr?His
455 460 465
Tyr?Pro?Ala?Ile?Ala?Pro?Ile?Ile?MET?Asp?Val?Cys?Asn?Lys?Tyr
470 475 480
Lys?Ile?Lys?Tyr?Thr?Val?Leu?Pro?Thr?Phe?Ser?Ala?Ala?Leu?Ala
485 90 495
Gly?His?Phe?Glu?His?Leu?Val?Val?MET?Gly?Lys?MET?Gly?Lys?Arg
500 05 510
Val?Thr?Val?His?MET?Gly
515
Downcut this fragment with EcoRI and XhoI from the pMD18-T carrier, be inserted into the downstream of the GAL1 promotor among the Yeast expression carrier pYES2.Change the Yeast expression carrier pYES2-PinD5 that builds over to yeast saccharomyces cerevisiae YPH500.Under the inducing of semi-lactosi, find that this cDNA encoded protein can change into dihomo-gamma-linolenic acid (DGLA) arachidonic acid (AA), have Δ 5 desaturase activity, so be PinD5 with this unnamed gene.
According to above-mentioned technology, the Δ 5 delta 8 desaturase genes PinD5 that from the potato phytophthora infestans, are separated to, its encoded protein has Δ 5 desaturase activity, DGLA can be changed into AA respectively, this is to utilize plant LA and ALA to produce essential step reaction in the metabolic pathway of synthesizing of overlength chain polyunsaturated fatty acids such as EPA, DHA by transgenic technology.Therefore, this enzyme is worth having important use by transgenic technology production overlength chain polyunsaturated fatty acid in oil crops, can be used for plant particularly oil crops produce fish oil.
(4) description of drawings
Fig. 1 is that the cDNA agarose gel electrophoresis picture M of pcr amplification potato phytophthora infestans Δ 5 delta 8 desaturase genes PinD5 represents molecular weight marker, and 1 represents PinD5 cDNA band
Fig. 2 is that Yeast expression carrier pYES-PinD5 makes up synoptic diagram and enzyme is cut proof diagram
A represents pYES2-PinD5 vector construction synoptic diagram, and on behalf of EcoRI and XboI enzyme, B cut proof diagram; M represents molecular weight marker, and 2 represent linearizing pYES2 carrier strap, and 3 represent PinD5 cDNA band
Fig. 3 be yeast saccharomyces cerevisiae YPH500 (containing pYES2-PinD5) add lipid acid substrate DGLA (20:3n6) and do not inducing/inductive condition under gas chromatographic detection figure
X-coordinate is represented retention time, ordinate zou expression signal intensity;-GAL+DGLA represents not add semi-lactosi and induces but add lipid acid substrate DGLA, and+GAL+DGLA represents to add semi-lactosi and induces and add lipid acid substrate DGLA; The detected fatty acid component of numeral on the chromatographic peak, 16:0, Palmiticacid; 16:1, consistent lubricant acid; 18:0, stearic acid; 18:1, oleic acid; 20:3, dihomo-gamma-linolenic acid; 20:4, arachidonic acid.
(5) concrete invention embodiment
Embodiment 1: the acquisition of potato phytophthora infestans Δ 5 delta 8 desaturase genes PIND5
1. the cultivation of potato phytophthora infestans: inoculation potato phytophthora infestans is to fresh oat medium, and 20 spend dark culturing.
2. the extraction of total RNA: collect the mycelium of growth about 15 days, utilize TRIZOL method (invitrogen) to extract total RNA.
3. get the total RNA of 2 micrograms, become strand cDNA with MMLV Reverse Transcriptase (Promega) reverse transcription.
4. the cDNA with the potato phytophthora infestans that obtained is a template, with primer 5 '-ATGACCACGGATGACATCAC-3 ' and 5 '-CTCGAGTTAGCCCATGTGGACGGT-3 '; PCR system: ddH
2O 35.5 μ l; 10xPCRbuffer (contains Mg
2+) 5 μ l; DNTP (2.5mM) 4 μ l; Each 2 μ l of primer (5 μ M); Template 1 μ l; Taq enzyme 0.5 μ l.Reaction conditions: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change 30 seconds, 65 ℃ of annealing 30 seconds, 72 ℃ were extended 35 circulations 1 minute 30 seconds; 72 ℃ were extended 10 minutes.After the fragment glue recovery with amplification, be connected among the cloning vector pMD18-T.
5. sequencing: originally be operated in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and carry out.Obtain the cDNA fragment of a 1551bp.
6. homology retrieval: utilize BLAST software that the aminoacid sequence of the cDNA fragment coding of above-mentioned acquisition and the sequence in the gene library are compared, find that it has cytochrome b5 structural domain HPGG of " front-end " desaturase and conservative region HxxHH, the HxxHH, the LNxQxxHHLFP that are rich in Histidine relevant with electron transport, determines that it belongs to the fatty acid desaturase gene.
Embodiment 2: potato phytophthora infestans Δ 5 delta 8 desaturase genes PinD5, and following sequence:
(1) information of SEQ ID NO.1
(a) sequence signature
* length: 1551 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: potato phytophthora infestans (Phytophthora infestans)
(f) sequence description: SEQ IN NO.1
1?ATGACCACGG?ATGACATCAC?ACTCCAATTC?GCACTCACGG?CCGGAAAGAC?CATCGGAACC
61?TCTGCTTCCC?CTGCATTGTC?ACCACTAATT?GACGAAGTAG?AAGCTGCCAT?GGCCCCTATC
121?GAGACCGAGA?AGACCATCGT?TAACGAGGGT?CTTTACCAGC?GCAAGACTGC?TGCTGATGCA
181?GCTACTAACA?AGTCTGCCGC?CACCTACACG?TGGCAGGACG?TGGCCAAGCA?CAACACAGAC
241?AACAGCGCTT?GGGTCATCAT?CCGCGGAATC?GTATACGACG?TAACTGAATG?GGCGGATCGC
301?CACCCTGGAG?GCCGTGAACT?CGTGCTGCTG?CACTCTGGTC?GCGAGTGCAC?CGACACATTC
361?GACTCGTACC?ACCCATTCTC?TAACCGCGCC?GAGAAGATCC?TGGCCAAATA?CGCCATTGGC
421?AAGCTTGTCG?GAGGCTCCGA?GTTCCCCACC?TACAAGCCTG?ACACAGGTTT?CTACAGGGAA
481?TGCTGCGACC?GTGTGCACCA?GTACTTCAAG?GACAACAACC?TGGACCCTCG?CAGTCCGTAC
541?TCGGGTCTGT?TGCGCATGCT?TTTCGTGGCT?ATCCTTGGTG?CGGTGTCCTA?CGTGGGTATG
601?AACCAGCTGC?TGTCCGACAA?CATCTACGCA?CACTACGCAT?GGGGCGCCCT?CTTCGGTGCG
661?TGCCAAGCTC?TACCTTTGCT?GCACGTGATG?CACGATGCCT?CACACGCGGC?CATTACCAGC
721?AGCCCTACAG?GCTGGAGACT?TATCGGGCGT?CTTGCAATGG?ACTGGGTGGC?CGGCGCCAAC
781?ATGGTGTCTT?GGCTCAACCA?GCACGTTGTG?GGCCACCACA?TCTACACGAA?CGTTGCGGGA
841?GCTGACCCTG?ACCTTCCCGT?GGACTTCAAG?AGCGACGTGC?GTCGCATAGT?GTACCGCCAG
901?GTGTTGCTTC?CAATCTACAA?GTTCCAACAC?ATCTACCTAC?CTCCGCTCTA?CGGCGTGCTC
961?GGCCTCAAGT?TCCGTGTTCA?GGACATCTTT?GAGACGTTCA?TCTCCCTCAC?CAACGGACCG
1021?CTACGTGTGA?ACCCCCTCTC?TATCGGTGAC?TGGGCTGAAA?TGATCATCTC?CAAGGCCTTC
1081?TGGGCGTTCT?ACCGCATCTA?CATCCCGCTG?GTTGTGCTGC?AGGTTAACCC?CGCTCGCTTC
1141?TGGGGTGTTT?TCCTTCTGGC?CGAGTTTGTG?ACGGGCTGGT?ACTTGGCCTT?CAACTTCCAA
1201?GTTAGTCACG?TGTCCACGGC?ATGCGACTAC?CCTGGTGGTG?ATGAGGAGGT?ATCGGCTATC
1261?GAAGACGAGT?GGGCTATCTC?GCAGATCAAG?TCATCGGTAG?ACTACGCGCA?CGGCTCGTTC
1321?CTCACGGCGT?TCCTCACGGG?TGCACTGAAC?TACCAGGTGA?CCCACCACCT?CTTCCCCGGC
1381?GTCTCGCAGT?ATCATTACCC?TGCTATCGCC?CCGATCATCA?TGGACGTGTG?CAACAAGTAC
1441?AAGATCAAGT?ACACGGTGCT?GCCTACGTTC?TCAGCGGCTC?TGGCTGGACA?CTTTGAGCAT
1501?CTCGTTGTCA?TGGGAAAGAT?GGGCAAGCGT?GTGACCGTCC?ACATGGGCTA?A
(3) information of SEQ IN NO.2
(a) sequence signature
* length: 516 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
Sequence description
MET?Thr?Thr?Asp?Asp?Ile?Thr?Leu?Gln?Phe?Ala?Leu?Thr?Ala?Gly
1 5 10 15
Lys?Thr?Ile?Gly?Thr?Ser?Ala?Ser?Pro?Ala?Leu?Ser?Pro?Leu?Ile
20 25 30
Asp?Glu?Val?Glu?Ala?Ala?MET?Ala?Pro?Ile?Glu?Thr?Glu?Lys?Thr
35 40 45
Ile?Val?Asn?Glu?Gly?Leu?Tyr?Gln?Arg?Lys?Thr?Ala?Ala?Asp?Ala
50 55 60
Ala?Thr?Asn?Lys?Ser?Ala?Ala?Thr?Tyr?Thr?Trp?Gln?Asp?Val?Ala
65 70 75
Lys?His?Asn?Thr?Asp?Asn?Ser?Ala?Trp?Val?Ile?Ile?Arg?Gly?Ile
80 85 90
Val?Tyr?Asp?Val?Thr?Glu?Trp?Ala?Asp?Arg?His?Pro?Gly?Gly?Arg
95 100 105
Glu?Leu?Val?Leu?Leu?His?Ser?Gly?Arg?Glu?Cys?Thr?Asp?Thr?Phe
110 115 120
Asp?Ser?Tyr?His?Pro?Phe?Ser?Asn?Arg?Ala?Glu?Lys?Ile?Leu?Ala
125 130 135
Lys?Tyr?Ala?Ile?Gly?Lys?Leu?Val?Gly?Gly?Ser?Glu?Phe?Pro?Thr
140 145 150
Tyr?Lys?Pro?Asp?Thr?Gly?Phe?Tyr?Arg?Glu?Cys?Cys?Asp?Arg?Val
155 160 165
His?Gln?Tyr?Phe?Lys?Asp?Asn?Asn?Leu?Asp?Pro?Arg?Ser?Pro?Tyr
170 175 180
Ser?Gly?Leu?Leu?Arg?MET?Leu?Phe?Val?Ala?Ile?Leu?Gly?Ala?Val
185 190 195
Ser?Tyr?Val?Gly?MET?Asn?Gln?Leu?Leu?Ser?Asp?Asn?Ile?Tyr?Ala
200 205 210
His?Tyr?Ala?Trp?Gly?Ala?Leu?Phe?Gly?Ala?Cys?Gln?Ala?Leu?Pro
215 220 225
Leu?Leu?His?Val?MET?His?Asp?Ala?Ser?His?Ala?Ala?Ile?Thr?Ser
230 235 240
Ser?Pro?Thr?Gly?Trp?Arg?Leu?Ile?Gly?Arg?Leu?Ala?MET?Asp?Trp
245 250 255
Val?Ala?Gly?Ala?Asn?MET?Val?Ser?Trp?Leu?Asn?Gln?His?Val?Val
260 265 270
Gly?His?His?Ile?Tyr?Thr?Asn?Val?Ala?Gly?Ala?Asp?Pro?Asp?Leu
275 280 285
Pro?Val?Asp?Phe?Lys?Ser?Asp?Val?Arg?Arg?Ile?Val?Tyr?Arg?Gln
290 295 300
Val?Leu?Leu?Pro?Ile?Tyr?Lys?Phe?Gln?His?Ile?Tyr?Leu?Pro?Pro
305 310 315
Leu?Tyr?Gly?Val?Leu?Gly?Leu?Lys?Phe?Arg?Val?Gln?Asp?Ile?Phe
320 325 330
Glu?Thr?Phe?Ile?Ser?Leu?Thr?Asn?Gly?Pro?Leu?Arg?Val?Asn?Pro
335 340 345
Leu?Ser?Ile?Gly?Asp?Trp?Ala?Glu?MET?Ile?Ile?Ser?Lys?Ala?Phe
350 355 360
Trp?Ala?Phe?Tyr?Arg?Ile?Tyr?Ile?Pro?Leu?Val?Val?Leu?Gln?Val
365 370 375
Asn?Pro?Ala?Arg?Phe?Trp?Gly?Val?Phe?Leu?Leu?Ala?Glu?Phe?Val
380 385 390
Thr?Gly?Trp?Tyr?Leu?Ala?Phe?Asn?Phe?Gln?Val?Ser?His?Val?Ser
395 400 405
Thr?Ala?Cys?Asp?Tyr?Pro?Gly?Gly?Asp?Glu?Glu?Val?Ser?Ala?Ile
410 415 420
Glu?Asp?Glu?Trp?Ala?Ile?Ser?Gln?Ile?Lys?Ser?Ser?Val?Asp?Tyr
425 430 435
Ala?His?Gly?Ser?Phe?Leu?Thr?Ala?Phe?Leu?Thr?Gly?Ala?Leu?Asn
440 445 450
Tyr?Gln?Val?Thr?His?His?Leu?Phe?Pro?Gly?Val?Ser?Gln?Tyr?His
455 460 465
Tyr?Pro?Ala?Ile?Ala?Pro?Ile?Ile?MET?Asp?Val?Cys?Asn?Lys?Tyr
470 475 480
Lys?Ile?Lys?Tyr?Thr?Val?Leu?Pro?Thr?Phe?Ser?Ala?Ala?Leu?Ala
485 490 495
Gly?His?Phe?Glu?His?Leu?Val?Val?MET?Gly?Lys?MET?Gly?Lys?Arg
500 505 510
Val?Thr?Val?His?MET?Gly
515
Embodiment 3: the structure of Yeast expression carrier pYES2-PinD5
1. the segmental pMD18-T carrier of PinD5 that contains that makes up from embodiment 1 with EcoRI and XhoI downcuts this fragment, is connected with the Yeast expression carrier pYES2 of same restrictions endonuclease digestion.Connect product transformed into escherichia coli XL10 competent cell, cultivate containing on the LB solid plate of penbritin then, bacterium colony is carried out the restriction analysis of PCR screening, evaluation and plasmid DNA, obtain to have the Yeast expression carrier pYES2-PinD5 of PinD5.
Embodiment 4: yeast abduction delivering PinD5
1. utilize the Lithium Acetate method that Yeast expression carrier pYES2-PinD5 is transformed among the yeast saccharomyces cerevisiae YPH500.
2. get positive yeast clone in 10ml SC-U (Synthetic Complete medium without Uracil) liquid nutrient medium, 28 ℃, incubated overnight.
3. next day, get the 1.5ml overnight culture and be inoculated in the fresh SC-U nutrient solution of 25ml (containing 1%NP40), 28 ℃, 180rpm.
4. work as nutrient solution OD600 between the 0.4-0.6, add 1ml 50% semi-lactosi and 0.5M 25 μ l fatty acid sodium salt substrate DGLA, 22 ℃, 180rpm continues to cultivate 2 days.
Embodiment 5: the 5 desaturase PinD5 determinations of activity of potato phytophthora infestans Δ
1. the yeast culture after centrifugal collection is induced, successively with contain 1% and the 0.5%NP40 deionized water rinsing once, and then with deionized water rinsing once, dry up with nitrogen;
2. add the 1ml solution (contain methyl alcohol 0.85ml, 2,2-dimethoxy propane 0.05ml, hydrochloric acid 0.1ml) that methylates, 85 ℃ of methylation reactions 1 hour;
3. after the cooling, add 1% sodium-chlor 1ml, normal hexane 0.5ml, vortex oscillation, room temperature was placed 10 minutes or the longer time then;
4. centrifuging and taking upper phase.Carry out gas chromatographic analysis by Shandong Forecasting and Analysis Center.The result shows that the Δ 5 desaturase PinD5 of abduction delivering can generate AA with the fatty acid sodium salt substrate DGLA catalysis that is added in the nutrient solution in yeast, and average conversion 10% proves that this enzyme has Δ 5 desaturase activity.
Claims (1)
1. the application of potato phytophthora infestans Δ 5 delta 8 desaturase genes PinD5, it is characterized in that the coded albumen of this gene cDNA can change into arachidonic acid with overlength chain polyunsaturated fatty acid dihomo-gamma-linolenic acid, can be used for plant particularly oil crops produce fish oil.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003012092A1 (en) * | 2001-07-31 | 2003-02-13 | Ipk - Institut Für Pflanzengenetik Und Kulturpflanzenforschung | Method for producing arachidonic acid in transgenic organisms |
| WO2007136671A2 (en) * | 2006-05-17 | 2007-11-29 | E. I. Du Pont De Nemours And Company | Delta5 desaturase and its use in making polyunsaturated fatty acids |
| CN101146911A (en) * | 2005-03-22 | 2008-03-19 | 巴斯福植物科学有限公司 | Method for producing polyunsaturated C20 and C22fatty acids with at least four double bonds in transgenic plants |
| US7736884B2 (en) * | 2004-06-04 | 2010-06-15 | Fluxome Sciences A/S | Metabolically engineered Saccharomyces cells for the production of polyunsaturated fatty acids |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003012092A1 (en) * | 2001-07-31 | 2003-02-13 | Ipk - Institut Für Pflanzengenetik Und Kulturpflanzenforschung | Method for producing arachidonic acid in transgenic organisms |
| US7736884B2 (en) * | 2004-06-04 | 2010-06-15 | Fluxome Sciences A/S | Metabolically engineered Saccharomyces cells for the production of polyunsaturated fatty acids |
| CN101146911A (en) * | 2005-03-22 | 2008-03-19 | 巴斯福植物科学有限公司 | Method for producing polyunsaturated C20 and C22fatty acids with at least four double bonds in transgenic plants |
| WO2007136671A2 (en) * | 2006-05-17 | 2007-11-29 | E. I. Du Pont De Nemours And Company | Delta5 desaturase and its use in making polyunsaturated fatty acids |
Non-Patent Citations (2)
| Title |
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| 《Nature》 20090917 Brian J. Haas er al. Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans 第1-6页 1 第461卷, * |
| 《NCBI Reference Sequence: XM_002908235.1》 20100622 Nusbaum,C. et al Phytophthora infestans T30-4 Delta(5) fatty acid desaturase, putative (PITG_01654) mRNA, complete cds 全文 1 , * |
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