CN102505018B - Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver - Google Patents
Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 152
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 title claims abstract description 35
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 241000208689 Eucommia ulmoides Species 0.000 title abstract 3
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- 230000037361 pathway Effects 0.000 title description 15
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 claims abstract description 14
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Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Provided are genes involved in the synthesis of isopentenyl diphosphate from mevalonic acid in Eucommia ulmoides Oliver. These genes encode acetyl-CoA C-acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase or mevalonate pyrophosphate decarboxylase in Eucommia ulmoides Oliver.
Description
The application is to be on June 20th, 2008 applying date, the divisional application of application for a patent for invention that application number is 200810125360.9, that denomination of invention is identical with the present invention.
Technical field
The present invention relates to the biosynthetic gene group of the isoprenoid compounds that participates in the bark of eucommia.
Background technology
Polyisoprene (rubber) as one of isoprenoid compounds is divided into cis and trans according to the difference of the polymerization methods of isoprene unit.Produce that the plant of long-chain cis-polyisoprene is known a lot of plants such as taraxacum, Herba Lactucae Indicae.The cis-polyisoprene that Para rubber tree (Hevea brasiliensis) is produced is as commercially widespread use of natural rubber.And about long-chain using trans-polyisoprene, known have the minority plant production such as bakelite in natural, but commercially do not utilize.At present, using trans-polyisoprene is chemosynthesis, is applied in golf ball crust, bandage, movement protection utensil etc.Using trans-polyisoprene is to show low melting point and elastomeric thermoplastic elastomer, can be used as isolator.
By being unit with the carbonatoms compound-isopentenylpyrophosphate (IPP) that is 5, condensation reaction generates isoprenoid compounds.Mevalonate pathway is one of initial stage route of synthesis in the biosynthesizing of isoprenoid compounds.In animal, plant, mushroom, can pass through mevalonate pathway biosynthesizing IPP.
As IPP biosynthetic pathway, except mevalonate pathway, also there is non-mevalonate pathway.In plant, in tenuigenin, be that mevalonate pathway plays a role, in chromatoplast, be non-mevalonate pathway plays a role.
The gene of the enzyme playing a role by mevalonate pathway is all known in each kind of plant.For example, in Kutki (Rhizoma Picrorhizae) (Picrorhiza kurrooa), (the more iridoid glycoside that contains of root) logined acetyl-CoA C-Transacetylase (base sequence has been DQ347964 in the accession number of GenBank, the accession number of aminoacid sequence is ABC74567), (accession number of base sequence is AF429389 in Para rubber tree (synthesizing cis polyisoprene), to have logined HMG-CoA synthetic enzyme, aminoacid sequence accession number is AAS46245), (base sequence accession number is X54659 to HMT-CoA reductase enzyme, the accession number of aminoacid sequence is P29057), (base sequence accession number is AF429384 to Mevalonic kinase, aminoacid sequence accession number is AAL18925), (base sequence accession number is AF429385 to Phosphomevalonic kinase, aminoacid sequence accession number is AAL18926), and mevalonic acid tetra-sodium decarboxylase (base sequence accession number AF429386, the accession number of aminoacid sequence is AAL18927).
The long-chain using trans-polyisoprene of the Chinese original xylophyta bark of eucommia (Eucommia ulmoides Oliver) producd fibers shape.In leaf, bark and the seed coat of the bark of eucommia, contain a large amount of using trans-polyisoprenes (non-patent literature 1).
The bark of the bark of eucommia, as nourishing strong and hypertensive medicine among the people, is used since ancient times always.In the bark of eucommia, contain isoprenoid compounds a kind of-glycosides (Geniposidic acid) (non-patent literature 2 and 3) of iridoid, it has hypotensive activity.
As mentioned above, the bark of eucommia is containing the isoprenoid compounds useful just like using trans-polyisoprene and iridoid glycoside etc.
In order to prepare the plant of rubber high-content, people wish to obtain the gene that participates in IPP biosynthetic pathway in the bark of eucommia.In the past, obtain the homologous gene with certain function from target organism kind time, be the polycondensation PCR of base sequence and the method for ensuing 5 '-RACE, 3 '-RACE and RP-PCR combination of utilizing the screening by hybridization of the DNA fragmentation that uses known gene or utilizing known gene.But the accuracy of these methods is low, pretty troublesome.When particularly the conservative region of target gene is indefinite, be difficult to adopt these methods.
The gene order of the bark of eucommia has been carried out analyzing (non-patent literature 4 and 5).The gene and the sequence thereof that participate in one of enzyme of mevalonate pathway-HMG-CoA reductase enzyme in non-patent literature 4, are reported.But the enzyme in addition that participates in mevalonate pathway has no report.
Non-patent literature 1:Bamba T, Fukusaki E, Nakazawa Y, and Kobatashi A, In-situ chemical analyses of tran-polyisoprene by histochemical staining and Fourier transform infrared microapectroscopy in a rubber-producing plant, Eucommia ulmoides Oliver.Planta 215:934-939 (2002)
Non-patent literature 2:Kawasaki, T., Uezono, K., and Nakazawa, Y., Antihypertensive mechanism of food for apecified health use: " Eucommia leaf glycoside " and its clinical application, J.Health Sci., 22,29-36 (2000)
Non-patent literature 3:Nakmura, T., Nakazawa, Y., Onozuka, S., Tanaka, C., Yahara, S.and Nohara, T., Studies on the constituents of Eucommia ulmoides iridoids from the leaves, Natural Medicines, 51,275277 (1997)
Non-patent literature 4:Jiang J, Kai G, Gao X, and Chen F, Molecular cloning of a HMG-CoA reductase gene from Eucommia ulmoides Oliver.Biosci Rep 26:171-181 (2006)
Non-patent literature 4:Hou H-W, Zhou Y-T, Mwang K-N, Li W-F, He X-Q, with Cui K-M, ABP1 expression regulated by IAA and ABA is associated with the cambium periodicity in Eucommia ulmoides Oliv.J Exp Bot 57:3857-3867 (2006)
Summary of the invention
The object of the present invention is to provide the biosynthetic gene group of the isoprenoid compounds that participates in the bark of eucommia.
The invention provides the gene participating in by the synthetic isopentenylpyrophosphate of mevalonic acid of the bark of eucommia.Said gene coding is selected from the protein of 1-408 amino acids of aminoacid sequence that contain SEQ ID NO.2, the protein of 1-408 amino acids of aminoacid sequence that contain SEQ ID NO.4, the protein of 1-463 amino acids of aminoacid sequence that contain SEQID NO.6, the protein of 1-466 amino acids of aminoacid sequence that contain SEQ ID NO.8, the protein of 1-629 amino acids of aminoacid sequence that contain SEQ ID NO.12, the protein of 1-591 amino acids of aminoacid sequence that contain SEQ ID NO.14, the protein of 1-387 amino acids of aminoacid sequence that contain SEQ ID NO.16, the protein of 1-506 amino acids of aminoacid sequence that contain SEQ ID NO.18, the protein of the protein of 1-418 amino acids of aminoacid sequence that contain SEQ ID NO.20.
In one embodiment, said gene is selected from the gene of 101-1327 Nucleotide of base sequence that contain SEQ ID NO.1, the gene of 128-1354 Nucleotide of base sequence that contain SEQ ID NO.3, the gene of 172-1563 Nucleotide of base sequence that contain SEQ ID NO.5, the gene of 216-1616 Nucleotide of base sequence that contain SEQ ID NO.7, the gene of 32-1921 Nucleotide of base sequence that contain SEQ IDNO.11, the gene of 60-1835 Nucleotide of base sequence that contain SEQ ID NO.13, the gene of 61-1224 Nucleotide of base sequence that contain SEQ ID NO.15, the gene of 682-2202 Nucleotide of base sequence that contain SEQ ID NO.17, the gene of 68-1324 Nucleotide of base sequence that contain SEQ ID NO.19.
In other embodiments, said gene is selected from the gene of 1-1516 Nucleotide of base sequence that contain SEQ ID NO.1, the gene of 1-1757 Nucleotide of base sequence that contain SEQ ID NO.3, the gene of 1-1892 Nucleotide of base sequence that contain SEQ ID NO.5, the gene of 1-1833 Nucleotide of base sequence that contain SEQ ID NO.7, the gene of 1-1825 Nucleotide of base sequence that contain SEQ ID NO.9, the gene of 1-2057 Nucleotide of base sequence that contain SEQ ID NO.11, the gene of 1-2225 Nucleotide of base sequence that contain SEQ ID NO.13, the gene of 1-1341 Nucleotide of base sequence that contain SEQ ID NO.15, the gene of 1-2432 Nucleotide of base sequence that contain SEQ ID NO.17, the gene of 1-1542 Nucleotide of base sequence that contain SEQ IDNO.19.
The invention provides the gene participating in by the synthetic isopentenylpyrophosphate of mevalonic acid of the bark of eucommia.
Embodiment
The invention provides the gene participating in by the synthetic isopentenylpyrophosphate (IPP) of mevalonic acid of the bark of eucommia.The synthetic of IPP undertaken by the biosynthetic initial stage route of synthesis-mevalonate pathway of isoprenoid compounds.Following enzyme participates in mevalonate pathway: (1) acetyl-CoA C-Transacetylase; (2) 3-hydroxy-3-methyl glutaryl-CoA (HMG-CoA) synthetic enzyme; (3) HMG-CoA reductase enzyme; (4) Mevalonic kinase; (5) Phosphomevalonic kinase; And (6) mevalonic acid tetra-sodium decarboxylase (also referred to as " tetra-sodium mevalonic acid decarboxylase ").In mevalonate pathway, by (1) acetyl-CoA C-Transacetylase, by acetyl-CoA synthesis of acetyl acetyl-CoA, by (2) HMG-CoA synthetic enzyme, by the synthetic HMG-CoA of acetoacetyl-CoA, by (3) HMG-CoA reductase enzyme, change HMG-CoA into mevalonic acid, by (4) Mevalonic kinase and (5) Phosphomevalonic kinase, change mevalonic acid into mevalonic acid 5-tetra-sodium, then generate isopentenylpyrophosphate (IPP) by (6) mevalonic acid tetra-sodium decarboxylase.
Genes encoding of the present invention is selected from the protein (below also referred to as " protein of SEQ ID NO.2 ") of 1-408 amino acids of aminoacid sequence that contain SEQ ID NO.2; The protein (below also referred to as " protein of SEQ IDNO.4 ") of 1-408 amino acids of aminoacid sequence that contain SEQ IDNO.4; The protein (below also referred to as " protein of SEQ ID NO.6 ") of 1-463 amino acids of aminoacid sequence that contain SEQ ID NO.6; The protein (below also referred to as " protein of SEQ ID NO.8 ") of 1-466 amino acids of aminoacid sequence that contain SEQ ID NO.8; The protein (below also referred to as " protein of SEQ ID NO.12 ") of 1-629 amino acids of aminoacid sequence that contain SEQ ID NO.12; The protein (below also referred to as " protein of SEQ ID NO.14 ") of 1-591 amino acids of aminoacid sequence that contain SEQ ID NO.14; The protein (below also referred to as " protein of SEQ ID NO.16 ") of 1-387 amino acids of aminoacid sequence that contain SEQ ID NO.16; The protein (below also referred to as " protein of SEQ ID NO.18 ") of 1-506 amino acids of aminoacid sequence that contain SEQ ID NO.18; The protein of the protein (below also referred to as " protein of SEQ IDNO.20 ") of 1-418 amino acids of aminoacid sequence that contain SEQ IDNO.20.The amino acid whose protein of 1-466 that contains SEQ ID NO.10 is identical with the protein of SEQ ID NO.8.
The protein of the protein of SEQ ID NO.2 and SEQ ID NO.4 is (1) acetyl-CoA C-Transacetylase.The protein of SEQ ID NO.2 is by the genes encoding gained of the Nucleotide of 101-1327 of the base sequences that contains SEQ ID NO.1.This gene for example can contain whole base sequences (1-1516) of SEQ ID NO.1.The protein of SEQ ID NO.4 can be obtained by the genes encoding of the Nucleotide of 128-1354 of the base sequences that contains SEQ IDNO.3.This gene for example can contain whole base sequences (1-1757) of SEQ ID NO.3.
The protein of the protein of SEQ ID NO.6 and SEQ ID NO.8 is (2) HMG-CoA synthetic enzyme.The protein of SEQ ID NO.6 is by the genes encoding gained of the Nucleotide of 172-1563 of the base sequences that contains SEQ ID NO.5.This gene for example can contain whole base sequences (1-1892) of SEQ ID NO.5.The protein of SEQ ID NO.8 can be obtained by the genes encoding of the Nucleotide of 216-1616 of the base sequences that contains SEQ ID NO.7 (or the base sequence of SEQ ID NO.9 216-1616).In this gene, comprise the gene of the Nucleotide of 1-1825 of the gene of Nucleotide of 1-1833 of base sequences that for example contain SEQ ID NO.7 and the base sequences that contain SEQ ID NO.9.There is the gene of base sequence shown in SEQ ID NO.9 in its 1689-1825 Wei districts, with respect to 1689-1833 Wei districts of gene with the base sequence shown in SEQ ID NO.7, there is the displacement at 6 places (1689,1709,1713,1745,1756 and 1820 of the base sequences of SEQ ID NO.7) and the shortening at the disappearance of 1 place (1777 of the base sequences of SEQ ID NO.7) and the poly-adenosine position of 3 ' end.
The protein of the protein of SEQ ID NO.12 and SEQ ID NO.14 is (3) HMG-CoA reductase enzyme.The protein of SEQ ID NO.12 is by the genes encoding gained of the Nucleotide of 32-1921 of the base sequences that contains SEQ ID NO.11.This gene for example can contain whole base sequences (1-2057) of SEQ ID NO.11.The protein of SEQ ID NO.14 can be obtained by the genes encoding of the Nucleotide of 60-1835 of the base sequences that contains SEQ IDNO.13.This gene for example can contain whole base sequences (1-2225) of SEQ ID NO.13.
The protein of SEQ ID NO.16 is (4) Mevalonic kinase.The protein of SEQ ID NO.16 is by the genes encoding gained of the Nucleotide of 61-1224 of the base sequences that contains SEQ ID NO.15.This gene for example can contain whole base sequences (1-1341) of SEQ ID NO.15.
The protein of SEQ ID NO.18 is (5) Phosphomevalonic kinase.The protein of SEQ ID NO.18 is by the genes encoding gained of the Nucleotide of 682-2202 of the base sequences that contains SEQ ID NO.17.This gene for example can contain whole base sequences (1-2432) of SEQ ID NO.17.
The protein of SEQ ID NO.20 is (6) mevalonic acid tetra-sodium decarboxylase.The protein of SEQ IDNO.20 is by the genes encoding gained of the Nucleotide of 68-1324 of the base sequences that contains SEQ ID NO.19.This gene for example can contain whole base sequences (1-1542) of SEQ ID NO.19.
As long as gene of the present invention can be expressed with the functional equal enzyme of the enzyme with above-mentioned aminoacid sequence or can be brought into play said function with the gene with above-mentioned base sequence, the different protein of one or more amino-acid residues of can encoding, or can have one or more base differences.The difference of above-mentioned sequence can and/or be inserted due to the displacement of base, disappearance and obtain, or the any-mode of natural mutagenesis or induced mutations (for example application of rite-directed mutagenesis introductory technique) occurs.The function of the gene basic skills that usual practice is recorded as following examples 2 as adopted really, the normally used method of those skilled in the art carries out.
Gene of the present invention is that those skilled in the art adopt the method conventionally using, and the sequence information of recording according to this specification sheets is prepared probe or primer, take the chromosomal DNA of the bark of eucommia or cDNA as template, obtains target fragment by PCR.Can certainly, take RNA as template, utilize reverse transcription PCR.Gene of the present invention, except the natural polynucleotide such as DNA, RNA, can be also the artificial molecule that contains artificial nucleotide derivative.Gene of the present invention can also be the chimeric molecule of DNA-RNA.
Gene of the present invention can use common the adopted method of those skilled in the art, imports in the microorganisms such as yeast or plant.Gene of the present invention can transform in host, with express one or more or all.For example its formation can be: express at least one in the one group of enzyme (above-mentioned (1) is to (6)) being present in mevalonate pathway.
Gene of the present invention is suitable for the genetically modified plants (for example bark of eucommia) that preparation contains using trans-polyisoprene or iridoid in a large number.Gene of the present invention for example, by transforming the bark of eucommia, can be used for preparing the more plant that contains rubber.
Embodiment
Below provide embodiment, further illustrate the present invention, but the present invention is not limited to this.
(embodiment 1: the EST in the bark of eucommia analyzes)
(material)
As bark of eucommia plant materials sample, use phloem (bark) and the xylem of the sprout then gathering in the bark of eucommia standard tree of late May, the growth of Sheng Ming village of Cong Aiyuan county.
The mutant strain library of yeast is used commercially available YKO Heterozygous Essential Strain Collection-Glycerol Stocks (Open Biosystems company).
(by extracting RNA in the bark of eucommia)
By about 4g bark of eucommia plant materials sample (phloem of current growth and xylem) limit cooled with liquid nitrogen, mortar, grinding rod fragmentation for limit, be suspended in 2 × CTAB solution (2% (w/v) cetyl trimethylammonium bromide (CTAB), 1% (w/v) 2 mercapto ethanol, 0.1M Tris-HCl (pH 9.5), 1.4M NaCl, the 20mM EDTA of 10 times of amounts (w/v) of sample.By its incubation 10 minutes at 65 ℃, then with chloroform/primary isoamyl alcohol processing (washing) (twice repeatedly).In the water layer reclaiming, add the 10M LiCl of 1/4 (v/v) amount and mix, incubation 2 hours at-20 ℃, carries out the selective precipitation of RNA.It is centrifugal, will be precipitated and dissolved in appropriate Tris-EDTA (TE) damping fluid, centrifugation, reclaims supernatant, gets rid of polyose.The supernatant of recovery is carried out to phenol processing, phenol/chloroform processing and the processing of chloroform/primary isoamyl alcohol, again carry out the selective precipitation of RNA by LiCl.To precipitate and use 70% washing with alcohol, drying under reduced pressure, is then dissolved in diethylpyrocarbonate (DEPC) and processes in water.By absorbance measurement, gained RNA is carried out quantitatively, confirming by electrophoresis.By obtaining 0.84mg RNA in about 4g xylem, by obtaining 2mgRNA (specific absorption under 260nm and 280nm is respectively 1.956 and 1.990) in about 4g phloem.
(preparation of bark of eucommia cDNA library)
By the G-キ Star ピ Application グ method of tester Service Co., Ltd of Hitachi, prepare cDNA library by the RNA sample from bark of eucommia phloem and xylem.Library size from the cDNA library of phloem is 3.8 × 10
5, insertion rate is 88% (24 sample/agarose gel electrophoresis), total length rate 86% (for the clone who inserts).Library size from the cDNA of xylem is 2.2 × 10
5, insertion rate is 79% (24 sample/agarose gel electrophoresis), total length rate 63% (with respect to the clone who has insertion).
(sequential analysis of est sequence)
In genome information science research department of North life science institute of North university, carry out sequential analysis for each approximately 20000 clones of the cDNA library from bark of eucommia phloem and xylem.According to the sequence information being obtained by sequential analysis, remove the clone who does not possess insertion and the clone that cannot read sequence, obtain the high sequence information of precision.For the library of phloem and xylem, obtain respectively the high est sequence (adding up to 32680) of precision of 16567 and 16113 length.
Divide into groups (cluster) (clustering) and explain (annotation) for institute's calling sequence." grouping " is in est sequence, and same sequence, similar sequence are divided into one group.In order to divide into groups, use the VISUALBIO clustering of NTT software." note " be by with known comparison, est sequence is explained.When note, use the homology search that adopts NCBI BLAST.The database using when retrieval is nr (All non-redundant GenBank CDS translations+PDB+SwissProt+PIR (peptide sequence database)).
According to the information of grouping and note gained, find to be estimated as the est sequence of coding acetyl-CoA C-Transacetylase, HMG-CoA synthetic enzyme, HMG-CoA reductase enzyme, Mevalonic kinase and mevalonic acid tetra-sodium decarboxylase.These enzymes are all the enzymes that participate in initial stage route of synthesis in the biosynthesizing of isoprenoid compounds of the bark of eucommia.
(embodiment 2: the gene 1 of coding acetyl-CoA C-Transacetylase)
(acquisition of full-length cDNA)
By 3 '-RACE (rapid amplifying of cDNA end, RACE), determined the sequence of 3 ' end one side by the sequence obtaining by the analysis of embodiment 1, obtain full-length cDNA.
3 '-RACE uses 3 '-Full RACE Core Set (preparation of タ カ ラ バ イ オ Co., Ltd.).In reverse transcription reaction, use oligo-dT primer.Pcr amplification use with the primer of oligo-dT and with a part for known array be the sense primer of identical sequence.Sense primer basis is estimated as the est sequence information design of coding acetyl-CoA C-Transacetylase of embodiment 1 gained.Take the RNA of embodiment 1 gained as template, for the 1st time to use N192-82-tree_1968_3R_S1 (SEQ IDNO.21) as sense primer, be for the 2nd time to use N192-82-tree_1968_3R_s2 (SEQ IDNO.22) as sense primer, carry out to specifications PCR.The amplified fragments TA obtaining by reverse transcription reaction with by PCR is cloned in pT7Blue carrier, carries out sequential analysis.
Gained full-length cDNA has the base sequence of SEQ ID NO.1.The base sequence in region and acetyl-CoA C-Transacetylase (GenBank accession number DQ347964) of Kutki shown in the Nucleotide that base sequence described in SEQ ID NO.1 is 146-1299 have 82% homology.Open reading frame is 101-1327.The aminoacid sequence of inferring of being encoded by this cDNA is as shown in SEQID NO.2.The full length amino acid sequence (1-408) of SEQ ID NO.2 has 89% homology with acetyl-CoA C-Transacetylase (accession number ABC74567) of Kutki.
(using the complementarity test in yeast mutant library)
Use the complementarity test of carrying out with yeast mutant library to confirm the function of gained cDNA.This complementarity test is carried out as follows.
Sense primer and antisense primer that it is identical sequence that use is attached with 40 bp of the multiple clone site of pYES2 carrier (invitrogen preparation), carry out PCR (at 95 ℃ using 5 minutes as 1 circulation; Then at 95 ℃ by 1 minute, 54 ℃ by 1 minute and 72 ℃ using 1 minute as 30 circulations; At 72 ℃ using 7 minutes as 1 circulation; And at 4 ℃ using ∞ as 1 circulation), amplification target gene translation region sequence.
This amplified fragments is imported to Ura with the pYES2 carrier that becomes straight chain shape by restriction enzyme treatment
-yeast strains Y22800 (EUROSCARF preparation) in.Acetyl-CoA C-acetyl transferase gene as analytic target is necessary for the growth of yeast, and therefore, as the background of complementary assay, this yeast uses the heterozygous diploid of the contraposition genetically deficient target gene that only has a side.Gene uses Frozen-EZ Yeast Transformation II (ZYMO RESEARCH preparation) while importing.Yeast-inoculated after transforming, in limit perfect medium (without uridylic), is cultivated at 30 ℃.The colony of growth is inoculated in new limit perfect medium (without uridylic), then at 30 ℃, cultivates.Yeast mutant is uridylic demand property, contains the sequence of URA3 gene (coding orotidine 5 '-phosphate decarboxylase) in pYES2 carrier, therefore only has the thalline of conversion to grow.The mattress body of growth checks by PCR whether it contains Insert Fragment, then, this thalline is transferred in YPDA substratum, at 30 ℃, cultivates.Transfer them to sporulation substratum, at 25 ℃, cultivate, gained spore is carried out to analysis of tetrad.
There is GAL1 promotor in the upstream being inserted on the target gene in the multiple clone site of pYES2 carrier, therefore, target gene is abduction delivering under semi-lactosi exists only.Therefore, determine whether on YPD substratum or YPG substratum (carbon source: semi-lactosi) and grow.In YPD substratum, lethal from the dyad of mutant strain, only grow from the dyad of wild-type.And upper at YPG substratum (carbon source: semi-lactosi), there is the expression that gene is inserted in the pYES2 carrier induction of GAL1 promotor, therefore, and if had complementary functions, from can the surviving of mutant strain, triad or tetrad growth.Like this, by carrying out complementary confirmation, can carry out the function of target gene and determine.
And, by obtained by analysis of tetrad from eight sporangial colonies (can think monoploid) photocopy to (A) YPG substratum (carbon source: semi-lactosi), (B) limit perfect medium (carbon source: semi-lactosi, do not contain uridylic), (C) YPG substratum (carbon source: semi-lactosi, contain microbiotic G418) or (D) on YPG substratum (carbon source: glucose).
Possess the cultivation strain of plasmid growth in the limit perfect medium of (B) (carbon source: semi-lactosi, does not contain uridylic).Mutant strain has G418 resistant gene, therefore can think growth in (C) YPG substratum (carbon source: semi-lactosi, contain microbiotic G418).Can be determined the monoploid mutant strain of possessing plasmid by the growth result in these substratum.In the YPD of (D) substratum (carbon source: glucose), possess the mutant strain (monoploid) of plasmid and should grow, but be in fact difficult to judge having or not of its growth.Therefore differentiate having or not of growth by screening.
In the present embodiment, use N219-36-tree_1968_S (SEQ ID NO.23) as sense primer, use N219-36-tree_1968_a (SEQ ID NO.24) as antisense primer, using the cDNA that obtains in above-mentioned " completely cDNA acquisition " as template, carry out PCR, the sequence in amplification target translation district.This amplified fragments is supplied with to above-mentioned complementary test.In analysis of tetrad, observe the growth on YPG substratum (carbon source: semi-lactosi).And by using the observation on Growth from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, does not contain uridylic).As a result, by complementation, determined the function of acetyl-CoA C-acetyl transferase gene.
(embodiment 3: the gene 2 of coding acetyl-CoA C-Transacetylase)
The N192-84-tree_11012_3R_s1 (SEQ ID NO.25) (the 1st time) of the information design of the est sequence that is estimated as coding acetyl-CoA C-Transacetylase that use obtains according to embodiment 1 and the sense primer that N192-84-tree_11012_3R_s2 (SEQ ID NO.26) (the 2nd time) uses as 3 '-RACE, in addition similarly to Example 2, obtain full-length cDNA.
Gained full-length cDNA has the base sequence of SEQ ID NO.3.The base sequence in region and acetyl-CoAC-Transacetylase (accession number DQ347964) of Kutki shown in the Nucleotide of 173-1305 of the base sequences of SEQ ID NO.3 have 83% homology.Open reading frame is 128-1354.The putative amino acid sequence of being encoded by this cDNA is as shown in SEQ ID NO.4.The full length amino acid sequence (1-408) of SEQID NO.4 has 87% homology with acetyl-CoA C-Transacetylase (accession number ABC74567) of Kutki.
In the present embodiment, use N219-36-tree_11012_s (SEQ ID NO.27) as sense primer, use N-219-36-tree_11012_a (SEQ ID NO.28) as antisense primer, take above-mentioned gained cDNA as template, in addition carry out similarly to Example 2 PCR, the sequence in amplification target translation district.This amplified fragments is supplied with to complementary test similarly to Example 2, obtained result similarly to Example 2.In analysis of tetrad, observe the growth on YPG substratum (carbon source: semi-lactosi).And, by using the observation on Growth from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, does not contain uridylic).As a result, determined the function of acetyl-CoA C-acetyl transferase gene by complementation.
(embodiment 4: the gene 1 of coding HMG-CoA synthetic enzyme).
The N192-71-tree_6098_3R_s1 (SEQ ID NO.29) (the 1st time) of the information design of the est sequence that be estimated as coding HMG-CoA synthetic enzyme of use based on embodiment 1 gained and N192-71-tree_6098_3R_s2 (SEQ ID NO.30) (the 2nd time) are as the sense primer of 3 '-RACE, in addition similarly to Example 2, obtain full-length cDNA.
The full-length cDNA of gained has the base sequence of SEQ ID NO.5.The base sequence in the region shown in the region shown in the Nucleotide of 172-1187 of the base sequences of SEQ ID NO.5 and 1390-1439 s' Nucleotide, has 83% and 86% homology with the HMG-CoA synthetic enzyme (accession number AF429389) of Para rubber tree respectively.Open reading frame is 172-1563.The putative amino acid sequence of being encoded by this cDNA is as shown in SEQ ID NO.6.The full length amino acid sequence (1-463) of SEQ ID NO.6 has 85% homology with the HMG-CoA synthetic enzyme (accession number AAS46245) of Para rubber tree.
In the present embodiment, sense primer is used N219-36-tree_6098_s (SEQ ID NO.31), antisense primer uses N219-36-tree_6098_a (SEQ ID NO.32), take above-mentioned gained cDNA as template, in addition carry out similarly to Example 2 PCR, the sequence in amplification target translation district.Similarly to Example 2, this amplified fragments is supplied with to complementary test.Mutant strain uses Ura
-the damaged strain Y26527 of HMG-CoA synthetic enzyme (EUROSCARF preparation).In analysis of tetrad, observe the growth on YPG substratum (carbon source: semi-lactosi).And, in the observation on Growth using from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, does not contain uridylic).As a result, determined the function of HMG-CoA synthase gene by complementation.
(embodiment 5: the gene 2 of coding HMG-CoA synthetic enzyme)
Use N192-36-tree_10370_3R_s1 (SEQ ID NO.33) (the 1st time) and the N192-36-tree_10370_3R_s2 (SEQ ID NO.34) (the 2nd time) of the information design of the est sequence of inferring coding HMG-CoA synthetic enzyme of embodiment 1 gained, the sense primer of using as 3 '-RACE, in addition obtains full-length cDNA similarly to Example 2.Two full-length cDNAs are obtained.
The first full-length cDNA of gained has the base sequence of SEQ ID NO.7.Shown in the Nucleotide of 234-1496 of the base sequences of SEQ ID NO.7, the base sequence in region and the HMG-CoA synthetic enzyme (accession number AF429389) of Para rubber tree have 81% homology.Gained the second full-length cDNA has the base sequence of SEQ ID NO.9.The 2nd cDNA is in the region of its 1689-1825, and the region of 1689-1833 of a corresponding cDNA has 6 places (cDNA 1689,1709,1713,1745,1756 and 1820) displacement and 1 place (cDNA 1777) to lack and the shortening at the poly-adenosine position of 3 ' end.The base sequence of SEQ ID NO.9 is also same with the base sequence of SEQ ID NO.7, has 81% homology with the HMG-CoA synthetic enzyme (accession number AF429389) of Para rubber tree.Open reading frame is 216-1616 at two base sequences.The putative amino acid sequence of being encoded by these cDNA is respectively as shown in SEQ ID NO.8 and 10, and they are identical sequences.This putative amino acid sequence, in the amino acid region of 6-452 of SEQ ID NO.8, has 84% homology with the HMG-CoA synthetic enzyme (accession number AAS46245) of Para rubber tree.
In the present embodiment, use N219-36-tree_10370_s (SEQ ID NO.35) as sense primer, use N219-36-tree_10370_a (SEQ ID NO.36) as antisense primer, using above-mentioned gained cDNA as template, in addition carry out similarly to Example 2 PCR, the sequence in amplification target translation district.Similarly to Example 2, this amplified fragments is supplied with to complementary test.Mutant strain uses Ura
-the damaged strain Y26527 of HMG-CoA synthetic enzyme (EUROSCARF preparation).In analysis of tetrad, observe the growth on YPG substratum (carbon source: semi-lactosi).And, in the observation on Growth using from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, does not contain uridylic).As a result, by complementation, determined the function of HMG-CoA synthase gene.
(embodiment 6: the gene 1 of coding HMG-CoA reductase enzyme)
About the gene of coding HMG-CoA reductase enzyme, in order to obtain full-length cDNA, adopt the method for 5 ' RACE, 3 ' RACE and reverse transcription reaction combination.In the method, use 5 '-FullRACE Core Set (TaKaRa preparation) and 3 '-Full RACE Core Set (TaKaRa preparation).The antisense primer of using as 5 ' RACE according to est sequence design N192-45-tree_8220_5R_a1 (SEQ ID NO.37) (for the first time) and the N192-45-tree_8220_5R_a2 (SEQ ID NO.38) (for the second time) that are estimated as coding HMG-CoA reductase enzyme of embodiment 1 gained, the sense primer that N192-45-tree_8220_5R_3R_s1 (SEQ ID NO.39) (for the first time) and N192-45-tree_8220_5R_3R_s2 (SEQ ID NO.40) (for the second time) use as 3 '-RACE, N192-45-tree_8220_5R_p (SEQ ID NO.41) uses as reverse transcription reaction.Take the RNA of embodiment 1 gained as template, similarly to Example 2, the amplified fragments TA of their reaction gained is cloned on pTCBlue carrier, carry out sequential analysis.
Gained full-length cDNA has the base sequence of SEQ ID NO.11.Shown in region shown in region shown in the Nucleotide of 260-303 of the base sequences of SEQ ID NO.11, the Nucleotide of 773-936 and 1148-1845 s' Nucleotide the base sequence in region there is 88%, 82% and 81% homology with the HMG-CoA reductase enzyme (accession number AF315713) of apple (Malus x domestica) respectively.Open reading frame is 32-1921.The putative amino acid sequence of being encoded by this cDNA is as shown in SEQ ID NO.12.In the amino acid region of 33-612 of SEQ ID NO.12, the HMG-CoA reductase enzyme (accession number P48021) of this putative amino acid sequence and camplotheca acuminata (Camptotheca acuminata) has 78% homology.The amino acid region of 33-612 of SEQ ID NO.12 and the HMG-CoA reductase enzyme (accession number AAV54051) of non-patent literature 4 have 73% homology.Therefore, can think that the base sequence of sequence number 11 is the HMG-CoA reductase genes from the locus different from the gene of non-patent literature 4.
(embodiment 7: the gene 2 of coding HMG-CoA reductase enzyme)
The antisense primer of using as 5 ' RACE according to est sequence design N192-47-tree_13453_5R_a1 (SEQ ID NO.42) (for the first time) and the N192-47-tree_13453_5R_a2 (SEQ ID NO.43) (for the second time) that are estimated as coding HMG-CoA reductase enzyme of embodiment 1 gained, the sense primer that N192-47-tree_13453_5R_3R_s1 (SEQ ID NO.44) (for the first time) and N192-47-tree_13453_5R_3R_s2 (SEQ ID NO.45) (for the second time) use as 3 '-RACE, N192-47-tree_13453_5R_p (SEQ ID NO.46) uses as reverse transcription reaction, in addition similarly to Example 6, obtain full-length cDNA.
Gained full-length cDNA has the base sequence of SEQ ID NO.13.Shown in region shown in region shown in the Nucleotide of 169-373 of the base sequences of SEQ ID NO.13, the Nucleotide of 751-1600 and 1637-1720 s' Nucleotide the base sequence in region, there is 79%, 80% and 89% homology with the HMG-CoA reductase enzyme (accession number U72145) of camplotheca acuminata respectively.Open reading frame is 60-1835.The putative amino acid sequence of being encoded by this cDNA is as shown in SEQ IDNO.14.In the amino acid region of 1-588 of SEQ ID NO.14, the HMG-CoA reductase enzyme (accession number AAB87727) of this putative amino acid sequence and tobacco (Nicotiana tabacum) has 78% homology.The amino acid region of 1-589 of SEQ ID NO.14 and the HMG-CoA reductase enzyme (accession number AAV54051) of non-patent literature 4 have 75% homology.Therefore, can think that the base sequence of sequence number 13 is also the HMG-CoA reductase gene from the locus different from the gene of non-patent literature 4.
(embodiment 8: the gene of coding Mevalonic kinase)
The sense primer that 3 '-RACE uses is used tree 1729 primers (SEQ ID NO.47) (for the first time) and the tree 1729 primer covers (SEQ ID NO.48) (for the second time) of the information design of the est sequence that is estimated as coding Mevalonic kinase based on embodiment 1 gained, in addition similarly to Example 2, obtain full-length cDNA.
Gained full-length cDNA has the base sequence of SEQ ID NO.15.Shown in the Nucleotide of 616-1104 of the base sequences of SEQ ID NO.15, the base sequence in region and the Mevalonic kinase of Para rubber tree (accession number AF429384) have 83% homology.Open reading frame is 61-1224.The putative amino acid sequence of being encoded by this cDNA is as shown in SEQ ID NO.16.SEQ IDNO.16 full length amino acid sequence (1-387) has 77% homology with the Mevalonic kinase (accession number AAL18925) of Para rubber tree.
In the present embodiment, use N219-36-tree_1729_s (SEQ ID NO.49) as sense primer, use N219-36-tree_1729_a (SEQ ID NO.50) as antisense primer, using above-mentioned gained cDNA as template, in addition carry out similarly to Example 2 PCR, the sequence in amplification target translation district.This amplified fragments is supplied with to complementary test similarly to Example 2.Mutant strain uses Ura
-the damaged strain Y20794 of Mevalonic kinase (EUROSCARF preparation).In analysis of tetrad, observe the growth on YPG substratum (carbon source: semi-lactosi).And, in the observation on Growth using from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, does not contain uridylic).As a result, by complementation, determined the function of Mevalonic kinase gene.
(embodiment 9: the gene of coding Phosphomevalonic kinase)
Using embodiment 1 gained cDNA library as template, use following primer to carry out PCR, obtain partial sequence.The primer using in this degenerate pcr is as follows: use for the first time S_No.243_P.10_1 (SEQ ID NO.51) as sense primer and use AS_No.243_P.10_1 (SEQ IDNO.52) as antisense primer, use for the first time S_No.243_P.10_2 (SEQ ID NO.53) as sense primer and use AS_No.243_P.10_2 (SEQ ID NO.54) as antisense primer, the design of primer is based on BAD93946 (Arabidopis thaliana, Arabidopsis thaliana), AAL18926 (Para rubber tree, Hevea brasiliensis), CAB52264 (schizosaccharomyces pombe, Schizosaccharomyces pombe), NP593421 (schizosaccharomyces pombe, Schizosaccharomyces pombe), P24521 (yeast saccharomyces cerevisiae, Saccharomyces cerevisiae) and XP329795 (Neurospora crassa, Neurospora crassa) the conservative region of six aminoacid sequences carry out.The condition of PCR is as follows:
(PCR for the first time)
95 ℃ are carried out a circulation for 5 minutes; Within 30 seconds, carry out 30 circulations by 95 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 point; 72 ℃ are carried out a circulation for 7 minutes; 4 ℃ of ∞ carry out a circulation;
(PCR for the second time)
95 ℃ are carried out a circulation for 5 minutes; Within 30 seconds, carry out 30 circulations by 95 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 point; 72 ℃ are carried out a circulation for 7 minutes; 4 ℃ of ∞ carry out a circulation;
In above-mentioned partial sequence, do not obtain the base sequence information of 3 ' end and 5 ' end, therefore, using the RNA of embodiment 1 gained as template, carry out 5 '-RACE, 3 '-RACE and reverse transcription reaction.In the method, use 5 '-Full RACE Core Set (Takara) and 3 '-Full RACE Core Set (Takara).The antisense primer of using as 5 '-RACE according to the primary No.243_P.51_race_a1 of the information design of gained Partial Fragment (SEQ ID NO.56) and secondary No.243_P.51_race_a2 (SEQ ID NO.55), the sense primer that primary No.243_P.51_race_s1 (SEQ IDNO.57) and secondary No.243_P.51_race_s2 (SEQ ID NO.58) use as 3 '-RACE, No.243_P.51_race_p (SEQ ID NO.59) uses as reverse transcription reaction.Obtain thus the base sequence information of 3 ' end and 5 ' end.
According to the base sequence information of 3 ' end and 5 ' end, design cDNA library screening probe, uses the primer of SEQ ID NO.60 as sense primer, uses the primer of SEQ ID NO.61 as antisense primer, carries out PCR.Using gained fragment as probe, the cDNA library of screening embodiment 1 gained.Gained fragment TA is cloned on pT7Blue carrier, carries out sequential analysis.
Whole base sequences of gained cDNA fragment are as shown in SEQ ID NO.17.Shown in region shown in region shown in region shown in region shown in the Nucleotide of 681-886 of the base sequences of SEQ IDNO.17, the Nucleotide of 922-1000, the Nucleotide of 1042-1285, the Nucleotide of 1339-1732 and 1807-2054 s' Nucleotide, the base sequence in region has 83%, 84%, 81%, 81% and 78% homology with the Phosphomevalonic kinase (accession number AF429385) of Para rubber tree respectively.Open reading frame is 682-2202.The putative amino acid sequence of being encoded by this cDNA is as shown in SEQ ID NO.18.The full length amino acid sequence (1-506) of SEQ ID NO.18 has 78% homology with the Phosphomevalonic kinase (accession number AAL18926) of Para rubber tree.
In the present embodiment, use the primer of SEQ ID NO.62 as sense primer, use the primer of SEQ IDNO.63 as antisense primer, using above-mentioned gained cDNA as template, in addition carry out similarly to Example 2 PCR, the sequence in amplification target translation district.This amplified fragments is supplied with to complementary test similarly to Example 2.Mutant strain uses Ura
-the damaged strain Y20806 of Phosphomevalonic kinase (EUROSCARF preparation).In analysis of tetrad, observe the growth on YPG substratum (carbon source: semi-lactosi).And, in the observation on Growth using from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, does not contain uridylic).As a result, by complementation, determined the function of Phosphomevalonic kinase gene.
(embodiment 10: the gene of coding mevalonic acid tetra-sodium decarboxylase)
The sense primer that uses N192-102-tree_13103_3R_s1 (SEQ ID NO.64) (for the first time) and the N192-102-tree_13103_3R_s2 (SEQ ID NO.65) (for the second time) of the information design of the est sequence that is estimated as mevalonic acid tetra-sodium decarboxylase based on embodiment 1 gained to use as 3 '-RACE, in addition similarly to Example 2, obtain full-length cDNA.
Gained full-length cDNA has the base sequence of SEQ ID NO.19.Shown in the Nucleotide of 100-1325 of the base sequences of SEQ ID NO.1, the base sequence in region and the mevalonic acid tetra-sodium decarboxylase (accession number AF429386) of Para rubber tree have 80% homology.Open reading frame is 68-1324.The putative amino acid sequence of being encoded by this cDNA is as shown in SEQ ID NO.20.In the amino acid region of 5-418 of SEQ ID NO.20, the mevalonic acid tetra-sodium decarboxylase (accession number AAL18927) of this putative amino acid sequence and Para rubber tree has 84% homology.
In the present embodiment, use N219-36-tree_13103_s (SEQ ID NO.66) as sense primer, use N219-36-tree_13103_a (SEQ ID NO.67) as antisense primer, using above-mentioned gained cDNA as template, in addition carry out similarly to Example 2 PCR, the sequence in amplification target translation district.This amplified fragments is supplied with to complementary test similarly to Example 2.Mutant strain uses Ura
-the damaged strain Y25418 of mevalonic acid tetra-sodium decarboxylase (EUROSCARF preparation).In analysis of tetrad, observe the growth on YPG substratum (carbon source: semi-lactosi).And, in the observation on Growth using from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, does not contain uridylic).As a result, by complementation, determined the function of mevalonic acid tetra-sodium decarboxylase gene.
Industrial applicability
The invention provides the gene of the various enzymes of initial stage route of synthesis one mevalonate pathway in the biosynthesizing of isoprenoid compounds of encoding eucommia bark.Gene of the present invention is suitable for the genetically modified plants (for example bark of eucommia) that preparation contains trans isoprene or iridoid in a large number.
Claims (3)
1. gene, this gene is the gene participating in by the synthetic isopentenylpyrophosphate of the mevalonic acid of the bark of eucommia, the Mevalonic kinase that this genes encoding is made up of the amino acid of the 1st-387 in the aminoacid sequence of SEQ ID NO.16.
2. gene as claimed in claim 1, wherein, 61-1224 Nucleotide of base sequence that said gene contains SEQ ID NO.15.
3. gene as claimed in claim 1, wherein, 1-1341 Nucleotide of base sequence that said gene contains SEQ ID NO.15.
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| JP2007-163872 | 2007-06-21 | ||
| JP2007163872A JP2009000046A (en) | 2007-06-21 | 2007-06-21 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
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| CN2008101253609A Division CN101328485B (en) | 2007-06-21 | 2008-06-20 | Genes of mevalonate pathway enzyme encoding eucommia bark |
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| CN201110358587XA Pending CN102505017A (en) | 2007-06-21 | 2008-06-20 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
| CN2011103585653A Expired - Fee Related CN102492701B (en) | 2007-06-21 | 2008-06-20 | Genes of mevalonate pathway enzyme encoding eucommia bark |
| CN2008101253609A Expired - Fee Related CN101328485B (en) | 2007-06-21 | 2008-06-20 | Genes of mevalonate pathway enzyme encoding eucommia bark |
| CN201110358647.8A Expired - Fee Related CN102505018B (en) | 2007-06-21 | 2008-06-20 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
| CN201110355190.5A Expired - Fee Related CN102453724B (en) | 2007-06-21 | 2008-06-20 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
| CN201110358667.5A Expired - Fee Related CN102505019B (en) | 2007-06-21 | 2008-06-20 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
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| CN2011103585653A Expired - Fee Related CN102492701B (en) | 2007-06-21 | 2008-06-20 | Genes of mevalonate pathway enzyme encoding eucommia bark |
| CN2008101253609A Expired - Fee Related CN101328485B (en) | 2007-06-21 | 2008-06-20 | Genes of mevalonate pathway enzyme encoding eucommia bark |
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| CN201110358667.5A Expired - Fee Related CN102505019B (en) | 2007-06-21 | 2008-06-20 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
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| JP2015515866A (en) * | 2012-05-11 | 2015-06-04 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Biosynthetic pathways, recombinant cells and methods |
| CN103571857B (en) * | 2012-07-27 | 2016-03-23 | 中国中医科学院中药研究所 | Chinese herbaceous peony acetyl-CoA acyltransferase (PLAACT) gene and coded product thereof and application |
| JP5870464B2 (en) * | 2014-08-12 | 2016-03-01 | 日立造船株式会社 | Long chain trans prenyl diphosphate synthase gene |
| CN105039369A (en) * | 2015-09-02 | 2015-11-11 | 江西中医药大学 | Mevalonate 5-diphosphate decarboxylase gene for andrographis paniculata |
| JP6090700B2 (en) * | 2015-12-25 | 2017-03-08 | 日立造船株式会社 | Long chain trans prenyl diphosphate synthase gene |
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| CN1611602A (en) * | 2003-10-28 | 2005-05-04 | 北京农业生物技术研究中心 | Method for regulating lignin in populus tomentosa |
| WO2005071072A1 (en) * | 2004-01-13 | 2005-08-04 | The Institute Of Biophysics Chinese Academy Of Sciences | The recombinant murine leukemia virus reverse transcriptase, the gene encoding it and the method for expressing it |
| WO2006063752A1 (en) * | 2004-12-14 | 2006-06-22 | Dsm Ip Assets B.V. | Improved mevalonate kinase |
| CN1806042A (en) * | 2003-06-12 | 2006-07-19 | 帝斯曼知识产权资产管理有限公司 | Feedback-resistant mevalonate kinases |
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| TWI250210B (en) * | 1998-05-06 | 2006-03-01 | Dsm Ip Assets Bv | An isolated DNA sequence coding for an enzyme involved in the mevalonate pathway or the pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate |
| US6916972B2 (en) * | 1998-11-05 | 2005-07-12 | E. I. Du Pont De Nemours And Company | Mevalonate synthesis enzymes |
| IL155599A0 (en) * | 2000-10-30 | 2003-11-23 | Maxygen Inc | Polynucleotides encoding proteins which catalyze acetylation of glyphosate |
| DE60228439D1 (en) * | 2001-06-06 | 2008-10-02 | Dsm Ip Assets Bv | IMPROVED ISOPRENOID PRODUCTION |
| KR100704776B1 (en) * | 2004-11-06 | 2007-04-10 | 경상대학교산학협력단 | Method for screening a substance capable of inhibiting either or both of the mevalonate pathway and nonmevalonate pathway using a host cell transformed with a DNA encoding all enzymes associated with a foreign mevalonate pathway and having an inactivated indogenous nonmevalonate pathway |
| UA94038C2 (en) * | 2005-03-18 | 2011-04-11 | Майкробиа, Инк. | Production of carotenoids in oleaginous yeast and fungi |
| CN1313614C (en) * | 2005-10-17 | 2007-05-02 | 中国农业科学院生物技术研究所 | Glyphosate acetyl transferase gene and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1806042A (en) * | 2003-06-12 | 2006-07-19 | 帝斯曼知识产权资产管理有限公司 | Feedback-resistant mevalonate kinases |
| CN1611602A (en) * | 2003-10-28 | 2005-05-04 | 北京农业生物技术研究中心 | Method for regulating lignin in populus tomentosa |
| WO2005071072A1 (en) * | 2004-01-13 | 2005-08-04 | The Institute Of Biophysics Chinese Academy Of Sciences | The recombinant murine leukemia virus reverse transcriptase, the gene encoding it and the method for expressing it |
| WO2006063752A1 (en) * | 2004-12-14 | 2006-06-22 | Dsm Ip Assets B.V. | Improved mevalonate kinase |
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| CN102492701A (en) | 2012-06-13 |
| CN102505017A (en) | 2012-06-20 |
| CN102492701B (en) | 2013-11-06 |
| CN102453724B (en) | 2014-01-01 |
| CN102453724A (en) | 2012-05-16 |
| CN102505019A (en) | 2012-06-20 |
| CN101328485B (en) | 2012-02-15 |
| CN102505019B (en) | 2015-03-11 |
| CN102505018A (en) | 2012-06-20 |
| CN101328485A (en) | 2008-12-24 |
| JP2009000046A (en) | 2009-01-08 |
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