CN102505019B - 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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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 the applying date is on June 20th, 2008, application number be 200810125360.9, the divisional application of application for a patent for invention 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 participating in the bark of eucommia.
Background technology
Cis and trans is divided into according to the difference of the polymerization methods of isoprene unit as the polyisoprene (rubber) of one of isoprenoid compounds.The plant producing long-chain cis-polyisoprene there will be a known a lot of plant such as taraxacum, Herba Lactucae Indicae.The cis-polyisoprene that Para rubber tree (Heveabrasiliensis) is produced is as natural rubber commercially widespread use.And about long-chain using trans-polyisoprene, known natural in have the minority plant production such as bakelite, but commercially not utilize.At present, using trans-polyisoprene is chemosynthesis, is applied in golf ball crust, bandage, movement protection utensil etc.Using trans-polyisoprene is display low melting point and elastomeric thermoplastic elastomer, can be used as isolator.
By being unit with carbonatoms compound one 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.By mevalonate pathway biosynthesizing IPP in animal, plant, mushroom.
As IPP biosynthetic pathway, except mevalonate pathway, also there is non-mevalonate pathway.In plant, be that mevalonate pathway plays a role in tenuigenin, be that non-mevalonate pathway plays a role in chromatoplast.
The gene of the enzyme played a role by mevalonate pathway is all known in each kind of plant.Such as, in Kutki (Rhizoma Picrorhizae) (Picrorhiza kurrooa) (root more containing iridoid glycoside) (base sequence is DQ347964 in the accession number of GenBank to have logged in acetyl-CoA C-Transacetylase, the accession number of aminoacid sequence is ABC74567), (accession number of base sequence is AF429389 to have logged in HMG-CoA synthetic enzyme in Para rubber tree (synthesizing cis polyisoprene), 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 mevalonate pyrophosphate decarboxylase (base sequence accession number AF429386, the accession number of aminoacid sequence is AAL18927).
The long-chain using trans-polyisoprene of the xylophyta bark of eucommia (Eucommia ulmoides Oliver) the producd fibers shape that China originates in.Containing a large amount of using trans-polyisoprene (non-patent literature 1) in the leaf of the bark of eucommia, bark and seed coat.
The bark of the bark of eucommia, as the strong and hypertensive folk therapy medicine of nourishing, is used since ancient times always.In the bark of eucommia containing 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, for example the useful isoprenoid compounds such as using trans-polyisoprene and iridoid glycoside.
In order to prepare the plant of rubber high-content, people wish to obtain the gene participating in IPP biosynthetic pathway in the bark of eucommia.In the past, when obtaining the homologous gene with certain function from target organism kind, be utilize the screening by hybridization using the DNA fragmentation of known gene or the method utilizing the polycondensation PCR of the base sequence of known gene and ensuing 5 '-RACE, 3 '-RACE and RP-PCR to combine.But the accuracy of these methods is low, pretty troublesome.When particularly the conservative region of target gene is indefinite, be then difficult to adopt these methods.
Carry out analyzing (non-patent literature 4 and 5) to the gene order of the bark of eucommia.Gene and the sequence thereof of one of enzyme participating in mevalonate pathway-HMG-CoA reductase is reported in non-patent literature 4.But the enzyme in addition participating in mevalonate pathway then 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 stainingand Fourier transform infrared microapectroscopy in a rubber-producingplant, 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: " Eucommialeaf 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 Eucommiaulmoides iridoids from the leaves, Natural Medicines, 51,275277 (1997)
Non-patent literature 4:Jiang J, Kai G, Gao X, and Chen F, Molecular cloningof a HMG-CoA reductase gene from Eucommia ulmoides Oliver.BiosciRep 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 withthe cambium periodicity in Eucommia ulmoides Oliv.J Exp Bot57:3857-3867 (2006)
Summary of the invention
The object of the present invention is to provide the biosynthetic gene group of the isoprenoid compounds participating in the bark of eucommia.
The invention provides the gene participating in being synthesized isopentenylpyrophosphate by the mevalonic acid of the bark of eucommia.Said gene coding is selected from the protein of aminoacid sequence 1-408 amino acids containing SEQ ID NO.2, the protein of aminoacid sequence 1-408 amino acids containing SEQ ID NO.4, the protein of aminoacid sequence 1-463 amino acids containing SEQID NO.6, the protein of aminoacid sequence 1-466 amino acids containing SEQ ID NO.8, the protein of aminoacid sequence 1-629 amino acids containing SEQ ID NO.12, the protein of aminoacid sequence 1-591 amino acids containing SEQ ID NO.14, the protein of aminoacid sequence 1-387 amino acids containing SEQ ID NO.16, the protein of aminoacid sequence 1-506 amino acids containing SEQ ID NO.18, the protein of the protein of aminoacid sequence 1-418 amino acids containing SEQ ID NO.20.
In one embodiment, said gene is selected from the gene of base sequence 101-1327 Nucleotide containing SEQ ID NO.1, the gene of base sequence 128-1354 Nucleotide containing SEQ ID NO.3, the gene of base sequence 172-1563 Nucleotide containing SEQ ID NO.5, the gene of base sequence 216-1616 Nucleotide containing SEQ ID NO.7, the gene of base sequence 32-1921 Nucleotide containing SEQ IDNO.11, the gene of base sequence 60-1835 Nucleotide containing SEQ ID NO.13, the gene of base sequence 61-1224 Nucleotide containing SEQ ID NO.15, the gene of base sequence 682-2202 Nucleotide containing SEQ ID NO.17, the gene of base sequence 68-1324 Nucleotide containing SEQ ID NO.19.
In further embodiment, said gene is selected from the gene of base sequence 1-1516 Nucleotide containing SEQ ID NO.1, the gene of base sequence 1-1757 Nucleotide containing SEQ ID NO.3, the gene of base sequence 1-1892 Nucleotide containing SEQ ID NO.5, the gene of base sequence 1-1833 Nucleotide containing SEQ ID NO.7, the gene of base sequence 1-1825 Nucleotide containing SEQ ID NO.9, the gene of base sequence 1-2057 Nucleotide containing SEQ ID NO.11, the gene of base sequence 1-2225 Nucleotide containing SEQ ID NO.13, the gene of base sequence 1-1341 Nucleotide containing SEQ ID NO.15, the gene of base sequence 1-2432 Nucleotide containing SEQ ID NO.17, the gene of base sequence 1-1542 Nucleotide containing SEQ IDNO.19.
The invention provides the gene participating in being synthesized isopentenylpyrophosphate by the mevalonic acid of the bark of eucommia.
Embodiment
The invention provides the gene participating in being synthesized isopentenylpyrophosphate (IPP) by the mevalonic acid of the bark of eucommia.The synthesis of IPP is 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; (4) Mevalonic kinase; (5) Phosphomevalonic kinase; And (6) mevalonate pyrophosphate 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, HMG-CoA is synthesized by acetoacetyl-CoA, by (3) HMG-CoA reductase, change HMG-CoA into mevalonic acid, by (4) Mevalonic kinase and (5) Phosphomevalonic kinase, mevalonic acid is changed into mevalonic acid 5-tetra-sodium, then generate isopentenylpyrophosphate (IPP) by (6) mevalonate pyrophosphate decarboxylase.
Genes encoding of the present invention is selected from the protein (hereinafter also referred to " protein of SEQ ID NO.2 ") of aminoacid sequence 1-408 amino acids containing SEQ ID NO.2; The protein (hereinafter also referred to " protein of SEQ IDNO.4 ") of aminoacid sequence 1-408 amino acids containing SEQ IDNO.4; The protein (hereinafter also referred to " protein of SEQ ID NO.6 ") of aminoacid sequence 1-463 amino acids containing SEQ ID NO.6; The protein (hereinafter also referred to " protein of SEQ ID NO.8 ") of aminoacid sequence 1-466 amino acids containing SEQ ID NO.8; The protein (hereinafter also referred to " protein of SEQ ID NO.12 ") of aminoacid sequence 1-629 amino acids containing SEQ ID NO.12; The protein (hereinafter also referred to " protein of SEQ ID NO.14 ") of aminoacid sequence 1-591 amino acids containing SEQ ID NO.14; The protein (hereinafter also referred to " protein of SEQ ID NO.16 ") of aminoacid sequence 1-387 amino acids containing SEQ ID NO.16; The protein (hereinafter also referred to " protein of SEQ ID NO.18 ") of aminoacid sequence 1-506 amino acids containing SEQ ID NO.18; The protein of the protein (hereinafter also referred to " protein of SEQ IDNO.20 ") of aminoacid sequence 1-418 amino acids containing SEQ IDNO.20.The amino acid whose protein of 1-466 containing SEQ ID NO.10 is identical with the protein of SEQ ID NO.8.
The protein of SEQ ID NO.2 and the protein of SEQ ID NO.4 are (1) acetyl-CoA C-Transacetylases.The protein of SEQ ID NO.2 is by the genes encoding gained of the Nucleotide of the base sequence containing SEQ ID NO.1 101-1327.This gene such as 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 the base sequence containing SEQ IDNO.3 128-1354.This gene such as can contain whole base sequences (1-1757) of SEQ ID NO.3.
The protein of SEQ ID NO.6 and the protein of SEQ ID NO.8 are (2) HMG-CoA synthetic enzyme.The protein of SEQ ID NO.6 is by the genes encoding gained of the Nucleotide of the base sequence containing SEQ ID NO.5 172-1563.This gene such as 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 the base sequence containing SEQ ID NO.7 216-1616 (or base sequence 216-1616 of SEQ ID NO.9).The gene of the Nucleotide of the gene of the Nucleotide of the base sequence 1-1833 such as containing SEQ ID NO.7 and the base sequence 1-1825 containing SEQ ID NO.9 is comprised in this gene.There is the gene of base sequence shown in SEQ ID NO.9 in its district of 1689-1825, relative to the district of 1689-1833 of gene with the base sequence shown in SEQ ID NO.7, there is the displacement at the 6 places base sequence 1689 of the SEQ ID NO.7 (, 1709,1713,1745,1756 and 1820) and the shortening at the disappearance of 1 place (base sequence of SEQ ID NO.7 1777) and the poly-adenosine position of 3 ' end.
The protein of SEQ ID NO.12 and the protein of SEQ ID NO.14 are (3) HMG-CoA reductase.The protein of SEQ ID NO.12 is by the genes encoding gained of the Nucleotide of the base sequence containing SEQ ID NO.11 32-1921.This gene such as 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 the base sequence containing SEQ IDNO.13 60-1835.This gene such as 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 the base sequence containing SEQ ID NO.15 61-1224.This gene such as 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 the base sequence containing SEQ ID NO.17 682-2202.This gene such as can contain whole base sequences (1-2432) of SEQ ID NO.17.
The protein of SEQ ID NO.20 is (6) mevalonate pyrophosphate decarboxylase.The protein of SEQ IDNO.20 is by the genes encoding gained of the Nucleotide of the base sequence containing SEQ ID NO.19 68-1324.This gene such as 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 enzyme with above-mentioned aminoacid sequence functional equal enzyme or can be played said function with the gene with above-mentioned base sequence, the protein that one or more amino-acid residue of can encoding is different, or one or more base can be had different.The difference of above-mentioned sequence due to the displacement of base, disappearance and/or can insert acquisition, or the any-mode of natural mutagenesis or induced mutations (such as the application of rite-directed mutagenesis introductory technique) occurs.The function of gene really usual practice as can adopt the basic skills described in following examples 2, 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 usually used, and the sequence information recorded according to this specification sheets prepares probe or primer, with the chromosomal DNA of the bark of eucommia or cDNA for template, obtains target fragment by PCR.Can certainly be template with RNA, utilize reverse transcription PCR.Gene of the present invention, except the native polynucleotide such as DNA, RNA, also can be the artificial molecule containing artificial nucleotide derivative.Gene of the present invention can also be the chimeric molecule of DNA-RNA.
Gene of the present invention can use usual 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.Such as its formation can be: express at least one in the one group of enzyme (above-mentioned (1) is to (6)) be present in mevalonate pathway.
Gene of the present invention is suitable for preparing a large amount of genetically modified plants (such as the bark of eucommia) containing using trans-polyisoprene or iridoid.Gene of the present invention, such as by transforming the bark of eucommia, can be used for preparing the more plant containing rubber.
Embodiment
Below provide embodiment, illustrate the present invention further, 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, the phloem (bark) of sprout be then used in late May, gathering from the bark of eucommia standard tree of Sheng Ming village of Ai Yuan county growth and xylem.
The mutant strain library of yeast uses commercially available YKO Heterozygous Essential StrainCollection-Glycerol Stocks (Open Biosystems company).
(extracting RNA by the bark of eucommia)
By about 4g bark of eucommia plant materials sample (phloem of current growth and xylem) limit cooled with liquid nitrogen, while with mortar, grinding rod fragmentation, be suspended in 2 × CTAB solution (2% (w/v) cetyl trimethylammonium bromide (CTAB), 1% (w/v) 2 mercapto ethanol, 0.1M Tris-HC1 (pH 9.5), 1.4M NaCl, the 20mM EDTA of 10 times amount (w/v) of sample.By its incubation 10 minutes at 65 DEG C, then use chloroform/primary isoamyl alcohol process (washing) (repeatedly twice).In the water layer reclaimed, add the 10M LiCl that 1/4 (v/v) measure and mix, at-20 DEG C, incubation 2 hours, carries out the selective precipitation of RNA.It is centrifugal, will be precipitated and dissolved in appropriate Tris-EDTA (TE) damping fluid, centrifugation, reclaim supernatant, get rid of polyose.The supernatant of recovery is carried out phenol process, phenol/chloroform process and chloroform/primary isoamyl alcohol process, again carried out the selective precipitation of RNA by LiCl.Precipitation is used 70% washing with alcohol, drying under reduced pressure, be then dissolved in diethylpyrocarbonate (DEPC) and process in water.Carry out quantitatively, being confirmed by electrophoresis to gained RNA by absorbance measurement.0.84mg RNA is obtained, by obtaining 2mgRNA (specific absorption under 260nm and 280nm is respectively 1.956 and 1.990) in about 4g phloem by about 4g xylem.
(preparation of bark of eucommia cDNA library)
By the G-キ ッ 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% (clone for inserting).Library size from the eDNA of xylem is 2.2 × 10
5, insertion rate is 79% (24 sample/agarose gel electrophoresis), total length rate 63% (clone relative to there being insertion).
(sequential analysis of est sequence)
In North university North life science institute genome informatics research room, each about 20000 clones for the cDNA library from bark of eucommia phloem and xylem carry out sequential analysis.According to the sequence information obtained by sequential analysis, removing is not possessed the clone of insertion and cannot be read the clone of sequence, obtains the sequence information that precision is high.For the library of phloem and xylem, obtain the high est sequence of the precision of 16567 and 16113 length (adding up to 32680) respectively.
Gained sequence is carried out dividing into groups (cluster) (clustering) and explaining (annotation)." 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 " is by comparing with known, explains est sequence.The homology search adopting NCBI BLAST is used during note.The database used during retrieval is nr (All non-redundant GenBank CDStranslations+PDB+SwissProt+PIR (peptide sequence database)).
According to grouping and the information explaining gained, find the est sequence being estimated as encoding acetyl-CoA C-Transacetylase, HMG-CoA synthetic enzyme, HMG-CoA reductase, Mevalonic kinase and mevalonate pyrophosphate decarboxylase.These enzymes are all enzymes of initial stage route of synthesis in the biosynthesizing of the isoprenoid compounds participating in the bark of eucommia.
(embodiment 2: the gene 1 of encoding acetyl-CoA C-Transacetylase)
(acquisition of full-length cDNA)
By 3 '-RACE (rapid amplifying of cDNA end, RACE), the sequence obtained by the analysis by embodiment 1 determines the sequence of 3 ' end side, obtains full-length cDNA.
3 '-RACE uses 3 '-Full RACE Core Set (preparation of タ カ ラ バ イ ォ Co., Ltd.).Oligo-dT primer is used in reverse transcription reaction.Pcr amplification uses with the primer of oligo-dT and is the sense primer of identical sequence with a part for known array.Sense primer is according to the est sequence information design of encoding acetyl-CoA C-Transacetylase being estimated as embodiment 1 gained.With the RNA of embodiment 1 gained for template, for 1st time use N192-82-tree_1968_3R_S1 (SEQ IDNO.21) as sense primer, be for 2nd time use N192-82-tree_1968_3R_s2 (SEQ IDNO.22) as sense primer, carry out PCR to specifications.Reverse transcription reaction and the amplified fragments TA that obtained by PCR are cloned in pT7Blue carrier, carry out sequential analysis.
Gained full-length cDNA has the base sequence of SEQ ID NO.1.The base sequence in the region shown in Nucleotide of the base sequence described in SEQ ID NO.1 146-1299 and the acetyl-CoA C-Transacetylase (GenBank accession number DQ347964) of Kutki have the homology of 82%.Open reading frame is 101-1327.The aminoacid sequence of the presumption 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 and acetyl-CoA C-Transacetylase (accession number ABC74567) of Kutki have the homology of 89%.
(using the complementary test in yeast mutant library)
The complementary test that use yeast mutant library is carried out confirms the function of gained cDNA.This complementary test carries out as follows.
It is sense primer and the antisense primer of identical sequence that use is attached with the 40bp of the multiple clone site of pYES2 carrier (prepared by invitrogen), carry out PCR (at 95 DEG C using 5 minutes as 1 circulation; Then at 95 DEG C by 1 minute, 54 DEG C by 1 minute and 72 DEG C using 1 minute as 30 circulations; At 72 DEG C using 7 minutes as 1 circulation; And at 4 DEG C using ∞ as 1 circulation), amplification target gene translated region sequence.
Become by this amplified fragments the pYES2 vector introduction of straight-chain to Ura with by restriction enzyme treatment
-yeast strains Y22800 (EUROSCARF preparation) in.As analytic target acetyl-CoA C-acetyl transferase gene for yeast growth be necessary, therefore, as the background of complementary assay, this yeast uses only has the heterozygous diploid of the contraposition genetically deficient target gene of side.Frozen-EZ Yeast Transformation II (prepared by ZYMO RESEARCH) is used during channel genes.By the yeast-inoculated after conversion in limit perfect medium (lacking uracil), cultivate at 30 DEG C.The colony of growth is inoculated in new limit perfect medium (lacking uracil), then cultivates at 30 DEG C.Yeast mutant is uridylic demand property, and the sequence containing URA3 gene (encodes orotidine 5 '-phosphate decarboxylase) in pYES2 carrier, therefore only has the thalline of conversion to grow.By PCR, the mattress body of growth checks that whether it is containing Insert Fragment, then, is transferred to this thalline in YPDA substratum, cultivates at 30 DEG C.Transfer them to sporulation substratum, cultivate at 25 DEG C, gained spore is carried out analysis of tetrad.
There is GAL1 promotor in the upstream be 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 to grow on YPD substratum or YPG substratum (carbon source: semi-lactosi).In YPD substratum, the dyad from mutant strain is lethal, and the dyad only from wild-type grows.And on YPG substratum (carbon source: semi-lactosi), the expression of gene is inserted in the pYES2 carrier induction with GAL1 promotor, therefore, if had complementary functions, then can survive from mutant strain, triad or tetrad growth.Like this, by carrying out complementary confirmation, the function can carrying out target gene is determined.
And, by obtained by analysis of tetrad from eight sporangial colonies (can monoploid be thought) photocopy on (A) YPG substratum (carbon source: semi-lactosi), (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic), (C) YPG substratum (carbon source: semi-lactosi, containing microbiotic G418) or (D) YPG substratum (carbon source: glucose).
Possess cultivation strain growth in the limit perfect medium (carbon source: semi-lactosi, not containing uridylic) of (B) of plasmid.Mutant strain has G418 resistant gene, therefore can think in the middle growth of (C) YPG substratum (carbon source: semi-lactosi, containing microbiotic G418).The monoploid mutant strain possessing plasmid can be determined by the growth result in these substratum.In the YPD substratum (carbon source: glucose) of (D), the mutant strain (monoploid) possessing plasmid should grow, but is in fact difficult to the presence or absence that judges that it grows.Therefore the presence or absence of growth is differentiated 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, the cDNA obtained in above-mentioned " acquisition of complete cDNA " is as template, carry out PCR, the sequence of amplification target translated region.This amplified fragments is supplied above-mentioned complementary test.The growth on YPG substratum (carbon source: semi-lactosi) is observed in analysis of tetrad.And by using the observation on Growth from eight sporangial colonies, observe the growth on (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic).As a result, by complementation, determine the function of acetyl-CoA C-acetyl transferase gene.
(embodiment 3: the gene 2 of encoding acetyl-CoA C-Transacetylase)
Use N192-84-tree_11012_3R_s1 (SEQ ID NO.25) (the 1st time) that be estimated as the information design of the est sequence of encoding acetyl-CoA C-Transacetylase and N192-84-tree_11012_3R_s2 (SEQ ID NO.26) (the 2nd time) that obtain according to embodiment 1 as the sense primer of 3 '-RACE, in addition similarly to Example 2, full-length cDNA is obtained.
Gained full-length cDNA has the base sequence of SEQ ID NO.3.The base sequence in the region shown in Nucleotide of the base sequence 173-1305 of SEQ ID NO.3 and the acetyl-CoAC-Transacetylase (accession number DQ347964) of Kutki have the homology of 83%.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 and acetyl-CoA C-Transacetylase (accession number ABC74567) of Kutki have the homology of 87%.
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, with above-mentioned gained cDNA for template, in addition PCR is carried out similarly to Example 2, the sequence of amplification target translated region.This amplified fragments is supplied complementary test similarly to Example 2, obtains result similarly to Example 2.The growth on YPG substratum (carbon source: semi-lactosi) is observed in analysis of tetrad.Further, by using the observation on Growth from eight sporangial colonies, the growth on (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic) is observed.As a result, the function of acetyl-CoA C-acetyl transferase gene is determined by complementation.
(embodiment 4: the gene 1 of coding HMG-CoA synthetic enzyme).
Use N192-71-tree_6098_3R_s1 (SEQ ID NO.29) (the 1st time) and N192-71-tree_6098_3R_s2 (SEQ ID NO.30) (the 2nd time) sense primer as 3 '-RACE being estimated as the information design of the est sequence of coding HMG-CoA synthetic enzyme based on embodiment 1 gained, in addition similarly to Example 2, full-length cDNA is obtained.
The full-length cDNA of gained has the base sequence of SEQ ID NO.5.The region shown in Nucleotide of base sequence 172-1187 of SEQ ID NO.5 and the base sequence in the region shown in Nucleotide of 1390-1439, have the homology of 83% and 86% 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 and the HMG-CoA synthetic enzyme (accession number AAS46245) of Para rubber tree have the homology of 85%.
In the present embodiment, sense primer uses N219-36-tree_6098_s (SEQ ID NO.31), antisense primer uses N219-36-tree_6098_a (SEQ ID NO.32), with above-mentioned gained cDNA for template, in addition PCR is carried out similarly to Example 2, the sequence of amplification target translated region.Similarly to Example 2, by this amplified fragments supply complementary test.Mutant strain uses Ura
-hMG-CoA synthetic enzyme defect strain Y26527 (EUROSCARF preparation).The growth on YPG substratum (carbon source: semi-lactosi) is observed in analysis of tetrad.Further, using in the observation on Growth from eight sporangial colonies, observing the growth on (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic).As a result, the function of HMG-CoA synthase gene is determined 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 N192-36-tree_10370_3R_s2 (SEQ ID NO.34) (the 2nd time) of the information design of the est sequence of the presumption coding HMG-CoA synthetic enzyme of embodiment 1 gained, as the sense primer of 3 '-RACE, in addition obtain full-length cDNA similarly to Example 2.Obtain two full-length cDNAs.
First full-length cDNA of gained has the base sequence of SEQ ID NO.7.The base sequence in region shown in the Nucleotide of the base sequence 234-1496 of SEQ ID NO.7 and the HMG-CoA synthetic enzyme (accession number AF429389) of Para rubber tree have the homology of 81%.Gained second full-length cDNA has the base sequence of SEQ ID NO.9.2nd cDNA is in its region of 1689-1825, and there is the shortening at the poly-adenosine position of 6 places (1689,1709,1713,1745,1756 of a cDNA and 1820) displacement and 1 place (1777 of a cDNA) disappearance and 3 ' end in the region of 1689-1833 of a corresponding cDNA.The base sequence of SEQ ID NO.9 is also same with the base sequence of SEQ ID NO.7, has the homology of 81% 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 the homology of 84% 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 PCR is carried out similarly to Example 2, the sequence of amplification target translated region.Similarly to Example 2, by this amplified fragments supply complementary test.Mutant strain uses Ura
-hMG-CoA synthetic enzyme defect strain Y26527 (EUROSCARF preparation).The growth on YPG substratum (carbon source: semi-lactosi) is observed in analysis of tetrad.Further, using in the observation on Growth from eight sporangial colonies, observing the growth on (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic).As a result, by complementation, determine the function of HMG-CoA synthase gene.
(embodiment 6: the gene 1 of coding HMG-CoA reductase)
About the gene of coding HMG-CoA reductase, 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).According to the antisense primer of est sequence design N192-45-tree_8220_5R_al (SEQ ID NO.37) (for the first time) and N192-45-tree_8220_5R_a2 (SEQ ID NO.38) (for the second time) as 5 ' RACE being estimated as coding HMG-CoA reductase of embodiment 1 gained, 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) are as the sense primer of 3 '-RACE, and N192-45-tree_8220_5R_p (SEQ ID NO.41) uses as reverse transcription reaction.With the RNA of embodiment 1 gained for template, similarly to Example 2, the amplified fragments TA of their reaction gained is cloned on pTCBlue carrier, carries out sequential analysis.
Gained full-length cDNA has the base sequence of SEQ ID NO.11.Shown in the Nucleotide of region shown in region shown in the Nucleotide of the base sequence 260-303 of SEQ ID NO.11, the Nucleotide of 773-936 and 1148-1845 the base sequence in region with the HMG-CoA reductase (accession number AF315713) of apple (Malus x domestica), there is the homology of 88%, 82% and 81% 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 (accession number P48021) of this putative amino acid sequence and camplotheca acuminata (Camptotheca acuminata) has the homology of 78%.The amino acid region of 33-612 of SEQ ID NO.12 and the HMG-CoA reductase (accession number AAV54051) of non-patent literature 4 have the homology of 73%.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)
According to the antisense primer of est sequence design N192-47-tree_13453_5R_a1 (SEQ ID NO.42) (for the first time) and N192-47-tree_13453_5R_a2 (SEQ ID NO.43) (for the second time) as 5 ' RACE being estimated as coding HMG-CoA reductase of embodiment 1 gained, 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) are as the sense primer of 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 the Nucleotide of region shown in region shown in the Nucleotide of the base sequence 169-373 of SEQ ID NO.13, the Nucleotide of 751-1600 and 1637-1720 the base sequence in region, with the HMG-CoA reductase (accession number U72145) of camplotheca acuminata, there is the homology of 79%, 80% and 89% 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 (accession number AAB87727) of this putative amino acid sequence and tobacco (Nicotiana tabacum) has the homology of 78%.The amino acid region of 1-589 of SEQ ID NO.14 and the HMG-CoA reductase (accession number AAV54051) of non-patent literature 4 have the homology of 75%.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 of 3 '-RACE uses tree 1729 primer (SEQ ID NO.47) (for the first time) being estimated as the information design of the est sequence of coding Mevalonic kinase based on embodiment 1 gained and tree 1729 primer cover (SEQ ID NO.48) (for the second time), in addition similarly to Example 2, full-length cDNA is obtained.
Gained full-length cDNA has the base sequence of SEQ ID NO.15.The base sequence in region shown in the Nucleotide of the base sequence 616-1104 of SEQ ID NO.15 and the Mevalonic kinase (accession number AF429384) of Para rubber tree have the homology of 83%.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 the homology of 77% 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 PCR is carried out similarly to Example 2, the sequence of amplification target translated region.This amplified fragments is supplied complementary test similarly to Example 2.Mutant strain uses Ura
-mevalonic kinase defect strain Y20794 (EUROSCARF preparation).The growth on YPG substratum (carbon source: semi-lactosi) is observed in analysis of tetrad.Further, using in the observation on Growth from eight sporangial colonies, observing the growth on (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic).As a result, by complementation, determine the function of Mevalonic kinase gene.
(embodiment 9: the gene of coding Phosphomevalonic kinase)
Using embodiment 1 gained cDNA library as template, following primer is used to carry out PCR, obtaining portion sub-sequence.The primer used in this degenerate pcr is as follows: first time uses S_No.243_P.10_1 (SEQ ID NO.51) as sense primer and uses AS_No.243_P.10_1 (SEQ IDNO.52) as antisense primer, first time uses S_No.243_P.10_2 (SEQ ID NO.53) as sense primer and uses 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, and XP329795 (Neurospora crassa Saccharomycescerevisiae), Neurospora crassa) the conservative region of six aminoacid sequences carry out.The condition of PCR is as follows:
(first time PCR)
95 DEG C are carried out a circulation for 5 minutes; 30 circulations within 30 seconds, are carried out by 95 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 point; 72 DEG C are carried out a circulation for 7 minutes; 4 DEG C of ∞ carry out a circulation;
(second time PCR)
95 DEG C are carried out a circulation for 5 minutes; 30 circulations within 30 seconds, are carried out by 95 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 point; 72 DEG C are carried out a circulation for 7 minutes; 4 DEG C of ∞ carry out a circulation;
Do not obtain the base sequence information of 3 ' end and 5 ' end in above-mentioned partial sequence, therefore, using the RNA of embodiment 1 gained as template, carry out 5 '-RACE, 3 '-RACE and reverse transcription reaction.5 '-Full RACE Core Set (Takara) and 3 '-Full RACE Core Set (Takara) is used in the method.According to the antisense primer of the primary No.243_P.51_race_al of the information design of gained Partial Fragment (SEQ ID NO.56) and secondary No.243_P.51_race_a2 (SEQ ID NO.55) as 5 '-RACE, primary No.243_P.51_race_s1 (SEQ IDNO.57) and secondary No.243_P.51_race_s2 (SEQ ID NO.58) is as the sense primer of 3 '-RACE, and No.243_P.51_race_p (SEQ ID NO.59) uses as reverse transcription reaction.Obtain the base sequence information of 3 ' end and 5 ' end thus.
According to the base sequence information of 3 ' end and 5 ' end, design cDNA library screening probe, the primer of use SEQ ID NO.60 is as sense primer, and the primer of use 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 the Nucleotide of the region shown in Nucleotide of the base sequence 681-886 of SEQ IDNO.17, the region shown in Nucleotide of 922-1000, the region shown in Nucleotide of 1042-1285, the region shown in Nucleotide of 1339-1732 and 1807-2054, the base sequence in region has the homology of 83%, 84%, 81%, 81% and 78% 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 and the Phosphomevalonic kinase (accession number AAL18926) of Para rubber tree have the homology of 78%.
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 PCR similarly to Example 2, the sequence of amplification target translated region.This amplified fragments is supplied complementary test similarly to Example 2.Mutant strain uses Ura
-phosphomevalonic kinase defect strain Y20806 (EUROSCARF preparation).The growth on YPG substratum (carbon source: semi-lactosi) is observed in analysis of tetrad.Further, using in the observation on Growth from eight sporangial colonies, observing the growth on (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic).As a result, by complementation, determine the function of Phosphomevalonic kinase gene.
(embodiment 10: the gene of coding mevalonate pyrophosphate decarboxylase)
Use 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) that are estimated as the information design of the est sequence of mevalonate pyrophosphate decarboxylase based on embodiment 1 gained as the sense primer of 3 '-RACE, in addition similarly to Example 2, full-length cDNA is obtained.
Gained full-length cDNA has the base sequence of SEQ ID NO.19.The base sequence in region shown in the Nucleotide of the base sequence 100-1325 of SEQ ID NO.1 and the mevalonate pyrophosphate decarboxylase (accession number AF429386) of Para rubber tree have the homology of 80%.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 mevalonate pyrophosphate decarboxylase (accession number AAL18927) of this putative amino acid sequence and Para rubber tree has the homology of 84%.
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 PCR is carried out similarly to Example 2, the sequence of amplification target translated region.This amplified fragments is supplied complementary test similarly to Example 2.Mutant strain uses Ura
-mevalonate pyrophosphate decarboxylase defect strain Y25418 (EUROSCARF preparation).The growth on YPG substratum (carbon source: semi-lactosi) is observed in analysis of tetrad.Further, using in the observation on Growth from eight sporangial colonies, observing the growth on (B) limit perfect medium (carbon source: semi-lactosi, not containing uridylic).As a result, by complementation, determine the function of mevalonate pyrophosphate decarboxylase gene.
Industrial applicability
The invention provides the gene of the various enzymes of the initial stage route of synthesis-mevalonate pathway in the biosynthesizing of the isoprenoid compounds of encoding eucommia bark.Gene of the present invention is suitable for preparing a large amount of genetically modified plants (such as the bark of eucommia) containing trans-isoprene or iridoid.
Claims (1)
1. gene, this gene is the gene participating in being synthesized by the mevalonic acid of the bark of eucommia isopentenylpyrophosphate, and this gene is the gene of the Phosphomevalonic kinase of aminoacid sequence 1-506 amino acids composition of coding SEQ ID NO.18.
2. gene as claimed in claim 1, wherein, said gene is made up of base sequence 682-2202 Nucleotide of SEQ ID NO.17.
3. gene as claimed in claim 1, wherein, said gene is made up of base sequence 1-2432 Nucleotide of SEQ ID NO.17.
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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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| 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 |
| 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 |
| CN2011103585653A Expired - Fee Related CN102492701B (en) | 2007-06-21 | 2008-06-20 | Genes of mevalonate pathway enzyme encoding eucommia bark |
| CN201110358587XA Pending CN102505017A (en) | 2007-06-21 | 2008-06-20 | Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver |
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| CN104520426A (en) * | 2012-05-11 | 2015-04-15 | 明尼苏达大学评议会 | 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 |
| CN1531594B (en) * | 2000-10-30 | 2011-05-25 | 弗迪亚股份有限公司 | Novel glyphosate N-acetyltransferase (GAT) genes |
| CN1630718A (en) * | 2001-06-06 | 2005-06-22 | Dsmip资产公司 | 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 |
| EA016258B1 (en) * | 2005-03-18 | 2012-03-30 | Микробиа, Инк. | Recombinant fungus producing carotenoids and methods of use thereof |
| 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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| 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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