CN101250542B - Salvia 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application - Google Patents
Salvia 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application Download PDFInfo
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Abstract
The invention discloses a salvia 3-hydroxy-3-methylglutaryl coenzyme A synthase gene, protein which is encoded by the salvia 3-hydroxy-3-methylglutaryl coenzyme A synthase gene and the use thereof. The 3-hydroxy-3-methylglutaryl coenzyme A synthase gene has a nucleotide sequence or a homologous sequence which adds, replaces, inserts or losses one or a plurality of nucleotides or allele thereof andthe nucleotide sequence which is derived from the 3-hydroxy-3-methylglutaryl coenzyme A synthase gene, which are displayed in the SED ID No.1. The protein which is encoded by the gene has an amino acid sequence or the homologous sequence which adds, replaces, inserts or losses one or a plurality of amino acids, which is displayed in the SEQ ID No.2. The 3-hydroxy-3-methylglutaryl coenzyme A synthase gene which is provided by the invention can increase the content of tanshinone which is a drug used active compound in the resource plants such as salvia and the like through the genetic engineering technology and can be used in research and industrialization for increasing the content of the tanshinone through utilizing the transgenic technology, which is helpful for accelerating to solve theproblem that the drug resources of the camptothecin are seriously scarce and has very good application prospect.
Description
Technical field
The invention belongs to biological technical field, specifically, the 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene that relates in the red sage root, express and encoded protein matter and application.
Background technology
TANSHINONES (tanshinone) is a kind of class active substance that obtains that separates from the traditional Chinese medicinal materials red sage root of China, mainly is made up of tanshinones such as fat-soluble Tanshinone I, Tanshinone II A, dihydrotanshinone, Cryptotanshinone.Suppurative tonsillitis, mazoitis, cellulitis, osteomyelitis, inflammation of external auditory canal and carbuncle that clinical medicine widely applies total tanshinone treatment streptococcus aureus and Resistant strain, suis etc. to cause are swollen, and surgical infection etc. are evident in efficacy.TANSHINONES is widely used in clinical medicine, and the world market demand is very huge at present.TANSHINONES in the modern industrialization production mainly obtains by extracting from natural plant such as the red sage root.Yet the extraction process of TANSHINONES is loaded down with trivial details, and waste is big, and TANSHINONES and analogue thereof are lower at the plant materials intensive amount, can not satisfy people's needs growing to TANSHINONES far away so extract the method for TANSHINONES from the natural red sage root.Therefore, the serious scarcity in TANSHINONES medicine source has become the biggest obstacle that restriction TANSHINONES related industries develops.
The develop rapidly of plant gene engineering technology and widespread use in recent years is for the content that utilizes modern biotechnology to improve TANSHINONES or its precursor has been opened up a brand-new approach.Utilizing modern biotechnology that the key gene in the TANSHINONES biosynthetic pathway is imported in the red sage root, obtain genetically modified clone, tissue or regeneration plant, and cultivate on a large scale, is the optimal path of realizing fundamentally improving TANSHINONES content.The 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme is an important metabolic enzyme in the MVA approach, can the catalysis acetyl-CoA and the acetoacetyl-CoA condensation generate 3-hydroxy-3-methylglutaryl-coenzyme A, under the effect of HMG-CoA reductase enzyme (HMGR), generate mevalonic acid (MVA) subsequently, mevalonic acid forms the IPP of C5 through pyrophosphorylation and decarboxylation, thereby provides general precursor for TANSHINONES is synthetic.By improving the activity or the content of 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme, the content of TANSHINONES or its precursor in the raising red sage root that can be indirect.
The medicinal plant red sage root (Salvia miltiorrhiza Bunge) is provinces such as Labiatae per nnial herb, main product Sichuan, Shandong, Zhejiang.At China's stasis-dispelling and pain-killing that is usually used among the people, promoting blood circulation to restore menstrual flow, relieving restlessness clears away heart-fire.Modern medicine study proves, the contained activeconstituents of the red sage root has vasodilation, reducing blood-fat, atherosclerosis, inhibition platelet aggregation, protection cardiac muscle, antisepsis and anti-inflammation and effect such as antitumor.Because the red sage root is as the herbaceous plant of important product TANSHINONES, it is short as the growth fast period to make it have more research advantage, be easy to genetic transformation and plant regeneration etc., this provides source and approach more efficiently for the biosynthesizing and the molecular regulation thereof of research TANSHINONES undoubtedly.
In analysis to existing document, " Scinece Asia (Asia science) 2002; 28:29-36 " reported and cloned the 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene from rubber tree, but the bibliographical information that clones and isolates the 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene from the medicine plant red sage root has not been arranged so far as yet.Because the enzyme of this genes encoding has material impact for the biosynthesizing of TANSHINONES, therefore, this step is to utilize genetic engineering technique to regulate and control the biosynthetic important point of penetration of TANSHINONES.
Summary of the invention
The object of the present invention is to provide a kind of salvia 3-hydroxy-3-methylglutaryl A synthase gene.
Second purpose of the present invention provides the protein of this genes encoding.
The present invention also provides recombinant vectors and the host cell that contains this gene.
Another object of the present invention is to provide the application of this gene and encoded protein matter thereof.
The nucleotide sequence of salvia 3-hydroxy-3-methylglutaryl A synthase gene provided by the present invention (DsHMGS) is shown in the 118th～1500 of SEQ ID No.1.
The protein of this coded by said gene is the salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme, and its aminoacid sequence is shown in SEQ ID No.2.
Contain the complete coding of salvia 3-hydroxy-3-methylglutaryl A synthase gene of the present invention and read the recombinant vectors of frame, all belong to protection scope of the present invention.These recombinant vectorss comprise plasmid and plant expression vector.
Contain the complete coding of salvia 3-hydroxy-3-methylglutaryl A synthase gene of the present invention and read the host cell of frame, as the host cell that contains above-mentioned recombinant vectors also belongs to protection scope of the present invention.
Host cell is selected from Bacillus coli cells, agrobatcerium cell, yeast cell, tobacco cell, red sage root cell or red sage root root of hair cell.
Preferred host cell is Bacillus coli cells or agrobatcerium cell or red sage root root of hair cell.
The application of salvia 3-hydroxy-3-methylglutaryl A synthase gene of the present invention comprises and uses described recombinant vectors, transforms red sage root cell as plant expression vector; Perhaps with described Agrobacterium and the red sage root co-culture of cells that contains this gene, obtain genetically modified red sage root root of hair system; Perhaps use described red sage root root of hair cell regeneration red sage root plant; Perhaps obtain transgenosis red sage root plant with described salvia 3-hydroxy-3-methylglutaryl A synthetase gene sequence genetic transformation.
The notion particular content that relates in the technical solution of the present invention is as follows:
The nucleotide sequence of the polypeptide that the dna molecular of the said salvia 3-hydroxy-3-methylglutaryl A synthase gene of the present invention has the salvia 3-hydroxy-3-methylglutaryl A synthase activity for encoding, and described nucleotide sequence is shown in the 118th～1500 of SEQ ID No.1.
The isolated salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme of the present invention polypeptide is the polypeptide of SEQ ID NO.2 aminoacid sequence.
Dna molecular among the present invention comprises 8-100 continuous nucleotide in the described dna molecular.In the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
Term among the present invention " salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme (or polypeptide) gene " refers to: coding has the nucleotide sequence of the polypeptide of salvia 3-hydroxy-3-methylglutaryl A synthase activity, as 118-1500 position nucleotide sequence among the SEQ ID NO.1.
Can also comprise its degenerate sequence, this degenerate sequence is meant, is arranged in the encoder block 118-1500 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 118-1500 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.Also comprising can be under the rigorous condition of moderate, better under highly rigorous condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 118-1500 position.Also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 118-1500 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.Also comprising to encode has the variant form of open reading frame sequence among the proteic SEQ ID NO.1 with natural salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
Term among the present invention " salvia 3-hydroxy-3-methylglutaryl A synthetase albumen or polypeptide " refers to: the SEQ ID NO.2 polypeptide of sequence with salvia 3-hydroxy-3-methylglutaryl A synthase activity.
Also comprise the variant form that has with the SEQ IDNO.2 sequence of natural salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme identical function.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme, also comprises operationally being connected in the derivative that signal peptide, promotor or ribosome bind site sequence are formed.
The variant form of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low rigorous condition can with the coded albumen of the DNA of salvia 3-hydroxy-3-methylglutaryl A synthetase dna hybridization and the polypeptide or the albumen that utilize the serum of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme polypeptide to obtain.
Salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme conservative property variation polypeptide refers among the present invention: compare with the aminoacid sequence of SEQ ID NO.2, there are 10 at the most, preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and are formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Replacement residue in the table 1. conservative property variation polypeptide
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Initial residue | Representational replacement | The preferred replacement |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Initial residue | Representational replacement | The preferred replacement |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also comprises the analogue of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme or polypeptide.The difference of these analogues and natural 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.Described modification (not changing primary structure usually) form comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its proteolysis performance or optimized solubility property by modifying.
In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme polypeptide of the present invention, the nucleotide sequence of salvia 3-hydroxy-3-methylglutaryl A synthase gene operationally can be connected in expression regulation sequence, thereby form salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme expression vector.Described " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
Host cell is prokaryotic cell prokaryocyte or eukaryotic cell among the present invention.Prokaryotic host cell commonly used comprises intestinal bacteria; Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.
Existence and the quantity of rna transcription thing in cell of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme is promptly analyzed in the expression of the also available Northern blotting of the present invention technical Analysis salvia 3-hydroxy-3-methylglutaryl A synthase gene product.
In addition, the nucleic acid molecule that can be used as probe among the present invention has 8-100 continuous nucleotide of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme.
The present invention relates to whether exist in the test sample method of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.In addition, according to salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme source gene or homologous protein.
In order to obtain the dot matrix of the red sage root cDNAs relevant with the salvia 3-hydroxy-3-methylglutaryl A synthase gene, can screen red sage root cDNA library with dna probe, these probes are under low rigorous condition, with 32P salvia 3-hydroxy-3-methylglutaryl A synthase gene all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from the red sage root.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be discerned the nucleotide sequence with the gene family of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme.
Salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco by direct peptide synthesis; MerrifieldJ. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.Utilize salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme of the present invention,, can filter out with the salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme interactional material takes place, perhaps acceptor, inhibitor or short of money dose etc. by various conventional screening methods.
3-hydroxy-3-methylglutaryl-coenzyme A synthase gene provided by the invention is to clone preparation first from the red sage root, has filled up the blank that clones and isolates the 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme from China's medicine plant red sage root.Can be used for improving the content of terpene activeconstituents TANSHINONES in the plants such as the red sage root by genetic engineering technique, transgene result shows, the salvia 3-hydroxy-3-methylglutaryl A synthase gene has obvious effect to the raising that promotes TANSHINONES content, the salvia 3-hydroxy-3-methylglutaryl A synthase gene can improve in TANSHINONES Study on content and the industrialization by transgenic technology, especially the quality-improving that can be used for the Chinese medicinal materials red sage root, have promoter action preferably for alleviating the serious deficient problem in TANSHINONES medicine source, so the present invention has good application prospects.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The clone of embodiment 1 salvia 3-hydroxy-3-methylglutaryl A synthase gene
1. separate tissue (isolation)
Red sage root plant derives from Sichuan, takes the children to place the freezing preservation of liquid nitrogen immediately after tender.
2.RNA separation (RNA isolation)
Get portion of tissue and grind, add the 1.5mL EP pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to after the homogenate in the 1.5mL EP pipe, and extracted total RNA (TRIzol Reagents, GIBCO BRL, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
3. the full-length clone of gene (Cloning of Full-length cDNA)
According to paragutta, Ramulus et folium taxi cuspidatae and other have been cloned the 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme amino acid conserved sequence that obtains, the design degenerated primer, utilize homologous genes clone principle, adopt Smart-RACE method (Clonetech test kit) to carry out the cDNA full-length clone, divide three phases to carry out:
(1)3’-RACE
PCR (UPM+F2) obtains DsHMGS F2 ' (746bp), reclaim, be connected on the T-Easy carrier, with SP6 or T7 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsingroup, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the result shows that the homology of its nucleotide sequence and proteins encoded and known 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene (as rubber tree 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene etc.) is very high, so think that tentatively it is a 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene.
(2)5’-RACE
According to 3 ' RACE result, design reverse special primer R2, obtain DsHMGS R2 ' (896bp) (process is with (1)) through PCR (UPM+R2).Reclaim, be connected on the T-Easy carrier, with SP6 or T7 as universal primer, adopt stop the thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA), (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result and 3 ' RACE result than preface and splice, are obtained the full length fragment sequence.
(3) with 5 ' RACE sequencing result and 3 ' RACE sequencing result than preface and splice, obtain the full length fragment sequence information, and design a pair of special primer DsHMGSKF1:5 '-ATGGCCAAGAATGTCGGG-3 ' (SEQ ID NO.3) and 5 '-TCAGTGGCCGTTCGCAA-3 ' DsHMGSKR1 (SEQ ID NO.4)) carry out pcr amplification DsHMGS coding region and obtain DsHMGS coding region (1383bp) (the same step of process (1)).
The gene that result's proof of BLAST newly obtains from the red sage root really is a 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene.Studies show that the 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme is an important metabolic enzyme in the MVA approach, have similar function so infer the gene of new clone.
By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of red sage root DsHMGS.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further DsHMGSF1:5 '-ACACCATTCAGCCACCCATTCT-3 ' is a forward primer to the design primer, oligonucleotide DsHMGSR1:5 '-CGATAAAGAAACTACAGATAAT-3 ' is a reverse primer, with total RNA is template, carry out the RT-PCR amplification, the PCR condition of F1/R2 be 94 ℃ 5 minutes, carried out 35 circulations in 2 minutes with 94 ℃ 1 minute, 60 ℃ 1 minute and 72 ℃ thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 1097bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ ID NO.1.
The sequence information and the homology analysis of embodiment 2 salvia 3-hydroxy-3-methylglutaryl A synthase genes
The length of the salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme full-length cDNA that the present invention is new is 1655bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 118-1500 position Nucleotide.Derive the aminoacid sequence of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme according to full-length cDNA, totally 460 amino-acid residues, molecular weight 50.6KD, pI are 6.04, detailed sequence is seen SEQ ID NO.2.
The full length cDNA sequence and the coded protein thereof of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that it and Para rubber tree gene (GenBank Accession No.AB294689) have 79% homology (seeing Table 2); On amino acid levels, the 1-460 amino acids residue of it and short and small Herba ophiorrhizae japonicae HMGS (GenBank Accession No.BAF98279) has 83% homogeny and 91% similarity (seeing Table 3).Therefore all there are higher homology in salvia 3-hydroxy-3-methylglutaryl A synthase gene and Para rubber tree 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene on nucleic acid still is protein level.So can think and have remarkable promoter action on the content of salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme TANSHINONES in improving resource plant.
The homology of the nucleotide sequence of table 2. red sage root DsHMGS of the present invention and Para rubber tree (Hevea brasiliensis) HbHMGS is (GAP) table relatively
Query 118 ATGGCCAAGAATGTCGGG-ATCCTCGCCATGGAAATCTACTTCCCTCCCA-CT-TGCATC 174
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Sbjct 302 ATGGCAAAGAATGT-GGGAATTCTCGCTATGGACATCTATTTCCCTCCTACCTATG--TT 358
Query 175 CAGCAGGAGGTATTGGAAGCTCACGATGGAGCAAGCAAAGGGAAGTACACAATTGGGCTT 234
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Sbjct 359 CAGCAGGAAGCATTGGAGGCTCATGATGGTGCAAGCAAAGGGAAGTATACCATTGGTCTT 418
Query 235 GGCCAAGATTGCATGGCATTTTGTTCGGAGGTTGAAGATGTCATTTCGATGAGCATGACA 294
|| || ||||||||| |||||||| | ||||| ||||||||||| || ||||| ||||
Sbjct 419 GGACAGGATTGCATGCCATTTTGTACTGAGGTGGAAGATGTCATCTCAATGAGTTTGACT 478
Query 295 GCGGTTACTTCGCTTCTAGGGAAGTACAATGTTGATCCGAAGCAGATTGGACGTCTTGAA 354
|| |||||||| || || | ||||| ||| ||||||| || || || || ||||| |||
Sbjct 479 GCAGTTACTTCACTCCTCGACAAGTATAATATTGATCCTAAACAAATCGGTCGTCTGGAA 538
Query 355 GTTGGAAGTGAGACCGT-ACTCGACAAGAGCAAATCCATTAAGACATTTC-TGATGCCGA 412
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Sbjct 539 GTTGGCAGTGAGACTGTGA-TCGACAAGAGCAAATCTATTAAAAC-CTTCTTGATGCAAA 596
Query 413 TCTTCGAGAAATGT-GGCAATATTGACATCGAAGGTGTTGACTCAAGC-AATGCTTGCTA 470
| |||||||||| | || || | |||||| ||||| |||||||||| | ||||| || ||
Sbjct 597 TTTTCGAGAAAT-TCGGAAACACTGACATTGAAGGCGTTGACTCAA-CAAATGCATGTTA 654
Query 471 TGGTGGGACTGCAGCACTATTTAACTGTGTCAATTGGGTGGAAAGTAGTTCTTGGGATGG 530
||| ||||||||||| |||| ||||||||||||||||| || || ||||| ||||||||
Sbjct 655 TGGGGGGACTGCAGCTTTATTCAACTGTGTCAATTGGGTTGAGAGCAGTTCATGGGATGG 714
Query 531 AAGATATGGGCTTGTTGTCTGCACAGACAGTGCGGTATATGCCGAGGGACCAGCTAGACC 590
| | ||||| ||||| || || || ||||||||||| ||||| ||||| ||||| ||||
Sbjct 715 ACGCTATGGACTTGTAGTGTGTACTGACAGTGCGGTCTATGCAGAGGGTCCAGCCCGACC 774
Query 591 TACTGGTGGGGCTGCAGCTATTGCCATGCTAATAGGACCAAATGCACCCATTGCTTTTGA 650
||||| || |||||||| ||||||||||||||||| ||| | ||||| |||||||||||
Sbjct 775 AACTGGAGGAGCTGCAGCCATTGCCATGCTAATAGGTCCAGAGGCACCTATTGCTTTTGA 834
Query 651 AAGCAAGCTTAGGGCGAATCACATGGCTCATGTTTATGATTTTTACAAGCCCGACCTTGC 710
|||||| |||||| || || ||| |||||| ||| ||||||||||||||| |||| ||
Sbjct 835 AAGCAAATTTAGGGGGAGCCATATGTCTCATGCTTACGATTTTTACAAGCCCAACCTGGC 894
Query 711 CAGTGAATATCCAGTTGTTGATGGCAAGCTTTCTCAGACTTGTTGCCTTACGGCACTGGA 770
|| |||||||||||||| || ||||| |||||||| || || | ||| | ||| || ||
Sbjct 895 TAGCGAATATCCAGTTGTGGACGGCAAACTTTCTCAAACATGCTACCTCATGGCTCTTGA 954
Query 771 TGCTTGTTACAAAGGCTTC--TGCCAAAAGTTTGAGAAGCAGGAGGGCAAGCAGTTCTCG 828
| | || |||||| ||| |||| |||| |||||| ||||||||| || ||||||
Sbjct 955 TTCCTGCTACAAACATTTCTGTGCC--AAGTATGAGAAATTGGAGGGCAAACAATTCTCT 1012
Query 829 ATCTT-GGATGCCGACTACTTTGTATTTCATTCTCCATACAACAAGCTTGTACAGAAAAG 887
|| || ||||| || || ||||||||||| ||||| |||||||||||||||||||||||
Sbjct 1013 AT-TTCTGATGCTGAATATTTTGTATTTCACTCTCCTTACAACAAGCTTGTACAGAAAAG 1071
Query 888 CTTCT-CTAGATTGTTGTTCAATGACTTT-TCGAGAAATGCCAGCTCCATCGATGAGGCT 945
||| | || | ||| |||||||||||||| | ||| ||||||||||| || ||||| |||
Sbjct 1072 CTT-TGCTCGTTTGGTGTTCAATGACTTTGT-GAGGAATGCCAGCTCTATTGATGACGCT 1129
Query 946 GCTAAAGAAAAGCTGGCACCATTTTCATCATTAAGCAAC-GAGGAAAGCTACCAAAGTCG 1004
||||||||||||||||||||||||||| | ||| | || ||||||||||||| ||
Sbjct 1130 GCTAAAGAAAAGCTGGCACCATTTTCAACTTTAT-CTGGTGATGAAAGCTACCAAAACCG 1188
Query 1005 TGATCTTGAGAAGGCATCTCAACAAGTCGCTAAACCTTTCTT-TGATACCAAGGTGCAAC 1063
|||||||| |||| || |||||||| || || || | || |||| | ||||||||||
Sbjct 1189 GGATCTTGAAAAGGTGTCCCAACAAGTTGCCAAGCCCCT-TTATGATGCAAAGGTGCAAC 1247
Query 1064 CATCTACTCTCG-TCCCAAAACAAGTGGGAAACATGTATACTGCATCGCTCTATGCTGCA 1122
|| | ||| | | | ||||| ||||| || || ||||| |||||||| | ||||| |||
Sbjct 1248 CAACCACTTT-GATACCAAAGCAAGTTGGCAATATGTACACTGCATCTTTGTATGCAGCA 1306
Query 1123 TTTGCTTCCCT-CATCCACAACAAGAGTAGCACT---TTGGCTGGGCAGAGGGTCATATT 1178
|| || ||||| | || |||| || | | |||| ||||| || || |||| ||| |
Sbjct 1307 TTCGCATCCCTACTTC-ACAATAA-AC-A-CACTGAATTGGCAGGTAAGCGGGTGATACT 1362
Query 1179 GTTTTCCTACGGCAGTGGTCTGTCAGCCATC-ATGTTCTCTCTTC-GTTT-CACTGAGGG 1235
|| || || || |||||| || |||||| | |||||||| || | | || ||| ||
Sbjct 1363 ATTCTCATATGGGAGTGGTTTGACAGCCA-CGATGTTCTCA-TTGAGACTACA-TGAAGG 1419
Query 1236 AGAACATCC-TTTCAGCCTGTCCAACATTGCATCTGTTATGAATGTTTCAGAGAA-GTTG 1293
||||||| ||| ||| |||| ||||||||| |||| ||||||||| ||| ||| ||||
Sbjct 1420 CCAACATCCCTTT-AGCTTGTCAAACATTGCAACTGTGATGAATGTTGCAG-GAAAGTTG 1477
Query 1294 AAGTCAAGGCACGAGTTCCCACCAGAGAAATTCGTC-GA-ACTGATGCAGCTCATGGAGC 1351
||| |||| ||||||||||| |||||||| || | | | | | ||| |||| |||||||
Sbjct 1478 AAGACAAGACACGAGTTCCCCCCAGAGAAGTTTG-CAGTTA-TCATGAAGCTAATGGAGC 1535
Query 1352 A-CAGATATGGAGGC-AAAGACTTCATCACAAGCAAGGACTGCAGCCTTCTTG-CACCAG 1408
| | |||| || ||| |||||||| | ||||||||||| |||||| | |||| | || |
Sbjct 1536 ATC-GATACGG-GGCTAAAGACTTTGTGACAAGCAAGGATTGCAGCAT-CTTGGCGCCTG 1592
Query 1409 GTACATACTATCTCACTGAAGTCGACTCCCAA-TACCGAAGATTCTATGCCAAGAAGGC 1466
| |||||||||||||| ||||| ||| || | || ||||||||||||||| |||||||
Sbict 1593 GAACATACTATCTCACAGAAGTTGACACC-ATGTATCGAAGATTCTATGCCCAGAAGGC 1650
Wherein: Query represents the nucleotide sequence of red sage root DsHMGS; Subject represents the nucleotide sequence (GenBank Accession No.AB294689) of short and small Herba ophiorrhizae japonicae HbHMGS2.
The result: both have 79% similarity in the comparison of 1250 Nucleotide.
The homology of table 3. red sage root DsHMGS of the present invention and Para rubber tree HbHMGS aminoacid sequence is (FASTA) table relatively
Query 1 MAKNVGILAMEIYFPPTCIQQEVLEAHDGASKGKYTIGLGQDCMAFCSEVEDVISMSMTA 60
MAKNVGILAM+IYFPPT +QQE LEAHDGASKGKYTIGLGQDCM FC+EVEDVISMS+TA
Sbjct 1 MAKNVGILAMDIYFPPTYVQQEALEAHDGASKGKYTIGLGQDCMPFCTEVEDVISMSLTA 60
Query 61 VTSLLGKYNVDPKQIGRLEVGSETVLDKSKSIKTFLMPIFEKCGNIDIEGVDSSNACYGG 120
VTSLL KYN+DPKQIGRLEVGSETV+DKSKSIKTFLM IFEK GN DIEGVDS+NACYGG
Sbjct 61 VTSLLDKYNIDPKQIGRLEVGSETVIDKSKSIKTFLMQIFEKFGNTDIEGVDSTNACYGG 120
Query 121 TAALFNCVNWVESSSWDGRYGLVVCTDSAVYAEGPARPTGGAAAIAMLIGPNAPIAFESK 180
TAALFNCVNWVESSSWDGRYGLVVCTDSAVYAEGPARPTGGAAAIAMLIGP APIAFESK
Sbjct 121 TAALFNCVNWVESSSWDGRYGLVVCTDSAVYAEGPARPTGGAAAIAMLIGPEAPIAFESK 180
Query 181 LRANHMAHVYDFYKPDLASEYPVVDGKLSQTCCLTALDACYKGFCQKFEKQEGKQFSILD 240
R +HM+H YDFYKP+LASEYPVVDGKLSQTC L ALD+CYK FC K+EK EGKQFSI D
Sbjct 181 FRGSHMSHAYDFYKPNLASEYPVVDGKLSQTCYLMALDSCYKHFCAKYEKLEGKQFSISD 240
Query 241 ADYFVFHSPYNKLVQKSFSRLLFNDFSRNASSIDEAAKEKLAPFSSLSNEESYQSRDLEK 300
A+YFVFHSPYNKLVQKSF+RL+FNDF RNASSID+AAKEKLAPFS+LS +ESYQ+RDLEK
Sbjct 241 AEYFVFHSPYNKLVQKSFARLVFNDFVRNASSIDDAAKEKLAPFSTLSGDESYQNRDLEK 300
Query 301 ASQQVAKPFFDTKVQPSTLVPKQVGNMYTASLYAAFASLIHNKSSTLAGQRVILFSYGSG 360
SQQVAKP +D KVQP+TL+PKQVGNMYTASLYAAFASL+HNK + LAG+RVILFSYGSG
Sbjct 301 VSQQVAKPLYDAKVQPTTLIPKQVGNMYTASLYAAFASLLHNKHTELAGKRVILFSYGSG 360
Query 361 LSAIMFSLRFTEGEHPFSLSNIASVMNVSEKLKSRHEFPPEKFVELMQLMEHRYGGKDFI 420
L+A MFSLR EG+HPFSLSNIA+VMNV+ KLK+RHEFPPEKF +M+LMEHRYG KDF+
Sbjct 361 LTATMFSLRLHEGQHPFSLSNIATVMNVAGKLKTRHEFPPEKFAVIMKLMEHRYGAKDFV 420
Query 421 TSKDCSLLAPGTYYLTEVDSQYRRFYAKKAIA----NGTVANGH 460
TSKDCS+LAPGTYYLTEVD+YRRFYA+KA+ NG +ANGH
Sbjct 421 TSKDCSILAPGTYYLTEVDTMYRRFYAQKAVGDTVENGLLANGH 464
Wherein: Query represents the aminoacid sequence of red sage root DsHMGS; Subject represents the aminoacid sequence (GenBank Accession No.BAF98279) of Para rubber tree HbHMGS; Identical amino acid marks with the amino acid monocase between two sequences.
The result: in 460 amino acid whose comparisons, both have 83% homogeny and 91% similarity respectively.
Embodiment 3 salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme or polypeptide carry out prokaryotic expression and purification in intestinal bacteria
In this embodiment, the red sage root DsHMGS encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.
1, construction of prokaryotic expression vector and transformed into escherichia coli
According to the nucleotide sequence of red sage root DsHMGS, design amplifies the primer of protein-coding region, and introduces restriction endonuclease sites (this decides according to pET32a (+) carrier of selecting for use) on positive anti-primer respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, red sage root DsHMGS gene is being guaranteed to be cloned into pET32a (+) carrier (Novagen) under the correct prerequisite of reading frame.Identify that good expression vector utilizes CaCl
2Method changes e. coli bl21 over to, and Screening and Identification obtains containing engineering bacteria BL21-pET32a (+)-DsHMGS of pET32a (+)-DsHMGS expression vector.
2, express the isolation identification of the engineering bacteria of Trx-DsHMGS recombinant protein
The BL21-pET32a (+) of picking list bacterium colony-DsHMGS engineering bacteria contains jolting overnight incubation in the LB substratum of 100 μ g/mL penbritins in 3mL, draw nutrient solution by the concentration of 1:100 and in new LB substratum (containing 100 μ g/mL penbritins), cultivated about 3 hours, to OD
600After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivated respectively 0,1,2,3 hours.It is centrifugal to get the different 1mL bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ L, distilled water 45 μ L, 3-mercaptoethanol 5 μ L), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the Trx-DsHMGS fusion rotein.
3, the extraction purifying of Trx-DsHMGS fusion rotein
The proteic engineering bacteria BL21-pET32a of abduction delivering Trx-DsHMGS amalgamation and expression (+)-DsHMGS as stated above, collect thalline through centrifugation, and come the purifying inclusion body with BugBuster reagent and Benzonase nuclease according to the specification sheets of producer (Novagen).Inclusion body can with the dissolving damping fluid (50mM CAPS, pH 11.0,0.3%N-lauroylsarcosine) dissolve, (200mM Tris-HCl pH8.5) dialyses to use dialysis buffer liquid again.Use Histidine to carry out affinity chromatography then, and collect the Trx-DsHMGS fusion rotein through elution buffer (1M imidazole, 500mMNaCl, 20mM Tris-HCl pH 7.9) wash-out in conjunction with (HisBind) resin.Fusion rotein is the expressing protein of the separable DsHMGS that obtains after 20 ℃ of enzymes of enteropeptidase are cut 16 hours.The molecular weight 50.6KD of this expressing protein, pI are 6.04.
4, the vitality test of the 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme of purifying
Press (Plant science such as Suwanmanee, 2004,166:531～537) method is carried out the mensuration of enzyme activity to the 3-hydroxy-3-methylglutaryl-coenzyme A synthetase albumen of expression and purification, studies the influence that it generates 3-hydroxy-3-methylglutaryl-coenzyme A.Reaction system contains 0.1M Tris-HCL (pH=8.0), the 0.2mM acetyl-CoA, and the 0.05mM acetoacetyl-CoA, 0.1mM EDTA and 10ul zymoprotein sample, cumulative volume is that the 100ul. reaction process is as follows: at first will
14The acetyl-CoA of C mark joins acyl group (the 67dps nmol that does not have mark
-1) in be diluted to mixture, 30 ℃ of pre-cultivations 2 minutes.After adding acetyl-CoA 2 and 4 minutes, the mixture of getting 40 microlitres of equivalent are transferred in the vial and are added the 6M HCL of 100 microlitres, then 95 ℃ of dryings.In the process of oven dry, thioesters is by hydroxylation, has neither part nor lot in reaction
14The ethanoyl of C mark has been evaporated, and has only
14The HMG acid of C mark still is retained in the bottle, adds the water of 500 microlitres, measures with the liquid scintillation register
14The content of the 3-hydroxy-3-methylglutaryl-coenzyme A of C mark.Do not remove clean in order to calculate at said procedure
14The residual volume of the acetyl-CoA of C mark has designed a parallel control in the experiment.The result shows that expressed proteins has the enzymic activity of catalysis acetyl-CoA and acetoacetyl-CoA generation 3-hydroxy-3-methylglutaryl-coenzyme A really.
Embodiment 4 salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme or polypeptide carry out TANSHINONES assay in eukaryotic cell expression and the transgenosis root of hair in the red sage root
The structure that contains the expression vector of goal gene (salvia 3-hydroxy-3-methylglutaryl A synthase gene), full length sequence (SEQ ID NO.1) according to the salvia 3-hydroxy-3-methylglutaryl A synthetic enzyme, design amplifies the primer that complete coding is read frame, and on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, salvia 3-hydroxy-3-methylglutaryl A synthase gene cDNA is cloned into binary expression vector (as pCAMBIA1304), guaranteeing to identify good expression vector under the correct prerequisite of reading frame, again it is changed in the Agrobacterium over to the genetic transformation resource plant red sage root:
1) get Agrobacterium rhizogenes A4, take out from refrigerator before using, go down to posterity 2 times, going down to posterity with solid medium is the YEB substratum.Bacterial classification is inoculated in the YEB liquid nutrient medium before use, 28 ℃ of overnight incubation.
2) get the aseptic leaflet tablet of the red sage root of growing about 8 weeks.
3) the Agrobacterium rhizogenes A4 bacterium liquid through spending the night and cultivating is 100 bacterium/mL with the conversion fluid dilution.Get aseptic red sage root blade, draw with "+" font wound with aseptic scalper, put into above-mentioned conversion, 60rpm/min shaking culture 8h takes out, with aseptic water washing 3 times, put into the B5 medium that contains 250-500mg/L kantlex and different concns 6-BA (0.5mg/L-3mg/L), transfer in per 2 weeks in the fresh culture 1 time, separate hairly root after waiting to grow hairly root, be transferred in the B5 medium that contains the 250-500mg/L kantlex and do not have hormone and cultivate, shift 4-5 time till no bacterium, and then be transferred in the no hormone B5 medium that does not contain kantlex and cultivate.
4) the secondary culture of the hairly root in solid medium, be inoculated in and 100mL is housed does not have hormone B5, in the 500mL triangular flask of substratum, culture condition such as culture temperature, illumination, rotating speed are identical with callus fluid suspension culture condition, cultivated 20 days, hairly root taken out from substratum puts into freeze drier and carry out drying, weigh then, be stored in-70 ℃ standby.
5) contain the TANSHINONES assay of the transgenosis root of hair of salvia 3-hydroxy-3-methylglutaryl A synthase gene
Press Shi etc. (Journal of Pharmaceutical and Biomedical Analysis, 2005, method 37:481-486) is carried out the TANSHINONES assay to the transgenosis root of hair of expressing the salvia 3-hydroxy-3-methylglutaryl A synthase gene.The result shows that TANSHINONES content improves 1.2 times (P＜0.05) than non-transgenic control group in the transgenosis root of hair of expressing the salvia 3-hydroxy-3-methylglutaryl A synthase gene.Therefore transgene result proves, the salvia 3-hydroxy-3-methylglutaryl A synthase gene has obvious effect to the raising that promotes TANSHINONES content, the salvia 3-hydroxy-3-methylglutaryl A synthase gene can be used for utilizing transgenic technology to improve in TANSHINONES Study on content and the industrialization, has good application prospects.
The nucleotides sequence tabulation
SEQUENCE LISTING
＜110〉Shanghai Normal University
＜120〉salvia 3-hydroxy-3-methylglutaryl A synthase gene and encoded protein matter and application
<160>4
<170>PatentIn version 3.3
<210>1
<211>1655
<212>DNA
＜213〉red sage root (Salvia miltiorrhiza Bunge)
<220>
<221>CDS
<222>(118)..(1500)
<223>
<400>1
acaccattca gccacccatt ctctctctac ctctctgatt cctgagctct gcgactactg 60
tgtgtgctgt ttaactgatc cgctatttcc ttcatttcgt caactcaaga agtcgag 117
atg gcc aag aat gtc ggg atc ctc gcc atg gaa atc tac ttc cct ccc 165
Met Ala Lys Asn Val Gly Ile Leu Ala Met Glu Ile Tyr Phe Pro Pro
1 5 10 15
act tgc atc cag cag gag gta ttg gaa gct cac gat gga gca agc aaa 213
Thr Cys Ile Gln Gln Glu Val Leu Glu Ala His Asp Gly Ala Ser Lys
20 25 30
ggg aag tac aca att ggg ctt ggc caa gat tgc atg gca ttt tgt tcg 261
Gly Lys Tyr Thr Ile Gly Leu Gly Gln Asp Cys Met Ala Phe Cys Ser
35 40 45
gag gtt gaa gat gtc att tcg atg agc atg aca gcg gtt act tcg ctt 309
Glu Val Glu Asp Val Ile Ser Met Ser Met Thr Ala Val Thr Ser Leu
50 55 60
cta ggg aag tac aat gtt gat ccg aag cag att gga cgt ctt gaa gtt 357
Leu Gly Lys Tyr Asn Val Asp Pro Lys Gln Ile Gly Arg Leu Glu Val
65 70 75 80
gga agt gag acc gta ctc gac aag agc aaa tcc att aag aca ttt ctg 405
Gly Ser Glu Thr Val Leu Asp Lys Ser Lys Ser Ile Lys Thr Phe Leu
85 90 95
atg ccg atc ttc gag aaa tgt ggc aat att gac atc gaa ggt gtt gac 453
Met Pro Ile Phe G1u Lys Cys Gly Asn Ile Asp Ile Glu Gly Val Asp
100 105 110
tca agc aat gct tgc tat ggt ggg act gca gca cta ttt aac tgt gtc 501
Ser Ser Asn Ala Cys Tyr Gly Gly Thr Ala Ala Leu Phe Asn Cys Val
115 120 125
aat tgg gtg gaa agt agt tct tgg gat gga aga tat ggg ctt gtt gtc 549
Asn Trp Val Glu Ser Ser Ser Trp Asp Gly Arg Tyr Gly Leu Val Val
130 135 140
tgc aca gac agt gcg gta tat gcc gag gga cca gct aga cct act ggt 597
Cys Thr Asp Ser Ala Val Tyr Ala Glu Gly Pro Ala Arg Pro Thr Gly
145 150 155 160
ggg gct gca gct att gcc atg cta ata gga cca aat gca ccc att gct 645
Gly Ala Ala Ala Ile Ala Met Leu Ile Gly Pro Asn Ala Pro Ile Ala
165 170 175
ttt gaa agc aag ctt agg gcg aat cac atg gct cat gtt tat gat ttt 693
Phe Glu Ser Lys Leu Arg Ala Asn His Met Ala His Val Tyr Asp Phe
180 185 190
tac aag ccc gac ctt gcc agt gaa tat cca gtt gtt gat ggc aag ctt 741
Tyr Lys Pro Asp Leu Ala Ser Glu Tyr Pro Val Val Asp Gly Lys Leu
195 200 205
tct cag act tgt tgc ctt acg gca ctg gat gct tgt tac aaa ggc ttc 789
Ser Gln Thr Cys Cys Leu Thr Ala Leu Asp Ala Cys Tyr Lys Gly Phe
210 215 220
tgc caa aag ttt gag aag cag gag ggc aag cag ttc tcg atc ttg gat 837
Cys Gln Lys Phe Glu Lys Gln Glu Gly Lys Gln Phe Ser Ile Leu Asp
225 230 235 240
gcc gac tac ttt gta ttt cat tct cca tac aac aag ctt gta cag aaa 885
Ala Asp Tyr Phe Val Phe His Ser Pro Tyr Asn Lys Leu Val Gln Lys
245 250 255
agc ttc tct aga ttg ttg ttc aat gac ttt tcg aga aat gcc agc tcc 933
Ser Phe Ser Arg Leu Leu Phe Asn Asp Phe Ser Arg Asn Ala Ser Ser
260 265 270
atc gat gag gct gct aaa gaa aag ctg gca cca ttt tca tca tta agc 981
Ile Asp Glu Ala Ala Lys Glu Lys Leu Ala Pro Phe Ser Ser Leu Ser
275 280 285
aac gag gaa agc tac caa agt cgt gat ctt gag aag gca tct caa caa 1029
Asn Glu Glu Ser Tyr Gln Ser Arg Asp Leu Glu Lys Ala Ser Gln Gln
290 295 300
gtc gct aaa cct ttc ttt gat acc aag gtg caa cca tct act ctc gtc 1077
Val Ala Lys Pro Phe Phe Asp Thr Lys Val Gln Pro Ser Thr Leu Val
305 310 315 320
cca aaa caa gtg gga aac atg tat act gca tcg ctc tat gct gca ttt 1125
Pro Lys Gln Val Gly Asn Met Tyr Thr Ala Ser Leu Tyr Ala Ala Phe
325 330 335
gct tcc ctc atc cac aac aag agt agc act ttg gct ggg cag agg gtc 1173
Ala Ser Leu Ile His Asn Lys Ser Ser Thr Leu Ala Gly Gln Arg Val
340 345 350
ata ttg ttt tcc tac ggc agt ggt ctg tca gcc atc atg ttc tct ctt 1221
Ile Leu Phe Ser Tyr Gly Ser Gly Leu Ser Ala Ile Met Phe Ser Leu
355 360 365
cgt ttc act gag gga gaa cat cct ttc agc ctg tcc aac att gca tct 1269
Arg Phe Thr Glu Gly Glu His Pro Phe Ser Leu Ser Asn Ile Ala Ser
370 375 380
gtt atg aat gtt tca gag aag ttg aag tca agg cac gag ttc cca cca 1317
Val Met Asn Val Ser Glu Lys Leu Lys Ser Arg His Glu Phe Pro Pro
385 390 395 400
gag aaa ttc gtc gaa ctg atg cag ctc atg gag cac aga tat gga ggc 1365
Glu Lys Phe Val Glu Leu Met Gln Leu Met Glu His Arg Tyr Gly Gly
405 410 415
aaa gac ttc atc aca agc aag gac tgc agc ctt ctt gca cca ggt aca 1413
Lys Asp Phe Ile Thr Ser Lys Asp Cys Ser Leu Leu Ala Pro Gly Thr
420 425 430
tac tat ctc act gaa gtc gac tcc caa tac cga aga ttc tat gcc aag 1460
Tyr Tyr Leu Thr Glu Val Asp Ser Gln Tyr Arg Arg Phe Tyr Ala Lys
435 440 445
aag gcc att gcg aat ggc aca gtt gcg aac ggc cac tga agatgtgata 1510
Lys Ala Ile Ala Asn Gly Thr Val Ala Asn Gly His
450 455 460
tgccataaag ttggctcttc ttctgtctag ttgattcagc acatagaaaa ataatctaat 1570
ctattttcat tgtttgatac aagcatttga gttggtttga cagatgaatt attatctgta 1630
gtttctttat cgaaaaaaaa aaaaa 1655
<210>2
<211>460
<212>PRT
＜213〉red sage root (Salvia miltiorrhiza Bunge)
<400>2
Met Ala Lys Asn Val Gly Ile Leu Ala Met Glu Ile Tyr Phe Pro Pro
1 5 10 15
Thr Cys Ile Gln Gln Glu Val Leu Glu Ala His Asp Gly Ala Ser Lys
20 25 30
Gly Lys Tyr Thr Ile Gly Leu Gly Gln Asp Cys Met Ala Phe Cys Ser
35 40 45
Glu Val Glu Asp Val Ile Ser Met Ser Met Thr Ala Val Thr Ser Leu
50 55 60
Leu Gly Lys Tyr Asn Val Asp Pro Lys Gln Ile Gly Arg Leu Glu Val
65 70 75 80
Gly Ser Glu Thr Val Leu Asp Lys Ser Lys Ser Ile Lys Thr Phe Leu
85 90 95
Met Pro Ile Phe Glu Lys Cys Gly Asn Ile Asp Ile Glu Gly Val Asp
100 105 110
Ser Ser Asn Ala Cys Tyr Gly Gly Thr Ala Ala Leu Phe Asn Cys Val
115 120 125
Asn Trp Val Glu Ser Ser Ser Trp Asp Gly Arg Tyr Gly Leu Val Val
130 135 140
Cys Thr Asp Ser Ala Val Tyr Ala Glu Gly Pro Ala Arg Pro Thr Gly
145 150 155 160
Gly Ala Ala Ala Ile Ala Met Leu Ile Gly Pro Asn Ala Pro Ile Ala
165 170 175
Phe Glu Ser Lys Leu Arg Ala Asn His Met Ala His Val Tyr Asp Phe
180 185 190
Tyr Lys Pro Asp Leu Ala Ser Glu Tyr Pro Val Val Asp Gly Lys Leu
195 200 205
Ser Gln Thr Cys Cys Leu Thr Ala Leu Asp Ala Cys Tyr Lys Gly Phe
210 215 220
Cys Gln Lys Phe Glu Lys Gln Glu Gly Lys Gln Phe Ser Ile Leu Asp
225 230 235 240
Ala Asp Tyr Phe Val Phe His Ser Pro Tyr Asn Lys Leu Val Gln Lys
245 250 255
Ser Phe Ser Arg Leu Leu Phe Asn Asp Phe Ser Arg Asn Ala Ser Ser
260 265 270
Ile Asp Glu Ala Ala Lys Glu Lys Leu Ala Pro Phe Ser Ser Leu Ser
275 280 285
Asn Glu Glu Ser Tyr Gln Ser Arg Asp Leu Glu Lys Ala Ser Gln Gln
290 295 300
Val Ala Lys Pro Phe Phe Asp Thr Lys Val Gln Pro Ser Thr Leu Val
305 310 315 320
Pro Lys Gln Val Gly Asn Met Tyr Thr Ala Ser Leu Tyr Ala Ala Phe
325 330 335
Ala Ser Leu Ile His Asn Lys Ser Ser Thr Leu Ala Gly Gln Arg Val
340 345 350
Ile Leu Phe Ser Tyr Gly Ser Gly Leu Ser Ala Ile Met Phe Ser Leu
355 360 365
Arg Phe Thr Glu Gly Glu His Pro Phe Ser Leu Ser Asn Ile Ala Ser
370 375 380
Val Met Asn Val Ser Glu Lys Leu Lys Ser Arg His Glu Phe Pro Pro
385 390 395 400
Glu Lys Phe Val Glu Leu Met Gln Leu Met Glu His Arg Tyr Gly Gly
405 410 415
Lys Asp Phe Ile Thr Ser Lys Asp Cys Ser Leu Leu Ala Pro Gly Thr
420 425 430
Tyr Tyr Leu Thr Glu Val Asp Ser Gln Tyr Arg Arg Phe Tyr Ala Lys
435 440 445
Lys Ala Ile Ala Asn Gly Thr Val Ala Asn Gly His
450 455 460
<210>3
<211>18
<212>DNA
＜213〉red sage root (Salvia miltiorrhiza Bunge)
<400>3
atggccaag aatgtcggg
<210>4
<211>17
<212>DNA
＜213〉red sage root (Salvia miltiorrhiza Bunge)
<400>4
tcagtggc cgttcgcaa
Claims (7)
1. a salvia 3-hydroxy-3-methylglutaryl A synthase gene is characterized in that, the nucleotide sequence of described gene is shown in the 118th～1500 of SEQ ID No.1.
2. a salvia 3-hydroxy-3-methylglutaryl A synthase gene encoded protein matter is characterized in that described proteinic aminoacid sequence is shown in SEQ ID No.2.
3. a recombinant vectors is characterized in that, the complete coding that contains the described salvia 3-hydroxy-3-methylglutaryl A synthase gene of claim 1 is read frame.
4. a host cell is characterized in that, the complete coding that contains the described salvia 3-hydroxy-3-methylglutaryl A synthase gene of claim 1 is read frame.
5. host cell according to claim 4 is characterized in that, described host cell is Bacillus coli cells, agrobatcerium cell, yeast cell, tobacco cell, red sage root root of hair cell or red sage root cell.
6. the described salvia 3-hydroxy-3-methylglutaryl A synthase gene of claim 1 is applied to prepare transgenosis red sage root plant.
7. the described salvia 3-hydroxy-3-methylglutaryl A synthase gene of claim 1 is applied to prepare transgenosis red sage root root of hair system.
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| CN103710315B (en) * | 2012-10-30 | 2016-03-23 | 浙江工业大学 | From the long-chain-acyl-CoA synthetase of Cordyceps sinensis, gene and application |
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| Pluang Suwanmanee et al..Molecular Cloning and Sequencing of a cDNA Encoding 3-Hydroxy -3-Methylglutaryl Coenzyme A Synthase from Heveabrasiliensis (HBK ) Mull Arg.ScienceAsia28.2002,2829-36. |
| Pluang Suwanmanee et al..Molecular Cloning and Sequencing of a cDNA Encoding 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase from Heveabrasiliensis(HBK)Mull Arg.ScienceAsia28.2002,2829-36. * |
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