CN101250544A - Salvia 3-hydroxy-3-methylglutaryl A reductase gene and its coding protein and application - Google Patents
Salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase gene, protein which is encoded by the salvia 3-hydroxy-3-methylglutaryl coenzyme A reductase gene and the use thereof, which fills a gap that the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene is separated and cloned from salvia which is precious traditional Chinese medicine in China. The 3-hydroxy-3-methylglutaryl coenzyme A reductase gene which is provided by the invention has a nucleotide sequence or a homologous sequence which adds, replaces, inserts or losses one or a plurality of nucleotides or allele thereof and the nucleotide sequence which is derived from the 3-hydroxy-3-methylglutaryl coenzyme A reductase 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 reductase gene which is provided by the invention can increase the content of tanshinone which is a terpenes active component in the salvia 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 improving the quality of salvia mi ltiorrhiza and has very good application prospect.
Description
Technical field
The invention belongs to biological technical field, specifically, the 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene that relates in the red sage root, express and encoded protein matter and application.
Background technology
Cardiovascular and cerebrovascular diseases and diabetes, cancer etc. are at present the mankind to be threatened maximum three major types disease.Wherein cardiovascular and cerebrovascular diseases is positioned at first of this three big disease.According to statistics, annual nearly 1,700 ten thousand people in the whole world die from cardiovascular and cerebrovascular diseases, account for 1/3 of the total death toll in the whole world; Annual nearly 2,600,000 people of China die from cardiovascular and cerebrovascular diseases, and cardiovascular and cerebrovascular diseases has become " the No.1 killer " who threatens universe's health and life.Therefore the clinical medicine of efficient, low toxicity of active research and exploitation and cheap treatment cardiovascular and cerebrovascular diseases has very profound significance to improving level of human health.
Salviamiltiorrhizabung is the dry root and rhizome of the Labiatae salvia red sage root (Salvia miltiorrhiza Bunge), and red and shape is gained the name " red sage root " like ginseng because of its look, is a kind of conventional Chinese medicine of mainly curing cardiovascular systemic disease.The red sage root is continued to use for a long time in China as a kind of traditional Chinese medicine material, and the beginning is stated from Shennong's Herbal, is listed in top grade.Also on the books in " Bencao Jingshu ", the Compendium of Material Medica.The tradition traditional Chinese medical science thinks that the red sage root has stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, the effect of the relieving restlessness that clears away heart-fire, and cures mainly coronary heart disease, stenocardia, dysphoria and insomnia, menoxenia, through closing disease such as dysmenorrhoea.Studies show that red sage root activeconstituents mainly is divided into two big classes: promptly fat-soluble diterpene-kind compound (Tanshinone I, Tanshinone II A, Cryptotanshinone etc.) and water-soluble phenolic acid compounds (salvianolic acid A, salvianolic acid B, alkannic acid, rosmarinic acid, rancinamycin IV, Salvianic acidA etc.).
Modern pharmacological research shows the red sage root to cardiovascular systems, and the curative effect of disease of blood system is very remarkable.Multiple compound preparation such as compound injection of red sage root, FUFANG DANSHEN PIAN, FUFANG DANSHEN JIAONANG and FUFANG DANSHEN DIWAN etc. based on the red sage root, by clinical treatment cardiovascular disorder, ephrosis, the hepatopathy and anti-infective etc. of being widely used in, find also that in recent years the red sage root has anti-tumor activity, therefore, the red sage root has purposes very widely clinically.Yet because the market requirement of the red sage root is huge, and wild resource reduces day by day, and the red sage root is a per nnial herb in addition, its growth cycle is longer, active pharmaceutical ingredients content is low, under traditional cultivation mode, is faced with quality serious degradation and breed breeding cost and crosses high many drawbacks.Therefore, the demand that how to make the supply of this raw medicinal material of the red sage root can satisfy clinical application better on quality and quantity has become a research focus.
The develop rapidly of modern biotechnology is for the genetic improvement that utilizes the biotechnology means to carry out red sage root quality provides new approach.Utilize the modern genetic engineering technology that the key gene in the red sage root active pharmaceutical ingredients biosynthetic pathway is imported in the red sage root, obtain genetically modified root of hair, clone or regeneration plant, and cultivate on a large scale, be to improve the content of red sage root active pharmaceutical ingredients and expand one of the optimal path in red sage root active result source.Tanshinone compound (as Tanshinone I, Tanshinone II A and Cryptotanshinone etc.) belongs to diterpene compound, is that mevalonic acid (MVA) approach or 1-deoxidation wood sugar-5-phosphoric acid (DXP) approach can provide precursor substance for the biosynthesizing of terpene substances (comprising TANSHINONES and Artemisinin etc.) no matter current research shows.3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (HMGR) is a most important key enzyme of generally acknowledging in the MVA approach, and key precursor MVA is synthesized in catalysis, so this step is the crucial target for modulation of terpene substances metabolic engineering.
In analysis to existing document, " Bioscience reports (bio-science report); 2006; 26 (2): 171-181 " reported from the bark of eucommia and cloned-hydroxy-3-methylglutaryl A reductase gene, but the bibliographical information that clones and isolates total length 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene from the medicine plant red sage root has not been arranged so far as yet.Because the enzyme of this genes encoding has remarkably influenced for the biosynthesizing of tanshinone two terpene components, therefore, this step is to utilize genetic engineering technique to regulate and control the biosynthetic crucial 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 reductase 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.
Salvia 3-hydroxy-3-methylglutaryl A reductase enzyme provided by the present invention (SmHMGR) gene is one of following nucleotide sequence:
(1) has the nucleotide sequence shown in the SEQ ID No.1;
(2) nucleotide sequence shown in the SEQ ID No.1 add, replace, insert or delete homologous sequence or its allelotrope and the deutero-nucleotide sequence thereof of one or more Nucleotide.
The protein of this coded by said gene is the salvia 3-hydroxy-3-methylglutaryl A reductase enzyme, is one of following aminoacid sequence:
(1) has the aminoacid sequence shown in the SEQ ID No.2;
(2) SEQ ID No.2 add, replace, insert or delete one or more amino acid whose homologous sequences.
Contain salvia 3-hydroxy-3-methylglutaryl A reductase gene complete sequence of the present invention or part fragment recombinant vectors, all belong to protection scope of the present invention as plasmid or plant expression vector.
Contain salvia 3-hydroxy-3-methylglutaryl A reductase gene complete sequence of the present invention or the segmental host cell of part, as the host cell that contains above-mentioned recombinant vectors also belongs to protection scope of the present invention.
Described host cell is Bacillus coli cells, agrobatcerium cell, yeast cell, red sage root cell, tobacco cell or red sage root root of hair cell.
The preferred Bacillus coli cells of described host cell, agrobatcerium cell or red sage root root of hair cell.
The application of salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase gene sequence genetic transformation.
The notion particular content that relates in the technical solution of the present invention is as follows:
The dna molecular of said salvia 3-hydroxy-3-methylglutaryl A reductase gene comprises: coding has the nucleotide sequence of the polypeptide of salvia 3-hydroxy-3-methylglutaryl A reductase activity, and shows at least 70% homology from the nucleotides sequence of Nucleotide 69-1766 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under 40-55 ℃ of condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 69-1766.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.2.More preferably, described sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 69-1766 position.
The isolated salvia 3-hydroxy-3-methylglutaryl A reductase enzyme of the present invention polypeptide comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of 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 reductase enzyme (or polypeptide) gene " refers to: coding has the nucleotide sequence of the polypeptide of salvia 3-hydroxy-3-methylglutaryl A reductase activity, as 69-1766 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 69-1766 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 69-1766 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 69-1766 position.Also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 69-1766 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 reductase 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 reductase enzyme protein or polypeptide " refers to: the SEQ ID NO.2 polypeptide of sequence with salvia 3-hydroxy-3-methylglutaryl A reductase activity.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase 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 reductase enzyme polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, the polypeptide or the albumen that can reduce the coded albumen of the DNA of enzyme dna hybridization and utilize the serum of salvia 3-hydroxy-3-methylglutaryl A reductase enzyme polypeptide to obtain with salvia 3-hydroxy-3-methylglutaryl A under high or low rigorous condition.
Salvia 3-hydroxy-3-methylglutaryl A reductase 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 can be replaced according to table 1 and produce.
Replacement residue in the table 1. conservative property variation polypeptide
| Initial residue | Representational replacement residue | The preferred residue that replaces |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Initial residue | Representational replacement residue | The preferred residue that replaces |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| 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 reductase enzyme or polypeptide.The difference of these analogues and natural 3-hydroxy-3-methylglutaryl-coenzyme A reductase 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 reductase enzyme polypeptide of the present invention, the nucleotide sequence of salvia 3-hydroxy-3-methylglutaryl A reductase gene operationally can be connected in expression regulation sequence, thereby form salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase 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 reductase 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 reductase 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 reductase enzyme.
The present invention relates to whether exist in the test sample method of salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase 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 reductase 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 reductase 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 reductase enzyme, can screen red sage root cDNA library with dna probe, these probes are under low rigorous condition, use
32P salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase enzyme.
Salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase enzyme of the present invention,, can filter out with the salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase gene provided by the present 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 reductase gene from China's medicine plant red sage root.Utilize the present invention can improve the content of terpene activeconstituents TANSHINONES in the plants such as the red sage root by genetic engineering technique, transgene result shows that the salvia 3-hydroxy-3-methylglutaryl A reductase gene has obvious effect to the raising that promotes TANSHINONES content.The salvia 3-hydroxy-3-methylglutaryl A reductase gene can be used to improve TANSHINONES Study on content and industrialization by transgenic technology, especially the quality-improving that can be used for the Chinese medicinal materials red sage root, can alleviate the serious deficient problem in TANSHINONES medicine source, have promoter action preferably to improving TANSHINONES output, have 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 having read the content that the present invention tells about, 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 reductase gene
1. separate tissue (isolation)
Red sage root plant derives from Henan, 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 (TRlzol 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)
Cloned the 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme amino acid conserved sequence that obtains according to the bark of eucommia, tobacco and other, 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 SmHMGR F2 ' (775bp), 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 (Wisconsin group, 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 HMGR gene (connecting HMGR gene etc. as punching) is very high, so think that tentatively it is a HMGR gene.
(2)5′-RACE
According to 3 ' RACE result, design reverse special primer R2, obtain SmHMGR R2 ' (1580bp) (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.
(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 SmHMGR KF1 (5 '-ATGGATATCCGCCGGAGGCCA-3 ') (SEQ ID NO.3) and SmHMGR KR1 (5 '-TCAGGAGCCAATCTTCGTGATG-3 ') (SEQ IDNO.4) and carry out pcr amplification SmHMGR coding region and obtain SmHMGR coding region (1698bp) (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 reductase gene.Studies show that the 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene has material impact for the metabolism of terpene substances such as TANSHINONES etc. is synthetic, has 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 SmHMGR.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, with SmHMGR F1 (5 '-CCCCTTCATTCTTTCTCTCTCTC-3 ') is forward primer, SmHMGR R1 (5 '-GAAGCAGAAGAAGATTGCATTAC-3 ') be reverse primer, with total RNA is template, carry out the RT-PCR amplification, the PCR condition be 94 ℃ 5 minutes, carry out 35 circulations with 94 ℃ 1 minute, 58 ℃ 1 minute and 72 ℃ of 2 fens halfs thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 2091bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ IDNO.1.
The sequence information and the homology analysis of embodiment 2 red sage root HMGR genes
The length of salvia 3-hydroxy-3-methylglutaryl A reductase enzyme full-length cDNA of the present invention is 1258bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 65-1126 position Nucleotide.Derive the aminoacid sequence of salvia 3-hydroxy-3-methylglutaryl A reductase enzyme according to full-length cDNA, totally 565 amino-acid residues, molecular weight 60.5KD, pI are 7.1, 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 reductase enzyme are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundantGenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and Herba Andrographis HMGR gene (GenBank Accession No.AY254389.2) have 80% homology (seeing Table 2); On amino acid levels, the 1-562 amino acids residue of it and Herba Andrographis HMGR (GenBank Accession No.AAP14352.2) has 85% homogeny and 91% similarity (seeing Table 3).Therefore all there are higher homology in salvia 3-hydroxy-3-methylglutaryl A reductase gene and Herba Andrographis 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene on nucleic acid still is protein level.So can think that the salvia 3-hydroxy-3-methylglutaryl A reductase enzyme has promoter action on the tanshinone diterpene component content in improving the red sage root.
The homology of the nucleotide sequence of table 2. red sage root SmHMGR of the present invention and Herba Andrographis (Andrographis paniculata) ApHMGR is (GAP) table relatively
Wherein: Query represents the nucleotide sequence of red sage root SmHMGR; Subject represents the nucleotide sequence (GenBank Accession No.AY254389.2) of Herba Andrographis ApHMGR.
The result: both have 80% similarity in the comparison of 1729 Nucleotide.
The homology of table 3. red sage root SmHMGR of the present invention and Herba Andrographis ApHMGR aminoacid sequence is (FASTA) table relatively
Wherein: Query represents the aminoacid sequence of red sage root SmHMGR; Subject represents the aminoacid sequence (GenBank Accession No.AAP14352.2) of Herba Andrographis ApHMGR; Identical amino acid marks with the amino acid monocase between two sequences.
The result: in 562 amino acid whose comparisons, both have 85% homogeny and 91% similarity respectively.
Embodiment 3 salvia 3-hydroxy-3-methylglutaryl A reductase enzymes or polypeptide carry out prokaryotic expression and purification in intestinal bacteria
In this embodiment, the red sage root SmHMGR 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 SmHMGR, 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 SmHMGR 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 (+)-SmHMGR of pET32a (+)-SmHMGR expression vector.
2, express the isolation identification of the engineering bacteria of Trx-SmHMGR recombinant protein
The BL21-pET32a (+) of picking list bacterium colony-SmHMGR engineering bacteria contains jolting overnight incubation in the LB substratum of 100 μ g/mL penbritins in 3mL, draw nutrient solution by 1: 100 concentration 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-SmHMGR fusion rotein.
3, proteic extraction purifying of SmHMGR and enzyme assay
The proteic engineering bacteria BL21-pET32a of abduction delivering Trx-SmHMGR amalgamation and expression (+)-SmHMGR 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 dissolve with dissolving damping fluid (50mMCAPS, pH 11.0,0.3% N-Iauroylsarcosine), and (200mM Tris-HCl pH8.5) dialyses to use dialysis buffer liquid again.Use Histidine to carry out affinity chromatography then, and collect the Trx-SmHMGR fusion rotein through elution buffer (1Mimidazole, 500mM NaCl, 20mM Tris-HCl pH 7.9) wash-out in conjunction with (HisBind) resin.Fusion rotein is the expressing protein of the separable SmHMGR that obtains after 20 ℃ of enzymes of enteropeptidase are cut 16 hours.This expressing protein molecular weight is 60.5KD, and pI is 7.1.Press (Plant Physiology and Biochemistry such as Bonfill, 2003, method 41:91-96) is carried out the mensuration of enzyme activity to the 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme protein of expression and purification, studies the influence that it generates mevalonic acid (MVA).The result shows that expressed proteins has the enzymic activity that the catalysis 3-hydroxy-3-methylglutaryl-coenzyme A generates mevalonic acid (MVA) really.
Embodiment 4 salvia 3-hydroxy-3-methylglutaryl A reductase enzymes 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 reductase gene): according to the full length sequence (SEQ ID NO.1) of salvia 3-hydroxy-3-methylglutaryl A reductase 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 reductase gene cDNA is cloned into binary expression vector (as pBI121), 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.
Utilize the genetic transformation of the red sage root of Agrobacterium rhizogenes mediation, material requested and operation steps are:
1) 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, 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 25 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 reductase gene
Press the method for Ge etc. (Plant Science, 2005) the transgenosis root of hair of expressing the salvia 3-hydroxy-3-methylglutaryl A reductase gene is carried out the TANSHINONES assay.The result shows that TANSHINONES content improves 1.4 times (P＜0.05) than non-transgenic control group in the transgenosis root of hair of expressing the salvia 3-hydroxy-3-methylglutaryl A reductase gene.Therefore transgene result proves, the salvia 3-hydroxy-3-methylglutaryl A reductase gene has obvious effect to the raising that promotes TANSHINONES content, the salvia 3-hydroxy-3-methylglutaryl A reductase 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
Claims (7)
1. a salvia 3-hydroxy-3-methylglutaryl A reductase gene is characterized in that, is one of following nucleotide sequence:
(1) has the nucleotide sequence shown in the SEQ ID No.1;
(2) nucleotide sequence shown in the SEQ ID No.1 add, replace, insert or delete homologous sequence or its allelotrope and the deutero-nucleotide sequence thereof of one or more Nucleotide.
2. a salvia 3-hydroxy-3-methylglutaryl A reductase enzyme is characterized in that, is one of following aminoacid sequence:
(1) has the aminoacid sequence shown in the SEQ ID No.2;
(2) SEQ ID No.2 add, replace, insert or delete one or more amino acid whose homologous sequences.
3. a recombinant vectors is characterized in that: contain claim 1 described salvia 3-hydroxy-3-methylglutaryl A reductase gene complete sequence or part fragment.
4. host cell, it is characterized in that: described host cell contains claim 1 described salvia 3-hydroxy-3-methylglutaryl A reductase gene complete sequence or part fragment.
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 cell, red sage root root of hair cell.
6. the salvia 3-hydroxy-3-methylglutaryl A reductase gene is applied to prepare transgenosis red sage root plant.
7. the salvia 3-hydroxy-3-methylglutaryl A reductase gene is applied to prepare transgenosis red sage root root of hair system.
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| CN101942467A (en) * | 2010-08-27 | 2011-01-12 | 上海师范大学 | Method for enhancing content of tanshinone in salvia miltiorrhiza hairy root by double-key enzyme genetic transformation |
| CN102337279A (en) * | 2011-06-22 | 2012-02-01 | 上海师范大学 | Method of increasing content of tanshinone in hairy roots of salvia miltiorrhiza bunge through cotransformation of SmHMGR and SmDXR double genes |
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| CN105002148A (en) * | 2015-08-06 | 2015-10-28 | 南京中医药大学 | Alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase antibody preparation and detection method |
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