CN103695441A

CN103695441A - Cytochrome P450 gene participated in anabolism of tanshinone compound as well as coding product and application thereof

Info

Publication number: CN103695441A
Application number: CN201310504475.XA
Authority: CN
Inventors: 黄璐琦; 蔡媛; 郭娟; 马晓惠; 马莹
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2013-10-24
Filing date: 2013-10-24
Publication date: 2014-04-02
Anticipated expiration: 2033-10-24
Also published as: CN103695441B

Abstract

The invention discloses a cytochrome P450 gene which participates in anabolism of tanshinone compound as well as coding product and application thereof, and the invention belongs to the field of medicinal plant gene engineering. The gene is obtained by clone from red sage root for the first time, and is a key enzyme gene in anabolism pathway of tanshinone compound, and can catalyze ferruginol into 11-hydroxy ferruginol. The CYP76AH3 gene provided by the invention has a nucleotide sequence shown in the SEQ ID No.1. The gene coding protein comprises an amino acid residue sequence shown in the SEQ ID No.2 of the sequence table and a protein derived from the SEQ ID No.2 whose activity is identical to that of the amino acid residue sequence shown in the SEQ ID No.2. The CYP76AH3 gene provided by the invention is closely related to anabolism of tanshinone compound, and has important theoretical and real meanings for regulating plant diterpenoids compounds and increasing contents of diterpene active component tanshinone in red sage root by biological technology.

Description

One participates in the biosynthetic CYP450 gene of TANSHINONES and coded product and application

Technical field

The present invention relates to tanshinone compound pathways metabolism relevant cell cytochrome p 450 gene, and utilize whole-cell catalytic strategy to carry out functionally active analysis to this gene coded protein, relate to cytochrome P450 gene and coded product and application in red sage root main active ingredient tanshinone compound biosynthetic pathway, belong to Gene Engineering of Medicinal Plants field.

Background technology

The formation of active components in medicinal plant is the product of peculiar gene in Secondary Metabolism of Plant approach.Along with plant functional genomics research extensively with deeply, show unique characteristics and have the research of the synthetic correlation function gene of medicinal plant secondary metabolism of broad prospect of application to become gradually the focus of research, the clone of these genes is by biosynthetic pathway and regulatory mechanism thereof for annotation active components in medicinal plant, for the formation of medical material quanlity is provided fundamental basis, for utilizing biotechnology to improve target component content or direct production effective constituent or intermediate, bring wide application space simultaneously.

The red sage root is the dry root and rhizome of Labiatae Salvia Salvia miltiorrhiza Bge (Salvia miltiorrhiza Bunge), there is stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, the effects such as relieving restlessness that clear away heart-fire, its main effective constituent comprises fat-soluble TANSHINONES and water miscible salvianolic acid.Wherein tanshinone mainly comprises Cryptotanshinone, Tanshinone II A, Tanshinone II B and dihydrotanshinone etc., there is vasodilation, antithrombotic, antisepsis and anti-inflammation and the multiple pharmacological effect such as antitumor, anti-oxidant, in fields such as pharmacy and beauty treatment, makeup, be used widely.Tanshinone compound is Diterpene quinones, its biosynthesizing is on the synthetic basis of terpenoid precursor substance, by prenyltransferases (Prenyl Transferases, PT) effect produces geranyl geranyl tetra-sodium (the Geranylgeranyl Diphosphate of 20 carbon atoms, GGPP), GGPP is further at red sage root Ke Baji pyrophosphate synthase (S. miltiorrhiza Copalyl Diphosphate Synthase, SmCPS) and components in danshen kaurene synthase (S. miltiorrhiza ent-kaurene Synthase, SmKS) under effect, form the basic framework miltirone diene (Miltiradiene) of components in danshen compound, on the basis of miltirone diene, through Hypermethylation and hydroxylation etc., form tanshinone compound, wherein cytochrome P 450 enzymes plays an important role in modification, the result of study in later stage shows that CYP76AH1 can generate ferruginol by catalysis miltirone diene.

Cytochrome P 450 enzymes is hemopexin, all has a conservative heme-binding domain in all known cytochrome P 450 enzymes, is a key character of identification of cell cytochrome p 450 gene.In arabidopsis gene group, have 272 cytochrome P450 genes, rice genome annotation shows 457 cytochrome P450 genes, and so huge gene family has reflected the complicated and changeable of Plant Secondary Metabolites.The reaction of various cytochrome P 450 enzymes catalysis is extensive and complicated, comprising: dealkylation, epoxy group(ing), deamination, desulfurization, dehalogenation and the peroxidation etc. of hydroxylation, N-, O-and S-end.Although catalyzed reaction is different, catalytic mechanism is identical, by NADPH or NADH, is that Cytochrome P450 transmits electronics, and excited oxygen molecule, is inserted into one of them oxygen on substrate, generates a part water simultaneously.Cytochrome P 450 enzymes is as a class important enzyme albumen of the metabolic pathway of synthesizing such as terpene, flavonoid, fatty acid and plant hormone, not only catalysis associated metabolic reaction, because most cytochrome p450 protein all has one section of endoplasmic reticulum positioning protein, therefore in the combined enzyme agent of metabolon (Metabolon), also there is the effect of immobilized enzyme complex body.Along with the development of molecular biology and correlation detection technology, the cytochrome P450 gene function that secondary metabolism approach is relevant is verified gradually.Eukaryotic expression, prokaryotic expression and RNA disturb technology such as (RNA Interference, RNAi) to bring into play vital role in the functional verification of cytochrome P450 gene.Utilize in recent years eukaryotic expression and RNAi technology to make the vital role of cytochrome P 450 enzymes in the pathways metabolisms such as taxol, Artemisinin, phytoalexin resolved gradually.But relatively low as the Cytochrome P450 expression amount playing a role in Secondary Metabolism of Plant, and cytochrome p450 protein has strict catalytic substrate structure specificity mostly, clone and functional verification that the cytochrome P450 gene simultaneously extensively existing in genome is secondary metabolism genes involved provide difficulty, make it to become one of study hotspot of international academic community in recent years.

This laboratory has been cloned into whole key genes of the upstream of TANSHINONES compou nd synthesis approach on the basis of early-stage Study, and by building engineering bacteria, has produced precursor miltirone diene and the ferruginol of TANSHINONES compound.What the present invention relates to is that catalysis ferruginol (ferruginol) produces tanshinone compound route of synthesis intermediate product 11-hydroxyl ferruginol (Abieta-8 in the hydroxylation of C11,11,13-triene-11, key cells cytochrome p 450 enzyme gene 12-diol), this gene is the synthetic key cells cytochrome p 450 gene of tanshinone component obtaining from the red sage root first, according to cell plain color P450 unnamed gene rule called after CYP76AH3.Before the present invention comes forth, not yet there is any CYP76AH3 gene and aminoacid sequence thereof relating in present patent application that disclose or reported.

Summary of the invention

The object of the present invention is to provide the cytochrome P450 gene CYP76AH3 of a TANSHINONES compou nd synthesis pathways metabolism, the albumen of its coding can generate 11-hydroxyl ferruginol by catalysis ferruginol in the strain of ferruginol saccharomyces cerevisiae engineered yeast.

The invention provides a kind of gene relevant with tanshinone compound anabolism: CYP76AH3, it is one of following nucleotide sequences:

1) DNA sequence dna of SEQ ID No.1 in sequence table;

2) with sequence table in the DNA sequence dna that limits of SEQ ID No.1 there is one or several base mutation, and coding identical function protein DNA sequence.

A protein of being encoded by said gene CYP76AH3, have SEQ ID No.2 in sequence table amino acid residue sequence or by the amino acid residue sequence of SEQ ID No.2 through the replacement of one or several amino-acid residue and have SEQ ID No.2 the identical activity of amino acid residue sequence by the derivative protein of SEQ ID No.2.

The DNA sequence dna of SEQ ID No.1 of the present invention is by 1485 based compositions, the protein sequence SEQ ID No.2 being comprised of 494 amino-acid residues in code sequence list.

The application of this gene of the expression vector that contains gene C YP76AH3 of the present invention, clone and Host Strains and use in adjusting and production plant diterpene-kind compound and red sage root breeding is also within protection scope of the present invention.SEQ ID No.1 gene clone, between the restriction enzyme EcoR I and Sep I site of carrier for expression of eukaryon pESC-His, is built to the recombinant expression vector pESC-CYP76AH3 with CYP76AH3 gene; Proceed to ferruginol saccharomyces cerevisiae engineered yeast strain YJ35 (BY4741, MATa; His3 Δ 1; Leu2 Δ 0; Met15 Δ 0; Ura3 Δ 0/LEU2/MET15/pYX212-(BST1-ERG20)+(SmKSL-SmCPS)/p424-tHMG1+CYP76AH1+SmCPR1), expressive host bacterium adopts the catalysis of semi-lactosi abduction delivering.By 15L fermentor tank amplification culture, n-hexane extraction reaction product, column chromatographic isolation and purification obtains target product, and high resolution mass spectrum and nuclear-magnetism characterize product structure.Analytical results shows: ferruginol is under the catalysis of CYP76AH3 proteolytic enzyme, and having generated molecular weight is the novel substance of 302 (m/z), by high resolution mass spectrum and the new product of nmr analysis, thinks that the structure of this product is 11-hydroxyl ferruginol.Result of study shows, the present invention's gene synthetic relevant with tanshinone compound has the feature structure territory of cytochrome P450 gene, whole-cell catalytic response analysis finds that the C11 position hydroxylation of this gene catalysis ferruginol forms tanshinone compound intermediate product 11-hydroxyl ferruginol, has important theory and practical significance for regulating and produce plant diterpene-kind compound and cultivating the high-quality red sage root.

Accompanying drawing explanation

The structural analysis of Fig. 1 CYP76AH3 gene coded protein whole-cell catalytic reaction product

A: carbon-13 nmr spectra figure; B: hydrogen nuclear magnetic resonance spectrogram C: product high resolution mass spectrum figure;

Fig. 2 CYP76AH3 gene coded protein catalysis ferruginol to 11-hydroxyl ferruginol illustrates

Embodiment

Embodiment 1: the clone of cytochrome P450 gene in the red sage root

1, get the red sage root of full-bloom stage, utilize Trizol method to extract total RNA of the red sage root, utilize 5 ' and 3 ' the RACE test kit of invitrogen to increase respectively, obtain 5 ' and 3 ' end sequence of CYP76AH3 gene.

2, the Cloning and sequencing of full-length cDNA

By 5 ' and 3'RACE result splice, search ORF region, design full-length gene primer, the cDNA of take increases as template.Agarose gel electrophoresis shows to occur specific fragment about 1500bp place, sepharose reclaims test kit (Takara) and reclaims target fragment, be cloned in pGEM-T easy carrier (Promega), identify positive colony and carry out sequence verification) (Beijing Hua Da gene), for the structure of expression vector.

The information biology of embodiment 2, CYP76AH3 gene order

The length of the red sage root diterpene metabolic pathway of synthesizing cytochrome P450 gene CYP76AH3 full length gene opening code-reading frame (ORF) the present invention relates to is 1485bp, 494 amino acid of encoding, detailed sequence is shown in SEQ ID No.1 and the SEQ ID No.2 in sequence table.CYP76AH3 total length opening code-reading frame is carried out in ncbi database to homology search with blast program, this gene is compare of analysis demonstration on amino acid levels, and the protein amino acid sequence of red sage root CYP76AH3 genes encoding and the homology of other species are lower.

Embodiment 4, CYP76AH3 gene eucaryon expression and functional analysis

1, the structure of Yeast expression carrier

Primer by design with EcoR I and Spe I restriction enzyme site, utilize the method for PCR, by the open reading frame of the red sage root cytochrome P450 gene being cloned into, be inserted between the EcoR I and Spe I restriction enzyme site of yeast expression expression vector pESC-His, obtain recombinant plasmid pESC-CYP76AH3, in Beijing, Hua Da genome company carries out sequence verification.

2, abduction delivering

The pESC-CYP76AH3 plasmid building is transformed and expresses ferruginol Yeast engineering bacteria YJ35, utilize semi-lactosi to carry out abduction delivering.

3, the activation analysis of red sage root CYP76AH3

CYP76AH3 expression vector is imported to the saccharomyces cerevisiae engineered yeast strain of producing ferruginol, 3 mono-clonals of picking are inoculated in 5ml SD liquid nutrient medium, and 30 ℃, 200rmin ^-1cultivate 24h; In the inoculum size access 50ml SD liquid nutrient medium of 1:50,30 ℃, 200rmin ^-1, being cultured to mid-log phase, 2000g is centrifugal, with in the SD inducing culture of transferring after deionized water wash thalline containing semi-lactosi, 30 ℃, 200rmin ^-1, cultivate 48h.Fermentation adds equal-volume n-hexane extraction catalysate completely.Found that and include that CYP76AH3 DNA recombinant expression carrier pESC-CYP76AH3 catalysis group contrasts with empty carrier and other expression vector reaction product have been compared novel substance and produced.Nuclear magnetic spectrogram is as Figure 1A/B, and high resolution mass spectrum figure is as Fig. 1 C, and product structure is accredited as 11-hydroxyl ferruginol.The albumen that CYP76AH3 genes encoding is described can generate 11-hydroxyl ferruginol by catalysis ferruginol in tanshinone compound metabolic pathway of synthesizing.

Claims

1. a cytochrome P450 gene CYP76AH3 relevant to tanshinone compound biosynthesizing, its nucleotide sequence is as shown in SEQ IDNo.1.

2. gene according to claim 1, is characterized in that: the reading frame of this gene is the 1-1485 position Nucleotide of SEQ ID No.1.

3. by a protein for gene C YP76AH3 coding claimed in claim 1, its amino acid residue sequence is as shown in SEQ ID No.2.

4. the expression vector that contains gene claimed in claim 1.

5. the transgenic cell line that contains gene claimed in claim 1.

6. the Host Strains that contains gene claimed in claim 1.

7. gene claimed in claim 1 is regulating and is producing the application in plant diterpene-kind compound 11-hydroxyl ferruginol.

8. the application of gene claimed in claim 1 in red sage root breeding.