CN102465135A

CN102465135A - Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (SbCCOMT2) gene, and coding product and application thereof

Info

Publication number: CN102465135A
Application number: CN2010105332217A
Authority: CN
Inventors: 袁媛; 黄璐琦; 杨兆春; 胡国强; 秦双双
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2010-11-05
Filing date: 2010-11-05
Publication date: 2012-05-23

Abstract

The invention discloses a Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (CCOMT) gene, and a protease coded by the gene and application thereof. The gene is for the first time cloned from Scutellaria baicalensis Georgi by constructing overall length cDNA library and fills the blank of separating and cloning caffeoyl-CoA-3-O-methyltransferase from traditional Chinese medicine Scutellaria baicalensis Georgi. The caffeoyl-CoA-3-O-methyltransferase provided by the invention has a nucleotide sequence shown as a SEQ ID NO.1 or a homologous sequence by adding, substituting, inserting or deleting one or more nucleotides, or an allele thereof, and a nucleotide sequence derived therefrom. The protein coded by the gene has an amino acid sequence shown as a SEQ ID NO.2 or a homologous sequence by adding, substituting, inserting or deleting one or more nucleotides. The caffeoyl-CoA-3-O-methyltransferase provided by the invention can increase a content of phenylalanine metabolite in Scutellaria baicalensis Georgi through biotechnology, so as to facilitate quality improvement of the medicinal material Scutellaria baicalensis Georgi; besides the caffeoyl-CoA-3-O-methyltransferase can be used for variety breeding and has good application prospect.

Description

Root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene and coded product and application

Technical field

The invention belongs to biological technical field; Relate generally to and utilize full-length cDNA library clone oxygen methyl transferase gene and coded product and application; Relating in particular to biosynthesizing has phenolic acid fermentoid gene and the coded product and the application of pharmacological component, belongs to medicinal plant genetically engineered field.

Background technology

The formation of active components in medicinal plant (secondary metabolite) is the product of peculiar gene group in the 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 the focus of research gradually; The clone of these genes will provide fundamental basis for the formation that the biosynthetic pathway and the regulatory mechanism thereof of parsing active components in medicinal plant are conciliate release material quality, bring wide application space for utilizing biotechnology to improve target component content or direct production effective constituent or midbody simultaneously.

Phenolic acid compound is one of secondary metabolite important in the plant, and a lot of activeconstituentss such as salvianolic acid, coffic acid, FLA etc. are phenolic acid compound in medicinal plant.Medicinal plant root of large-flowered skullcap Scutellaria baicalensis Georgi is conventional Chinese medicine simply, has heat-clearing and damp-drying drug, and cool blood is antiabortive, and the effect of detoxicating functions is the staple of numerous compounds, healthcare products.The main active ingredient of the root of large-flowered skullcap comprises baicalin, scutellarin, wogonoside, wogonin etc.; Wherein FLA is formed by caffeoyl coenzyme A-3-O-Methyl transporters enzyme catalysis, and the variation of this enzymic activity also can influence the accumulation of other activeconstituentss such as scutellarin, naringenin.

Therefore the clone and the analysis of caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene is for the content that improves root of large-flowered skullcap efficient part with genetic engineering means provides important foundation.Before the present invention comes forth, any disclose or reported root of large-flowered skullcap phenolic acid fermentoid gene and aminoacid sequence thereof mentioned in the present patent application are not arranged as yet.

Summary of the invention

The object of the present invention is to provide a kind of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) 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.

Root of large-flowered skullcap caffeoyl coenzyme A provided by the present invention-3-O-methyltransgerase (SbCCOMT) gene is characterized in that,

It is one of following nucleotide sequences:

(1) dna sequence dna of SEQ ID No.1;

(2) under stringent condition, has the dna molecular of the protein molecule of caffeoyl coenzyme A-3-O-methyl transferase activity with hybridization of the dna sequence dna of SEQ ID No.1 and coding;

(3) nucleotide sequence of aminoacid sequence shown in the coding SEQ IDNo.2;

(4) coding to SEQ ID No.2 through add, replace, insert or delete one or more amino acid and having the proteinic nucleotide sequence of caffeoyl coenzyme A-3-O-methyl transferase activity.

The protein of this coded by said gene is root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2), is one of following aminoacid sequence:

(1) has the aminoacid sequence shown in the SEQ ID NO.2;

(2) SEQ ID No.2 is through add, replace, insert or delete one or more amino acid and having the sequence of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyl transferase activity.

The recombinant vectors that contains root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2) gene complete sequence or partial sequence, like protokaryon class carrier, eucaryon class expression vector and RNAi carrier all belong to protection scope of the present invention.

Contain the host cell of root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2) gene complete sequence or partial sequence, as the host cell that contains above-mentioned recombinant vectors also belongs to protection scope of the present invention.

Said host cell is valuable living materials.

The application of root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2) gene comprises and uses described recombinant vectors, like the plant expression vector transformed plant cells; Perhaps cultivate altogether, obtain genetically modified plant rooting system with said Agrobacterium and the vegetable cell that contains this gene; Perhaps use described root of hair cell regeneration plant; Perhaps use described root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene complete sequence or partial sequence to transform and obtain transgenic organism.

The notion particular content that relates in the technical scheme of the present invention is following:

The dna molecular of said root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene comprises: coding has the nucleotide sequence of the polypeptide of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene activity, and shows at least 70% homology from the nucleotides sequence of Nucleotide 1-818 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 1-818.Described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.2.The isolated root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2) gene 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.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 " root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) " gene refers to: coding has the nucleotide sequence of root of large-flowered skullcap caffeoyl coenzyme A-active polypeptide of 3-O-methyltransgerase (SbCCOMT2), like 1-818 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 1-818 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 1-818 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 1-818 position.Also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 1-818 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 root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) identical function.These variant forms comprise (but being not limited to): several (be generally, 1-90, preferably; 1-60, more preferably, 1-20; 1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and (being generally in 60, preferably is in 30 to add several at 5 ' and/or 3 ' end; More preferably being in 10, is in 5 best) Nucleotide.

Term among the present invention " root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) albumen or polypeptide " refers to: have the root of large-flowered skullcap caffeoyl coenzyme A-active SEQ ID of 3-O-methyltransgerase (SbCCOMT2) NO.2 polypeptide of sequence.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) identical function.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30; 1-20 more preferably, 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 root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2), also comprises operationally being connected in the verivate that signal peptide, promotor or ribosome bind site sequence are formed.The variant form of red sage root root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2) polypeptide 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 root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) DNA hybridization and the polypeptide or the albumen that utilize the serum of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) polypeptide to obtain.

Root of large-flowered skullcap caffeoyl coenzyme A among the present invention-3-O-methyltransgerase (SbCCOMT2) conservative property variation polypeptide refers to: compare with the aminoacid sequence of SEQ ID NQ.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 propertys variation polypeptide can be according to table 1, replaces and produces.

The present invention also comprises the analogue of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) or polypeptide.The difference of these analogues and natural root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) 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 through various technology, as through 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-(like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.Said 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, in the synthetic and processing of polypeptide or further, carries out glycosylation modified and polypeptide that produce in the procedure of processing like those.This modification can be carried out glycosylated enzyme (like mammiferous glycosylase or deglycosylating enzyme) and accomplishes through polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its proteolyze performance or optimized solubility property.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.

Table 1: the replacement residue in the conservative property variation polypeptide

Initial residue	Representational replacement residue	The preferred residue that replaces
			Ala(A)	Val；Leu；IIe	Val
Arg(R)	Lys；GIn；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			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	Leu
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

When producing the root of large-flowered skullcap caffeoyl coenzyme A of the present invention-former enzyme polypeptide of 3-O-methyltransgerase (SbCCOMT2); Can the nucleotide sequence of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene operationally be connected in expression regulation sequence, thereby form root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) expression vector.Said " 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 root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) is promptly analyzed in the expression of the also available Northern blotting of the present invention technical Analysis root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene product.

In addition, the nucleic acid molecule that can be used as probe among the present invention has 8-100 continuous nucleotide of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) 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 root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2).The present invention relates to whether exist in the test sample method of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) 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 root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) 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 root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2) nucleotide sequence and aminoacid sequence; Can be on the homology basis of nucleic acid homology or marking protein, screening root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) source gene or homologous protein.

In order to obtain the dot matrix with root of large-flowered skullcap caffeoyl coenzyme A-root of large-flowered skullcap cDNAs that 3-O-methyltransgerase (SbCCOMT2) is relevant, can screen root of large-flowered skullcap cDNA library with dna probe, these probes are under low rigorous condition, use ³²P root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from the root of large-flowered skullcap.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 of the gene family of root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2).

The Nucleotide full length sequence of the gene family of root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2) or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain 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 need carries 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 come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through 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-hase Peptide Synthesis, WH Freeman Co., San Francisco through direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can use 431A type peptide synthesizer (Foster City, CA) the next automatically synthetic peptide of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, connect to produce the molecule of total length with chemical process then.Utilize root of large-flowered skullcap caffeoyl coenzyme A of the present invention-3-O-methyltransgerase (SbCCOMT2); Through various conventional screening methods; Can filter out with root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) interactional material takes place, perhaps acceptor, suppressor factor or antagonist etc.

Root of large-flowered skullcap caffeoyl coenzyme A provided by the present invention-3-O-methyltransgerase (SbCCOMT2) gene is from the root of large-flowered skullcap, to clone preparation first; Utilize the present invention can improve the content of phenolic acids activeconstituents asafoetide acid in the plants such as the root of large-flowered skullcap through genetic engineering technique; Transgene result shows that root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene pairs promotes the raising of asafoetide acids content that obvious effect is arranged.Root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT2) gene can be used to improve the research and the industrialization of ferulaic acid content through transgenic technology; Especially the quality-improving that can be used for the Chinese medicinal materials root of large-flowered skullcap; Can alleviate the weary problem of the serious plaque of root of large-flowered skullcap resource; Have promoter action preferably to improving asafoetide acid output, have good application prospects.

Description of drawings:

Fig. 1: the pcr amplification electrophorogram of root of large-flowered skullcap RNA, cDNA, cDNA library clone;

Fig. 2: SmFPS domain forecast analysis (deriving from ncbi database);

Fig. 3: SmFPS systematic evolution tree (adjacent method);

Fig. 4: plant expression vector pC1301 physical map;

Embodiment

The structure in embodiment 1, root of large-flowered skullcap full-length cDNA library

1, the separation of the total RNA of the root of large-flowered skullcap and detection

Get the root of large-flowered skullcap (Scutellaria baicalensis Georgi) root 2g, with the quick grind into powder of liquid nitrogen, the 10mL that is transferred to 65 ℃ of preheatings fast extracts (CTAB (W/V) 2%, Tris-HCl (pH8.0) 100mmolL in the damping fluid in mortar ^-1, EDTA 25m molL ^-1, NaCl 2.0molL ^-1, PVP40 2%, spermidine 0.5g/L, mercaptoethanol 2%), mixing fully vibrates; With equal-volume chloroform extracting twice, centrifugal 15 minutes of 7500g.Supernatant adds the 10M LiCl of 1/4 volume, places 4 ℃ of depositions behind the mixing and spends the night; Centrifugal 20 minutes of 7500g, (SDS 0.5%, NaCl 1molL with 500 μ L SSTE for deposition ^-1, Tris-HCl (pH8.0) 10mmolL ^-1, EDTA 1mmolL ^-1, 65 ℃ of dissolvings 5 minutes.With the extracting of equal-volume chloroform, centrifugal 5 minutes of 13000g; Supernatant adds 2 times of volume absolute ethyl alcohols, places 2h for-70 ℃; Centrifugal 20 minutes of 4 ℃ of 13000g, the deposition drying at room temperature is dissolved in after 10 minutes in the water that 100 μ L DEPC handle, and detects the integrity of RNA with 1.0% agarose electrophoresis, with GenQuant nucleic acid quantification appearance mensuration A260, A280 ratio and concentration.Place-80 ℃ of refrigerators subsequent use.

2, the structure in cDNA library

Adopt mRNA purification kit (QuichprepTM Micro mRNA PurifiCation Kit; Pharmacia company) behind the separating mRNA; Adopt the Creator Smart cDNA Library Construction Kit (Cat.No.634903) of Clontech company to build the storehouse, principle is SMART (switch mechanism at 5 ' end of mRNA template).

3, the preparation of cDNA chip

3.1PCR amplification

Picking separates good plaque and is dissolved in the 100 μ L SM damping fluids (Amresco company) behind the mixing as pcr template.The pcr amplification primer is the partial sequence of λ ZAP Express MCS both sides: the M13-20 primer: 5 '-GTAAAACGACGGCCAGTG-3 ', the BK reverse primer: 5 '-GGAAACAGCTATGACCTTG-3 '.96 orifice plate PCR reaction systems are prepared following mixture: dNTP (25mmolL ^-1), 600 μ L; Mg ²⁺600 μ L, 10 * damping fluid, 1000 μ L; Taq (Takara Ex Taq, 5U μ L ^-1), 40 μ L; M13-20 primer (12.5 μ molL-1), 200 μ L; BK reverse primer (12.5 μ molL ^-1) 200 μ L; High purity water complements to 10mL.After mixing, be filled to 96 hole PCR plates with volley of rifle fire branch.Respectively add 6 μ L dna profilings then, mix.In the reaction of the enterprising performing PCR of ABI9700 type gene-amplificative instrament, reaction conditions is 94 ℃ of preparatory sex change 3 minutes, carries out 35 circulations then, is 94 ℃ of 30s, 54 ℃ of 30s, 72 ℃ 2 minutes, extended 5 minutes after 72 ℃ after the loop ends.Get 3 μ L reaction product containing electrophoresis on 1.5% the sepharose of EB, under SYNGENE type gel imaging system, observe, take pictures.

Embodiment 2: the clone of root of large-flowered skullcap genes involved

5000 mono-clonals of picking carry out bacterium colony PCR evaluation at random.Get an amount of PCR thin-walled tube, place on ice, every pipe adds the aqua sterilisa of 17.3ul earlier.10ul lancet choicest with the bacterium of going out is got the mono-clonal hickie to aqua sterilisa, the vibration mixing.Add successively: Taq buffer 2.5 μ L, MgCl ₂(25mM) 1.8 μ L, dNTP (2.5mM) 1 μ L, M13+ primer (10pmol) 1 μ L, M13-primer (10pmol) 1 μ L, Taq enzyme 0.4u L.Each reagent gets rid of on the whizzer after all adding well, makes it to sink to the bottom, and places on the PCR appearance.The PCR reaction conditions is 94 ℃ of preparatory sex change after 5 minutes, 94 ℃ 40 seconds, 54 ℃ 40 seconds, 72 ℃ 4 minutes, 35 circulations were extended 4 ℃ of preservations 10 minutes for back 72 ℃.After treating that the PCR reaction gets into 4 ℃, take off the PCR thin-walled tube, get the 7ulPCR product and add the leakage of electricity swimming of 3ul bromine Finland, take a picture after half a hour, observe glue figure, identify segmental size and the small segment rate (Fig. 1) inserted roughly according to glue figure.

Select the single amplified production of band to deliver to the big genome company of China and check order, obtain root of large-flowered skullcap genes involved sequence.

The bioinformatic analysis of embodiment 3, SbCCOMT2 gene

The length of the root of large-flowered skullcap oxygen methyl transferase gene full length gene cDNA that the present invention relates to is 873bp, and detailed sequence is seen the sequence 1 in the sequence table, and wherein opening code-reading frame is positioned at 59～818bp.Root of large-flowered skullcap full length cDNA sequence is carried out the nucleotide homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR DB.This gene on amino acid levels with other species in CCOMT higher homology is arranged, have typical caffeoyl-CoA O-methyltransferase structural domain simultaneously.Like Fig. 2 and 3.

The research of embodiment 4, SbCCOMT2 gene function

1. the structure of plant expression vector

With SbCCOMT2 cDNA is template; Utilize primer P1:5 '-AAGCAGAGCTCAAAACCAGAAC-3 '; P2:5 '-AGCTTGAAGGGGCTAATGAATC-3 ' carries out the PCR reaction, gets the 5ul amplified production and adds the leakage of electricity swimming of 3ul bromine Finland, takes a picture after half a hour; Observe glue figure, amplified fragments is 800bp.Cut amplified production 2 hours with BglII and BstEII at 37 ℃ of following enzymes, utilize recovery test kit (Takara company, China) purifying enzyme to cut product.Utilize BglII and BstEII to cut the pCAMBIA1301 carrier 2 hours simultaneously, add 5ul bromine Finland and carry out agarose gel electrophoresis, observe glue figure, and utilize and reclaim test kit recovery 10000bp fragment at 37 ℃ of following enzymes.

Two fragments that reclaim are mixed, under the effect of dna ligase,, connect product utilization CaCl in 16 ℃ of reactions 12 hours ₂Method changes in the bacillus coli DH 5 alpha, and the picking mono-clonal is also cultivated in containing the LB substratum of 50mg/L, utilizes the SDS method to extract genomic dna.Utilize BglII and BstEII to cut DNA 2 hours at 37 ℃ of following enzymes, add 5ul bromine Finland and carry out agarose gel electrophoresis, observe glue figure, there are two fragments in the result after showing complete degestion, and size is respectively 10000 and 800bp.This plant expression vector called after pC1301 (Fig. 4).

2. transgene tobacco

Utilize Gibco BRL electroporation that plant expression vector pC1301 is imported tumorigenesis Agrobacterium LBA4404.Adopt leaf dish method with the agrobacterium tumefaciens transformation of tobacco (Nicotiana tabacum) that contains pC1301.The substratum that screening transforms seedling is that MS adds 200mg/L Totomycin, 500mg/L carboxylic benzyl mycin and 1.0mg/l 6-BA, 0.2mg/l NAA; The root induction substratum is MS.Extract the total DNA of T0 for transgenic alfalfa; Utilize primer P1:5 '-AAGCAGAGCTCAAAACCAGAAC-3 ', P2:5 '-AGCTTGAAGGGGCTAATGAATC-3 ' to carry out pcr amplification; The agarose gel electrophoresis result shows that amplified fragments is 800bp, explains that the SbCCOMT2 gene has been incorporated in the genome of tobacco.

3. the transgene tobacco meta-bolites is analyzed

According to (Lin such as Lin and Dence; S.Y.; And Dence, C.W. (1992) .Methods in Lignin Chemistry.Springer Series in Wood Science.Berlin, Heidelberg; Springer-Verlag) method is got the content of the dry measuring blade xylogen of 200m.Structure shows that the content of lignin in the transgene tobacco is 2 times of non-transgenic tobacco.

The functional analysis of embodiment 5, sudden change SbCCOMT2 gene

According to SEQ ID No.1 sequences Design mutant primer, and add the PstI restriction enzyme site at 5 ' end, 3 ' end adds the BglII restriction enzyme site, gives birth to worker biotech company synthetic primer by Shanghai, and sequence is following: 5 '-AAGC CGAGCTCA TAAC GAGAAC-3 ' obtains 1 SbCCOMT2 gene that contains 3 base mutations through the PCR method amplification.Mutator gene is cloned in the Pst I/Bgl II restriction enzyme site of p1301 carrier; Obtain the series vegetable expression vector; Adopt leaf dish method will contain agrobacterium tumefaciens (Agrobacterium tumefaciens) the difference transformation of tobacco (Nicotiana tabacum) of deleted carrier; Extract the total flavones of transgene tobacco, concrete grammar is with embodiment 4.The result shows that the transcriptional activity of mutator gene and SbCCOMT2 gene do not have significant difference.

Claims

1. root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT) gene is characterized in that it is one of following nucleotide sequences:

(1) dna sequence dna of SEQ ID No.1;

(3) nucleotide sequence of aminoacid sequence shown in the coding SEQ ID No.2;

2. root of large-flowered skullcap caffeoyl coenzyme A-3-O-methyltransgerase (SbCCOMT) is characterized in that, is one of following aminoacid sequence:

(1) has the aminoacid sequence shown in the SEQ ID No.2;

3. recombinant vectors is characterized in that: contain the described root of large-flowered skullcap caffeoyl coenzyme A of claim 1-3-O-methyltransgerase (SbCCOMT) gene complete sequence or partial sequence.

4. host cell, it is characterized in that: it contains the described root of large-flowered skullcap caffeoyl coenzyme A of claim 1-3-O-methyltransgerase (SbCCOMT) gene complete sequence or partial sequence.