CN102465133B - Lonicera japonica chalcone synthase (LjCHS) gene, protein coded by LjCHS gene and application of LjCHS gene - Google Patents
Lonicera japonica chalcone synthase (LjCHS) gene, protein coded by LjCHS gene and application of LjCHS gene Download PDFInfo
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Abstract
The invention discloses a chalcone synthase gene, protease coded by the gene and application of the gene. The gene is extracted from a cDNA library of Lonicera japonica for the first time, which fills a gap in separation and cloning of a chalcone synthase gene from the traditional Chinese medicinal material Lonicera japonica in China. The chalcone synthase gene provided in the invention has a nucleotide sequence as represented by SEQ ID No: 1, a homologous sequence which is obtained through addition, replacement, insertion or deletion of one or more nucleotide of the nucleotide sequence, an allele of the nucleotide sequence or a derived nucleotide sequence of the nucleotide sequence. Protein coded by the gene has an amino acid sequence as represented by SEQ ID No: 2 or a homologous sequence which is obtained through addition, replacement, insertion or deletion of one or more amino acid of the amino acid sequence. The chalcone synthase gene provided in the invention can increase the content of the flavonoid active component Lonicera japonica flavonoids in Lonicera japonica by using a biological technology, is beneficial for improvement of the quality of the medicinal material Lonicera japonica, can be used for variety breeding and has a good application prospect.
Description
Technical field
The invention belongs to biological technical field, relate generally to by making up Japanese Honeysuckle cDNA library technology and obtain chalcone synthase gene and coded product thereof, belong to the Gene Engineering of Medicinal Plants 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 gradually the focus of research, these gene clonings will be provided fundamental basis for the formation that 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 intermediate simultaneously.
Flavonoid compound is one of secondary metabolite important in the plant, and flavones ingredient is also by the quality control standard of pharmacopeia in 2010 as Japanese Honeysuckle.At present in the research of Flavonoids biosynthetic pathway, chalcone synthase is first enzyme in the plant flavonoids material route of synthesis, being the synthetic rate-limiting enzyme of flavonoid, also is one of key enzyme in the Secondary Metabolism of Plant approach, and plant is had very important physiological significance.Therefore the acquisition of chalcone synthase (LjCHS) gene is for the content that improves honeysuckle effective part with genetic engineering means provides important foundation.Before the present invention comes forth, any disclose or reported Japanese Honeysuckle flavones fermentoid gene and aminoacid sequence thereof mentioned in the present patent application are arranged not yet.
Summary of the invention
The object of the present invention is to provide a kind of Japanese Honeysuckle chalkane synthetase (LjCHS) 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.
Japanese Honeysuckle chalkane synthetase provided by the present invention (LjCHS) is one of following nucleotide sequence:
(1) dna sequence dna of SEQ ID No.1;
(2) under stringent condition, has the dna molecular of the protein molecule of Japanese Honeysuckle chalkane synthetase activity with the hybridization of the dna sequence dna of SEQ ID No.1 and coding;
(3) nucleotide sequence of aminoacid sequence shown in the coding SEQ ID No.2;
(4) coding to SEQ ID No.2 through add, replace, insert or delete one or more amino acid and having the nucleotide sequence of the protein of Japanese Honeysuckle chalkane synthetase activity.
Japanese Honeysuckle chalkane synthetase provided by the present invention (LjCHS) 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 is through add, replace, insert or delete one or more amino acid and having the sequence of Japanese Honeysuckle chalkane synthetase.
The recombinant vectors that contains Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS) gene complete sequence or partial sequence, such as protokaryon class carrier, eucaryon class expression vector and RNAi carrier all belong to protection scope of the present invention
Contain the host cell of Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS) 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.
Described host cell is valuable living materials.
The application of Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS) gene comprises and uses described recombinant vectors, such as the plant expression vector transformed plant cells; Perhaps cultivate altogether with described Agrobacterium and the vegetable cell that contains this gene, obtain genetically modified plant rooting system; Perhaps use described root of hair cell regeneration plant; Perhaps use described Japanese Honeysuckle chalkane synthetase (LJCHS) gene complete sequence or partial sequence to transform and obtain transgenic organism.
The concept particular content that relates in the technical solution of the present invention is as follows:
The dna molecular of said Japanese Honeysuckle chalkane synthetase (LjCHS) gene comprises: coding has the nucleotide sequence of the active polypeptide of Japanese Honeysuckle chalkane synthetase (LjCHS), and shows at least 70% homology from the nucleotides sequence of Nucleotide 1-980 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 1-980.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 1-980 position.The isolated Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS) 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.Preferably, this polypeptide is the polypeptide with SEQ ID NO.2 sequence.Dna molecular among the present invention comprises 8-100 continuous nucleotide in the described dna molecular.In the present invention, " separation ", " purifying " DNA refer to, 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 " Japanese Honeysuckle chalkane synthetase (LjCHS) (or polypeptide) " gene refers to: coding has the nucleotide sequence of the active polypeptide of Japanese Honeysuckle chalkane synthetase (LjCHS), such as 1-980 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence refers to, is arranged in the encoder block 1-980 position Nucleotide of SEQ ID NO.1 sequence, the sequence that has one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces.Because the degeneracy of codon, thus with SEQ ID NO.1, in 1-980 position nucleotide sequence homology be low to moderate approximately 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 1-980 position.Also comprise with SEQ IDNO.1 in from the homology at least 70% of the nucleotide sequence of Nucleotide 1-980 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 SEQ ID NO.1 with the albumen of natural Japanese Honeysuckle chalkane synthetase (LjCHS) 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 (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 " Japanese Honeysuckle chalkane synthetase (LjCHS) albumen or polypeptide " refers to: the polypeptide with the active SEQ ID NO.2 sequence of Japanese Honeysuckle chalkane synthetase (LjCHS).This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural Japanese Honeysuckle chalkane synthetase (LjCHS) 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 with the close or similar amino acid of performance, usually can not change the function of protein.Again such as, add the function that or several amino acid also can not change protein usually at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of Japanese Honeysuckle chalkane synthetase (LjCHS), also comprises operationally being connected in the derivative that signal peptide, promotor or ribosome bind site sequence form.The variant form of Japanese Honeysuckle Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS) 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 Japanese Honeysuckle chalkane synthetase (LjCHS) DNA hybridization and the polypeptide or the albumen that utilize the serum of Japanese Honeysuckle chalkane synthetase (LjCHS) polypeptide to obtain.
Japanese Honeysuckle chalkane synthetase (LjCHS) conservative property variation polypeptide refers among the present invention: 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 Japanese Honeysuckle chalkane synthetase (LjCHS) or polypeptide.The difference of these analogues and natural Japanese Honeysuckle chalkane synthetase (LjCHS) polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise genetic variant natural or that induce.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-(such as D-amino acid), and the analogue with that non-natural exists or synthetic amino acid (such 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 (usually not changing primary structure) form comprises: 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 such as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (such 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 (such 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, 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 Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS) polypeptide, the nucleotide sequence of Japanese Honeysuckle chalkane synthetase (LjCHS) gene operationally can be connected in expression regulation sequence, thereby form Japanese Honeysuckle chalkane synthetase (LjCHS) expression vector.Described " operationally being connected in " refers to a kind of like this situation, and namely some part of linear DNA sequence can affect 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.
The present invention is the expression of available Northern blotting technical Analysis Japanese Honeysuckle chalkane synthetase (LjCHS) gene product also, namely analyzes existence and the quantity of rna transcription thing in cell of Japanese Honeysuckle chalkane synthetase (LjCHS).
In addition, the nucleic acid molecule that can be used as probe among the present invention has 8-100 continuous nucleotide of Japanese Honeysuckle chalkane synthetase (LjCHS) 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 Japanese Honeysuckle chalkane synthetase (LjCHS).The present invention relates to whether exist in the test sample method of Japanese Honeysuckle chalkane synthetase (LjCHS) nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing, and then whether detection probes combination has occured.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to Japanese Honeysuckle chalkane synthetase (LjCHS) nucleotide coding sequence, and can be positioned at both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.In addition, according to Japanese Honeysuckle chalkane synthetase (LjCHS) nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening Japanese Honeysuckle chalkane synthetase (LjCHS) source gene or homologous protein.
In order to obtain the dot matrix of the Japanese Honeysuckle cDNAs relevant with Japanese Honeysuckle chalkane synthetase (LjCHS), can screen the Japanese Honeysuckle cDNA library with dna probe, these probes are under low rigorous condition, use
32P Japanese Honeysuckle chalkane synthetase (LjCHS) gene all or part of cooked the radioactivity mark and.The cDNA library that is best suited for screening is the library from Japanese Honeysuckle.Structure is that biology field is well-known from the method for 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 identified the nucleotide sequence of the gene family of Japanese Honeysuckle chalkane synthetase (LjCHS).
The Nucleotide full length sequence of the gene family of Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS) 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 in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell over to again, then separates obtaining relevant sequence from the host cell after the propagation by ordinary method.In addition, also can will suddenly change by chemosynthesis and introduce in the protein sequence of the present invention.Except producing with recombination method, the fragment of albumen of the present invention is available solid phase technique also, is produced (people such as Stewart, (1969) Solid-hase Peptide Synthesis by direct peptide synthesis, WHFreeman Co., San Francisco; Merrifield J. (1963) J.Am Chem.Soc85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can come automatic pressing to become peptide with the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems.Can distinguish each fragment of chemosynthesis albumen of the present invention, then be connected to produce the molecule of total length with chemical process.Utilize Japanese Honeysuckle chalkane synthetase of the present invention (LjCHS), by various conventional screening methods, can filter out with Japanese Honeysuckle chalkane synthetase (LjCHS) interactional material occurs, perhaps acceptor, inhibitor or antagonist etc.
Japanese Honeysuckle chalkane synthetase provided by the present invention (LjCHS) gene is to clone first preparation from Japanese Honeysuckle, has filled up from the blank of China's medical herb Japanese Honeysuckle chalkane synthetase (LjCHS) gene.Utilize the present invention can improve by genetic engineering technique the content of Flavonoid substances in the plants such as Japanese Honeysuckle, transgene result demonstration, Japanese Honeysuckle Japanese Honeysuckle chalkane synthetase (LjCHS) gene pairs promote the raising of Japanese Honeysuckle Flavone content that obvious effect is arranged.Japanese Honeysuckle chalkane synthetase (LjCHS) gene can be used for improving by transgenic technology research and the industrialization of Japanese Honeysuckle flavones content, especially the quality-improving that can be used for the Chinese medicinal materials Japanese Honeysuckle, have preferably promoter action to improving Japanese Honeysuckle Flavonoid substances output, have good application prospect.
Description of drawings:
Fig. 1: Japanese Honeysuckle RNA, cDNA, cDNA library clone's pcr amplification electrophorogram;
Fig. 2: LjCHS functional domain forecast analysis (deriving from ncbi database);
Fig. 3: LjCHS systematic evolution tree (adjacent method);
Fig. 4: plant expression vector pC1301 physical map;
Embodiment
The structure of embodiment 1, Japanese Honeysuckle cDNA library
1, the separation and detection of the total RNA of Japanese Honeysuckle
Extracting honeysuckle (Lonicera japonica Thunb) bud 2g, in mortar with the quick grind into powder of liquid nitrogen, (CTAB (W/V) 2%, Tris-HCl (pH8.0) 100mmolL in the 10mL Extraction buffer of fast transfer to 65 ℃ preheating
-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 liquor adds the 10M LiCl of 1/4 volume, places 4 ℃ of precipitations behind the mixing and spends the night; Centrifugal 20 minutes of 7500g, (SDS 0.5%, NaCl 1molL with 500 μ L SSTE for precipitation
-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 liquor adds 2 times of volume dehydrated alcohols, places 2h for-70 ℃; Centrifugal 20 minutes of 4 ℃ of 13000g, the precipitation drying at room temperature is dissolved in after 10 minutes in the water that 100 μ L DEPC process, and detects the integrity of RNA with 1.0% agarose electrophoresis, with GenQuant nucleic acid quantification instrument mensuration A260, A280 ratio and concentration.Place-80 ℃ of refrigerators for subsequent use.
2, the structure of 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).
Embodiment 2: the clone of Japanese Honeysuckle genes involved
Random 5000 mono-clonals of picking carry out bacterium colony PCR to be identified.Get an amount of PCR thin-walled tube, place on ice, every pipe adds first the aqua sterilisa of 17.3ul.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 0.4 μ 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 instrument.The PCR reaction conditions is 94 ℃ of denaturations after 5 minutes, 94 ℃ 40 seconds, 54 ℃ 40 seconds, 72 ℃ 4 minutes, 4 ℃ of preservations were extended in rear 72 ℃ of 35 circulations 10 minutes.After the PCR reaction enters 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 an hour, observe glue figure, identify roughly size and the small segment rate (Fig. 1) of Insert Fragment according to glue figure.
Select the single amplified production of band to deliver to the large genome company of China and check order, obtain Japanese Honeysuckle genes involved sequence.
The bioinformatic analysis of embodiment 3, LjCHS gene
The length of the Japanese Honeysuckle chalcone synthase gene full-length cDNA that the present invention relates to is 980bp, and detailed sequence is seen the sequence 1 in the sequence table, and wherein opening code-reading frame is positioned at 85～798bp.The Japanese Honeysuckle 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 database.This gene on amino acid levels with other species in CHS higher homology (2 and 3) is arranged.
Embodiment 4, LjCHS gene functional research
1. the structure of plant expression vector
Take LjCHS cDNA as template, utilize primer P1:5 '-TACCACCTCTACAAGCATC-3 ', P2:5 '-GCGAAGATGCCCGTCAAT-3 ' carries out the PCR reaction, get the 5ul amplified production and add the leakage of electricity swimming of 3ul bromine Finland, take a picture after half an hour, observe glue figure, amplified fragments is 800bp.Cut amplified production 2 hours with BglII and BstEII at 37 ℃ of lower enzymes, utilize recovery test kit (Takara company, China) purifying enzyme to cut product.Utilize simultaneously BglII and BstEII to cut the pCAMBIA1301 carrier 2 hours at 37 ℃ of lower enzymes, add 5ul bromine Finland and carry out agarose gel electrophoresis, observe glue figure, and utilize and reclaim test kit recovery 10000bp fragment.
Two fragments that reclaim are mixed, under the effect of dna ligase, in 16 ℃ of reactions 12 hours, connect product utilization CaCl
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 lower 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 the tumorigenesis Agrobacterium LBA4404.Adopt the leaf dish method agrobacterium tumefaciens transformation of tobacco (Nicotiana tabacum) that contains pC1301.The substratum of screening transformation seedlings 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 T0 for total DNA of transgenic alfalfa, utilize primer P1:P1:5 '-TACCACCTCTACAAGCATC-3 ', P2:5 '-GCGAAGATGCCCGTCAAT-3 ' carries out pcr amplification, the agarose gel electrophoresis result shows that amplified fragments is 800bp, illustrates that the LjCHS gene has been incorporated in the genome of tobacco.
3. transgene tobacco Methanogenesis
According to (Hildegard Kaulen such as Hildegard Kaulen, Jeff Schell and Fritz Kreuzaler.Light-induced expression of the chimeric chalcone synthase-NPTII gene in tobaccocells) method is got the content of the dry measuring blade chalkane synthetase of 200m.Structure shows, the chalkane synthetase content in the transgene tobacco is 3 times of non-transgenic tobacco.
Embodiment 5, sudden change LjCHS functional analyses of genes
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, give birth to worker biotech company synthetic primer by Shanghai, sequence is as follows: 5 '-TACGACCTCGACATGCATC-3 ' obtains 1 LjCHS gene that contains 3 base mutations by the PCR method amplification.Mutator gene is cloned in the Pst I/BglII 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 LjCHS gene do not have significant difference.
Claims (6)
1. a Japanese Honeysuckle chalkane synthetase is characterized in that, its aminoacid sequence is shown in SEQ ID No.2.
2. the encoding gene of the described Japanese Honeysuckle chalkane synthetase of claim 1.
3. gene as claimed in claim 2, its nucleotide sequence is shown in SEQ ID No.1.
4. recombinant vectors, it is characterized in that: this recombinant vectors contains Japanese Honeysuckle chalcone synthase gene sequence claimed in claim 2.
5. host cell, it is characterized in that: it contains Japanese Honeysuckle chalcone synthase gene sequence claimed in claim 2.
6. gene claimed in claim 2 is in the application that improves by transgenic technology in the Japanese Honeysuckle flavones content.
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| CN103045621B (en) * | 2013-01-25 | 2014-05-21 | 山东大学 | P-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and application thereof |
| CN106591333B (en) * | 2016-12-12 | 2018-04-13 | 北京农学院 | Early lilac flower chalcone synthase gene and its application |
| CN108239650A (en) * | 2018-01-19 | 2018-07-03 | 海南省农业科学院粮食作物研究所 | A kind of sweet potato enzyme, namely chalcone isomerase IbCHIL1 and its encoding gene and application |
| CN110343680B (en) * | 2019-08-01 | 2020-03-17 | 济南广盛源生物科技有限公司 | Honeysuckle chalcone synthase mutant and application thereof |
| CN114410666A (en) * | 2021-12-28 | 2022-04-29 | 安徽中医药大学 | Polygonum multiflorum chalcone synthase gene FmCHS1, and encoding product and application thereof |
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| CN1603412A (en) * | 2004-10-27 | 2005-04-06 | 武汉大学 | Gene sequence for lotus root chalcone synthase and its use |
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| Title |
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| AJ413277;De Schepper,S.R.;《GENBANK》;20050415;序列全长 * |
| De Schepper,S.R..AJ413277.《GENBANK》.2005,序列全长. |
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