CN107190016B - A kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and application - Google Patents
A kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and application Download PDFInfo
- Publication number
- CN107190016B CN107190016B CN201710334743.6A CN201710334743A CN107190016B CN 107190016 B CN107190016 B CN 107190016B CN 201710334743 A CN201710334743 A CN 201710334743A CN 107190016 B CN107190016 B CN 107190016B
- Authority
- CN
- China
- Prior art keywords
- dcchil1
- enzyme
- gene
- chalcone isomerase
- cambodinna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y505/00—Intramolecular lyases (5.5)
- C12Y505/01—Intramolecular lyases (5.5.1)
- C12Y505/01006—Chalcone isomerase (5.5.1.6)
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Nutrition Science (AREA)
- Cell Biology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and applications.The nucleotide sequence of the gene includes: nucleotide sequence shown in (a) SEQ ID NO:1；Or (b) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases nucleotide sequence that is one or more and expressing identical function protein；Or nucleotide sequence (c) with (a) or (b) has 90% or more homology and expresses the nucleotide sequence of identical function protein.Described its amino acid sequence of enzyme, namely chalcone isomerase DcCHIL1 is as shown in SEQ ID NO:2；Or the amino acid sequence with same function is formed through replacement, missing or one or several amino acid of addition for SEQ ID NO:2.The present invention clones for the first time obtains Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 gene, obtained DcCHIL1 albumen has enzyme, namely chalcone isomerase activity, the gene is transferred to the yield that Dracaena cambodinna Flavonoids Accumulation and dragon's blood can be improved in Dracaena cambodinna using biotechnology, is had broad application prospects and great economic value.
Description
Technical field
The present invention relates to plant biotechnology fields, and in particular to a kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1
And its encoding gene and application.
Background technique
Flavonoids is small molecule secondary metabolites, wide participation flower color formation, UV protection,
Plant fertility, resists the biological processes such as osmotic stress, cell cycle regulating at Auxin transport.In addition, flavonoids is also
It is a kind of very strong antioxidant of bioactivity, has and the doctor such as adjust immunity of organisms, anti-oxidant, anti-aging and resist virus
Treat value.Enzyme, namely chalcone isomerase (Chalcone isomerase, CHI) is the key that speed limit in flavonoid biosynthesis pathway
Enzyme, catalysis chalcone are isomerized to flavanones, and flavanones is then converted into various types of flavonoids.CHI superfamily includes four
Seed type albumen: I type is prevalent in vascular plant, is responsible for the synthesis of flavonoids；II type is primarily present in leguminous plant, ginseng
It is synthesized with isoflavones；III type is the fatty acid binding protein being widely present in terrestrial plant；It has been found that IV type albumen do not have
It is active there is enzyme, namely chalcone isomerase, but can be used as enhancer, promotes flavonoids synthesis.
Dragon's blood is a kind of traditional rare Chinese medicine, is a kind of red resin, there is the good reputation of " panacea of promoting blood circulation ".Modern pharmacology
Learn studies have shown that dragon's blood have promote blood circulation, hemostasia effect, hypoglycemic, blood lipid, strengthen immunity, antibacterial, promote skin
The multiple pharmacological effects such as reparation, anti-spasm, anti-inflammatory, analgesia, anti-diabetic, antitumor.Dracaena cambodinna is the master of domestic Dragon Blood
Original plant is wanted, dragon's blood main chemical compositions are flavonoids.At present to the research of dragon's blood concentrate on chemical component and
Pharmacological activity, biosynthesis pathway and induction generation mechanism to dragon's blood understand few.
CHI gene, such as arabidopsis, soybean, apple, grape are cloned into from various plants at present.Utilize gene
Engineering technology can promote the biosynthesis of flavonoids in CHI gene transfered plant, have development and application prospect.It such as will be short
It leads a cow in CHI Gene into Tomato, Flavone content improves 78 times in transgene tomato pericarp, and flavonols increases 21 in pulp
Times；After Saussurea medusa CHI channel genes Xinjiang Saussurea involucrate, general flavone amount can be improved 4 times in transgenosis saussurea involucrata root of hair.But for sea
The clone of southern dragon tree CHI gene, expression pattern, protein expression and effect in flavonoids biosynthesis are unclear.
Summary of the invention
The purpose of the present invention is to provide a kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 gene and Hainan dragon's bloods
Enzyme, namely chalcone isomerase DcCHIL1 is set, the deficiency of Flavonoid Content in Dracaena cambodinna can not be improved to solve the prior art.
To achieve the above object, the method for the present invention is to be achieved by the following technical programs:
Present invention firstly provides a kind of Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1, the nucleotides sequences of the gene
Column include:
(a) nucleotide sequence shown in SEQ ID NO:1；Or
(b) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more and expression phase
The nucleotide sequence of congenerous protein；Or
(c) nucleotide sequence with (a) or (b) has 90% or more homology and expresses the nucleosides of identical function protein
Acid sequence.
The present invention also provides a kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1, amino acid sequence such as SEQ ID
Shown in NO:2；Or the ammonia with same function is formed through replacement, missing or one or several amino acid of addition for SEQ ID NO:2
Base acid sequence.
Dracaena cambodinna is the main Original plant of domestic Dragon Blood, and dragon's blood main chemical compositions are flavonoids.The present invention
The weak status of basic research is synthesized and regulated and controled for Dracaena cambodinna flavonoids, and clone obtains Dracaena cambodinna chalcone for the first time
The gene is transferred in Dracaena cambodinna by isomerase DcCHIL1 gene and enzyme, namely chalcone isomerase DcCHIL1 using biotechnology
The yield that Dracaena cambodinna Flavonoids Accumulation and dragon's blood can be improved has broad application prospects and great economic value.
The cloning process of the Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1, comprises the following steps:
(1) special primer P1 forward primer and P2 reverse primer are designed；
(2) PCR amplification is carried out by CDS sequence of the template to DcCHIL1 gene of Dracaena cambodinna stem the first chain cDNA；
(3) PCR product for obtaining amplification connects pMD18-T carrier, converts competent escherichia coli cell, and screening is positive
It clones and is sequenced.
Preferably, as shown in SEQ ID NO:3, the P2 reversely draws the sequence of P1 forward primer described in step (1)
The sequence of object is as shown in SEQ ID NO:4.
Preferably, PCR expansion condition described in step (2) are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C
Anneal 30sec, 70 DEG C of extension 1min, totally 35 circulations；70 DEG C of extension 8min.
The present invention also provides a kind of recombinant expression carrier or expression cassette, the recombinant expression carrier or expression cassette are containing having the right
It is required that Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 described in 1.
The present invention also provides a kind of transgenic cell line or recombinant bacterium, the transgenic cell line or recombinant bacterium are containing having the right
It is required that Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 described in 1.
The present invention also provides a kind of host cell, the host cell, which contains Dracaena cambodinna described in claim 1, to be looked into
You are ketone isomerase gene DcCHIL1.
The present invention also provides the applications of above-mentioned Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1, by the gene application
In eukaryon or prokaryotic expression.
The present invention also provides the applications of Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1, by the gene by turning base
Because technology is used to improve the yield of Dracaena cambodinna Flavonoids Accumulation and dragon's blood.
The method of the present invention has the advantages that the present invention provides a kind of completely new genes: Dracaena cambodinna chalcone is different
Structure enzyme gene DcCHIL1, the gene order can encode key enzyme chalcone in Dracaena cambodinna flavonoid biosynthesis pathway
Isomerase DcCHIL1；The gene is transferred to Escherichia coli and carries out inducing expression, obtaining DcCHIL1 albumen has chalcone isomery
Chalcone can be changed into flavanones by enzymatic activity.The Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 can regulate and control
Dracaena cambodinna flavonoids biosynthesis, the gene, which is transferred in Dracaena cambodinna, using biotechnology can be improved Hainan dragon's blood
The yield for setting Flavonoids Accumulation and dragon's blood, has broad application prospects and great economic value.
Detailed description of the invention
Fig. 1 is the phylogenetic analysis of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1.
Fig. 2 is the prokaryotic expression and activity analysis figure of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1.
Wherein, A is the prokaryotic expression and protein purification of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1；Swimming lane 1-4:
IPTG induces Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 protein expression after 0h, 1h, 3h, 5h；Swimming lane 5 is Hainan of purifying
Dragon tree enzyme, namely chalcone isomerase DcCHIL1；B is the activity analysis of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1；a,2',4,
4', 6'- tetrahydroxy chalcone (NC) standard items；B, 4', 5,7- trihydroxyflavone (NA) standard items；C: Dracaena cambodinna chalcone
The HPLC result that isomerase DcCHIL1 is reacted with naringenin chalcone.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
The clone of 1 Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 of embodiment
By analyzing Dracaena cambodinna stem transcript profile sequencing result early period, special primer P1 forward primer is designed
5 '-ATGGGCTCTGAGATAGTGATGGTG-3 ' (SEQ ID NO:3) and P2 reverse primer 5 '-
GCAGCAGATAACATAGTCCCAAG-3 ' (SEQ ID NO:4), using Dracaena cambodinna stem the first chain cDNA as template pair
The CDS sequence of DcCHIL1 gene carries out PCR amplification, amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C
Anneal 30sec, 70 DEG C of extension 1min, totally 35 circulations；70 DEG C of extension 8min.The PCR product that amplification is obtained connects pMD18-T
Carrier converts competent escherichia coli cell, and screening positive clone is simultaneously sequenced, and obtains Dracaena cambodinna enzyme, namely chalcone isomerase gene
The complete encoding sequence 642bp of DcCHIL1.
The above-mentioned recombinant vector containing Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 is named as pMD18-
DcCHIL1。
The sequence of 2 Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 of embodiment is analyzed
The overall length open reading frame of Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 is 642bp, and detailed sequence is shown in
SEQ ID NO:1.The amino acid sequence of DcCHIL1 is derived according to open reading frame sequence, totally 213 amino acid residues are (detailed
Thin sequence is shown in SEQ ID NO:2), molecular weight 23.8kDa, theoretical isoelectric point is 4.97.
Respectively from the different plants such as arabidopsis, corn, picking 31 different classes of CHI sequence (plant species and CHI
Protein sequence number is detailed in Fig. 1) it is used as template, to analyze the phyletic evolution status of DcCHIL1.Using MEGA7 software to this 32
CHI sequence carries out Multiple sequence alignments, and phylogenetic tree construction (such as Fig. 1).It is upper it can be clearly seen that these CHI from figure
4 branches can be divided into, DcCHIL1 gets together with IV type CHI member of plant, belongs to IV type CHI.
The building of 3 PET28a-DcCHIL1 recombinant expression carrier of embodiment
Utilize the restricted of 5 software of the Premier analysis code area Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1
Endonuclease digestion site determines that two restriction enzymes for carrier construction are EcoRI and SalI.Design Hainan dragon's blood
Set the code area enzyme, namely chalcone isomerase gene DcCHIL1 primer PE1:5 '-GCGAATTCATGGGCTCTGAGATAGTGATGGTG-3 '
(forward primer is marked as EcoRI restriction enzyme site) and PE2:5 '-GCGTCGACAGCAGCAGATAACATAGTCCC-3 ' are (reversed
Primer is marked as SalI restriction enzyme site), it is that template carries out PCR amplification with pMD18-DcCHIL1 (embodiment 1 is prepared), expands
Increasing condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 58 DEG C of annealing 30sec, 70 DEG C of extension 1min, totally 35 recycle；
70 DEG C of extension 8min.The PCR product that amplification is obtained connects pMD18-T carrier, obtains pMD18-E DcCHIL1 recombinant plasmid,
The correctness of aim sequence is determined through being sequenced.EcoRI and SalI is bis- in 37 DEG C of water-baths cuts pMD18-E DcCHIL1 recombinant plasmid,
Recycle the target fragment of 650bp or so.By between EcoRI the and SalI restriction enzyme site of segment insertion PET28a plasmid, obtain
Recombinant vector is identified through digestion and sequencing, confirms that the sequence of insertion is correct.Building is different containing Dracaena cambodinna chalcone
The expression vector of structure enzyme gene DcCHIL1 coding region sequence is named as pET28a-DcCHIL1.
The inducing expression and protein purification of 4 DcCHIL1 of embodiment
Expression vector plasmid pET28a-DcCHIL1 (embodiment 3 is prepared) is transformed into e. coli bl21 (DE3)
In, it screens and identifies recon.Recon is inoculated into the LB culture medium containing kanamycins (50 μ g/mL of final concentration), 37 DEG C
Shake culture overnight after, be transferred in new culture medium in 1:100 (V/V) ratio, when culture is to OD600=0.6-0.8, addition
Thalline were collected by centrifugation after IPTG to final concentration of 1Mm, culture 1h, 3h and 5h, not add IPTG culture medium to be control, extracts cell
Total protein carries out 12%SDS-PAGED electrophoresis detection.The result shows that DcCHIL1 obtains efficient heterogenous expression in Escherichia coli,
And the apparent molecular weight for expressing albumen (Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1) is close with theoretical molecular weight, about
28kDa.Fusion protein occurs in the form of inclusion body, the Protein Refolding of the separation reference MERCK company of albumen
Kit specification carries out, protein product (Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1) (specific method ginseng after ni-sepharose purification
According to the Protino Ni-NTA Columns product description of MN company) it is a clear single band, it is consistent with expected size, it can
It is analyzed for external enzyme activity.
The activity analysis of 5 recombinant protein of embodiment
Using HPLC method measurement recombinant protein, (i.e. the protein product Dracaena cambodinna chalcone that is prepared of embodiment 4 is different
Structure enzyme DcCHIL1) activity.Enzyme activity reaction system is 200 μ l, includes 10 μ l of recombinant protein, substrate 2', 4,4', 6'- tetrahydroxy
Chalcone (5mM) 2 μ l, Tris-HCl (100mM, pH 7.5) supply 200 μ l.Reaction condition: 200 μ are added in 30 DEG C of water-bath 5min
The ethyl acetate of l reacts to terminate.It is mixed after ethyl acetate is added, 10000rmp is centrifuged 10min, and solution is in stratification state, takes
Top ethyl acetate portion, repetition are extracted twice, and are volatilized ethyl acetate with vacuum pump, and the chromatography methanol of 100 μ l is added, are mixed
It is even.External enzyme activity determination is carried out using HPLC (High performance liquid chromatography).It draws above-mentioned
Reactant loading, 20 μ L of applied sample amount system default.40 DEG C of column temperature, flow velocity 0.8m L/min, Detection wavelength 270nm.As the result is shown
Recombinant protein (Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1) is shown can be by all substrate 2', 4,4', 6'- tetrahydroxys
Chalcone is fully converted into product 4', and the ability (as shown in Figure 2) of 5,7- trihydroxyflavones, this illustrates recombinant protein (Hainan dragon
Blood tree enzyme, namely chalcone isomerase DcCHIL1) activity with enzyme, namely chalcone isomerase.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
SEQUENCE LISTING
<110>China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute
<120>a kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and application
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 642
<212> DNA
<213>artificial sequence
<400> 1
atgggctctg agatagtgat ggtggatgaa tttcctttcc cgactgagat cactacaaca 60
gaggctttgc ctctcttggg ttctggcatc acggatattg aaatccattt ccttcaaatc 120
aagcacagcg caatcggtat ttatatggaa cgaagaattg ttgagcatct caggaactgg 180
gagggctgga aggggaaaac gggcagcgag cttgcagcgg acgatgattt cttctatgct 240
atcgtgtcag ctcctgtggc aaagtacttt cgagttgtgg tgatcaagga gataaaaggg 300
gctcaattcg gcctgcagat cgaaactgca gtgagagata ggttggtggc ttgcgataag 360
tttgaagaca aggaagaaga agagctagaa aagattgttc agtttttcca aacaccgtac 420
ttgaaaaagg gttcagttat cacatttcac ttccctgcga gttcttatac tgcagagata 480
tcgtatgcga tcgaaggtaa agatgaggcg aagatcaagg tggagaatgc gaatgtagct 540
gaaatgattc agaaatggta tttgggtggg tctacagctg tgtctccaag cactgtgcag 600
agcttggctg aaagacttgg gactatgtta tctgctgctt aa 642
<210> 2
<211> 213
<212> PRT
<213>artificial sequence
<400> 2
Met Gly Ser Glu Ile Val Met Val Asp Glu Phe Pro Phe Pro Thr Glu
1 5 10 15
Ile Thr Thr Thr Glu Ala Leu Pro Leu Leu Gly Ser Gly Ile Thr Asp
20 25 30
Ile Glu Ile His Phe Leu Gln Ile Lys His Ser Ala Ile Gly Ile Tyr
35 40 45
Met Glu Arg Arg Ile Val Glu His Leu Arg Asn Trp Glu Gly Trp Lys
50 55 60
Gly Lys Thr Gly Ser Glu Leu Ala Ala Asp Asp Asp Phe Phe Tyr Ala
65 70 75 80
Ile Val Ser Ala Pro Val Ala Lys Tyr Phe Arg Val Val Val Ile Lys
85 90 95
Glu Ile Lys Gly Ala Gln Phe Gly Leu Gln Ile Glu Thr Ala Val Arg
100 105 110
Asp Arg Leu Val Ala Cys Asp Lys Phe Glu Asp Lys Glu Glu Glu Glu
115 120 125
Leu Glu Lys Ile Val Gln Phe Phe Gln Thr Pro Tyr Leu Lys Lys Gly
130 135 140
Ser Val Ile Thr Phe His Phe Pro Ala Ser Ser Tyr Thr Ala Glu Ile
145 150 155 160
Ser Tyr Ala Ile Glu Gly Lys Asp Glu Ala Lys Ile Lys Val Glu Asn
165 170 175
Ala Asn Val Ala Glu Met Ile Gln Lys Trp Tyr Leu Gly Gly Ser Thr
180 185 190
Ala Val Ser Pro Ser Thr Val Gln Ser Leu Ala Glu Arg Leu Gly Thr
195 200 205
Met Leu Ser Ala Ala
210
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<400> 3
atgggctctg agatagtgat ggtg 24
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<400> 4
gcagcagata acatagtccc aag 23
Claims (10)
1. a kind of Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1, which is characterized in that the nucleotide sequence of the gene is such as
Nucleotide sequence shown in SEQ ID NO:1.
2. a kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1, which is characterized in that its amino acid sequence such as SEQ ID NO:2
It is shown.
3. the cloning process of Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 described in claim 1, which is characterized in that
It comprises the following steps:
(1) special primer P1 forward primer and P2 reverse primer are designed；
(2) PCR amplification is carried out by CDS sequence of the template to DcCHIL1 gene of Dracaena cambodinna stem the first chain cDNA；
(3) PCR product for obtaining amplification connects pMD18-T carrier, converts competent escherichia coli cell, screening positive clone
And it is sequenced.
4. cloning process according to claim 3, which is characterized in that the sequence of P1 forward primer described in step (1)
As shown in SEQ ID NO:3, the sequence of the P2 reverse primer is as shown in SEQ ID NO:4.
5. cloning process according to claim 3, which is characterized in that PCR amplification condition described in step (2) are as follows: 94
DEG C initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 70 DEG C of extension 1min, totally 35 recycle；70 DEG C of extensions
8min。
6. a kind of recombinant expression carrier or expression cassette, which is characterized in that the recombinant expression carrier or expression cassette contain such as right
It is required that Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 described in 1.
7. a kind of transgenic cell line or recombinant bacterium, which is characterized in that the transgenic cell line or recombinant bacterium contain such as right
It is required that Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 described in 1.
8. a kind of host cell, which is characterized in that the host cell contains Dracaena cambodinna Cha Er as described in claim 1
Ketone isomerase gene DcCHIL1.
9. the application of Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 described in claim 1, which is characterized in that by this
Gene is applied to eukaryon or prokaryotic expression.
10. the application of Dracaena cambodinna enzyme, namely chalcone isomerase gene DcCHIL1 described in claim 1, which is characterized in that by this
Gene is used to improve the yield of Dracaena cambodinna Flavonoids Accumulation and dragon's blood by transgenic technology.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710334743.6A CN107190016B (en) | 2017-05-12 | 2017-05-12 | A kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710334743.6A CN107190016B (en) | 2017-05-12 | 2017-05-12 | A kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107190016A CN107190016A (en) | 2017-09-22 |
| CN107190016B true CN107190016B (en) | 2019-10-18 |
Family
ID=59874075
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710334743.6A Expired - Fee Related CN107190016B (en) | 2017-05-12 | 2017-05-12 | A kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107190016B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117327B (en) * | 2018-02-06 | 2022-02-25 | 中国科学院遗传与发育生物学研究所 | Xanthohumol-related protein and application thereof in preparation of product in xanthohumol synthesis pathway |
| CN110117326B (en) * | 2018-02-06 | 2022-03-01 | 中国科学院遗传与发育生物学研究所 | Xanthohumol-related protein and application thereof in stabilizing product in xanthohumol synthesis pathway |
| CN110804672B (en) * | 2019-11-11 | 2023-03-21 | 中国医学科学院药用植物研究所云南分所 | PCR method for identifying variety of dragon tree |
| CN111218462B (en) * | 2020-03-04 | 2021-09-10 | 广西壮族自治区药用植物园 | Gene for coding suberect spatholobus stem chalcone synthetase and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283515A (en) * | 2013-05-29 | 2013-09-11 | 中国热带农业科学院热带生物技术研究所 | Method for artificially inducing dragon's blood |
| CN103614358A (en) * | 2012-10-31 | 2014-03-05 | 上海交通大学 | Tulip chalcone isomerase TfCHI protein and encoding gene thereof |
| CN103804479A (en) * | 2014-01-24 | 2014-05-21 | 中国热带农业科学院热带生物技术研究所 | Samphire aquaporins SpAQP1 and application of coding gene thereof in improving salt tolerance of plants |
| CN105950644A (en) * | 2016-06-23 | 2016-09-21 | 江西省农业科学院蔬菜花卉研究所 | Chalcone isomerase gene of asparaguses, protein encoded by chalcone isomerase gene and application of chalcone isomerase gene |
-
2017
- 2017-05-12 CN CN201710334743.6A patent/CN107190016B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614358A (en) * | 2012-10-31 | 2014-03-05 | 上海交通大学 | Tulip chalcone isomerase TfCHI protein and encoding gene thereof |
| CN103283515A (en) * | 2013-05-29 | 2013-09-11 | 中国热带农业科学院热带生物技术研究所 | Method for artificially inducing dragon's blood |
| CN103804479A (en) * | 2014-01-24 | 2014-05-21 | 中国热带农业科学院热带生物技术研究所 | Samphire aquaporins SpAQP1 and application of coding gene thereof in improving salt tolerance of plants |
| CN105950644A (en) * | 2016-06-23 | 2016-09-21 | 江西省农业科学院蔬菜花卉研究所 | Chalcone isomerase gene of asparaguses, protein encoded by chalcone isomerase gene and application of chalcone isomerase gene |
Non-Patent Citations (4)
| Title |
|---|
| De Novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon’s blood;Jia-Hong Zhu et al.;《Scientific Reports》;20161206;第6卷;第1-10页 * |
| Ornithogalum longebracteatum chalcone-flavanone isomerase (CHI) mRNA, complete cds，GenBank: KR822738.1;Guo,L.et al.;《GenBank》;20160310;第1页 * |
| 海南龙血树查尔酮合酶基因（DcCHS）的克隆及表达分析;王佳媛等;《热带植物学报》;20141231;第35卷(第8期);第1539-1545页 * |
| 海南龙血树查尔酮异构酶基因（DcCHI）的克隆及其启动子功能分析;赵婉;《中国优秀硕士学位论文全文数据库 基础科学辑》;20170815(第08期);A006-31 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107190016A (en) | 2017-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107190016B (en) | A kind of Dracaena cambodinna enzyme, namely chalcone isomerase DcCHIL1 and its encoding gene and application | |
| CN109554414B (en) | Application of flammulina velutipes genes Fvegt1, Fvegt2 and Fvegt3 in synthesis of ergothioneine | |
| CN110885846B (en) | Microorganism for synthesizing baicalein and scutellarin, preparation method and application thereof | |
| CN107955067A (en) | Participate in two myb transcription factors and its application of peach flavonols biosynthetic controlling | |
| CN110982830A (en) | Glycosyl transferase gene RyUGT3A, and coding protein and application thereof | |
| CN112852843B (en) | Flavonol 3-O-galactosyltransferase gene, encoding protein and application thereof | |
| CN104357419A (en) | Erigeron breviscapus glycosyl transferase, preparation method and application thereof | |
| CN111454344A (en) | Soybean MYB transcription factor, coding gene and application thereof | |
| CN110616205A (en) | Flavone synthase for synthesis and preparation of flavone glycoside | |
| CN115992109A (en) | Gelidine glycosyltransferase protein, and coding gene and application thereof | |
| CN110106189A (en) | VbMYB gene and its coding albumen and application | |
| KR101157116B1 (en) | 3' -Methyltransferse, Gene thereof and Production method of chrisoeriol and isorhamnetin by using the same | |
| CN114231545B (en) | Rhamnus rhamnoides glycosyltransferase gene, preparation method, expression and application thereof | |
| CN105671015B (en) | Fructus Crataegi Cyanidin -3- hydroxyl glycosyl transferase and its encoding gene and application | |
| CN107698672A (en) | A kind of male sterility of rice GAP-associated protein GAP and its encoding gene and application | |
| CN114058628A (en) | Gene PnWRKY1 and application thereof in regulating and controlling synthesis of notoginsenoside | |
| CN106967735B (en) | Safflower CtCHS1 gene, and coding protein and application thereof | |
| CN115678952A (en) | Highly specific glycosyltransferase for rhamnose and application thereof | |
| CN106636145A (en) | Nemesia strumosa KLM anthocyanin biosynthesis gene, and encoded protein and application thereof | |
| CN108359652A (en) | Glycosyl transferase and its application | |
| KR101902848B1 (en) | Fusion flavonoid O-methyltransferase and use thereof | |
| CN100370027C (en) | High-molecular glutelin By8 gene of flint wheat and use thereof | |
| CN110218711B (en) | Enzyme protein and coding gene and application thereof | |
| CN110423733B (en) | Protein in andrographis paniculata, and coding gene and application thereof | |
| CN114317494B (en) | alpha-L-rhamnosidase mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191018 Termination date: 20210512 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |