CN104745561A - Eggplant chalcone isomerase SmCHI protein and coding gene thereof - Google Patents
Eggplant chalcone isomerase SmCHI protein and coding gene thereof Download PDFInfo
- Publication number
- CN104745561A CN104745561A CN201510112447.2A CN201510112447A CN104745561A CN 104745561 A CN104745561 A CN 104745561A CN 201510112447 A CN201510112447 A CN 201510112447A CN 104745561 A CN104745561 A CN 104745561A
- Authority
- CN
- China
- Prior art keywords
- eggplant
- gene
- seq
- protein
- sequence
- 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.)
- Granted
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
- C12Y—ENZYMES
- C12Y505/00—Intramolecular lyases (5.5)
- C12Y505/01—Intramolecular lyases (5.5.1)
- C12Y505/01006—Chalcone isomerase (5.5.1.6)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses eggplant chalcone isomerase SmCHI protein and a coding gene thereof. The protein comprises (a) protein consisting of an amino acid sequence as shown in SEQ ID NO.3; or (b) protein derived from the amino acid sequence in (a), which is substituted, lost or added by one or more amino acids and has the activity of eggplant chalcone isomerase. The cDNA and gDNA of the coding gene respectively have base sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; and the gene is expressed in roots, stems, leaves, flowers, peels and pulp, and has the expression quantity in peels remarkably higher than that in other tissues. Under stress of low temperature, the expression quantity of the gene reaches the maximal value in 48 hours, which is 3.65 times that of an unprocessed gene. The eggplant chalcone isomerase SmCHI protein and the coding gene thereof provide a theoretical basis for improving plant quality by utilizing the genetic engineering technology to obtain high oxidation resistance medicines or foods in the future, thereby having great application values.
Description
Technical field
The present invention relates to the key enzyme in eggplant anthocyanidin route of synthesis and encoding gene thereof, be specifically related to a kind of eggplant enzyme, namely chalcone isomerase SmCHI albumen and encoding gene thereof, belong to biological technical field.
Background technology
Because metabolic activity inevitably produces a large amount of free radical in human body, anthocyanidin, as a kind of strong reducible agent, can be removed the free radical in human body, delay body aging.The route of synthesis of anthocyanidin is a part for flavonoid route of synthesis, and be also the widely deep Secondary Metabolism of Plant approach of research at present, it is elaborated in Main Patterns plant is as Arabidopis thaliana, petunia.The biosynthesizing of anthocyanidin is mainly divided into three phases: the first stage, this was the step that many secondary metabolisms have by phenylalanine to coumaric acyl CoA; Subordinate phase is by coumaric acyl CoA to flavanonol, and this is the committed step of flavonoid metabolism, the precursor of its synthesis anthocyanidin and other flavonoid substances; Phase III is the synthesis of all kinds of anthocyanidin.
Enzyme, namely chalcone isomerase is second key enzyme in catalysis flavonoid route of synthesis, cinnamophenone cyclisation is formed the skeleton flavanone of anthocyanidin.An important feature of this reaction is that the cinnamophenone of yellow is transformed to colourless flavanone.This single step reaction of plant also can spontaneously carry out when not having CHI, but under the catalysis of CHI speed of reaction be spontaneous carry out 10
7doubly.CHI gene is led people in plant by using gene engineering technique, and its expression product can promote the biosynthesizing of the flavonoid compound being of value to HUMAN HEALTH, changes pattern, fruit look, improves mouthfeel etc., have the prospect of very large Application and Development.By the CHI transgenation in carnation petal, the flower yellowing of white can be made; Proceed in tomato by petunia CHI gene, in its pericarp, Flavone content adds 78 times, and in pulp, Flavonol adds 2l doubly.CHS justice gene and CHI inverted defined gene are led people in sweet orange, and the resultant quantity of bitter substance reduces, and mouthfeel is better.
From a lot of plant, clone obtains CHI gene, as Arabidopis thaliana, apple, petunia, grape, soybean etc. at present.Eggplant is one of anthocyanidin content vegetable crop of enriching the most, but it be not immediately clear for the clone of eggplant CHI gene, expression pattern and promoter sequence thereof.At present, do not have and anyly to report with the pertinent literature of eggplant CHI gene and proteins encoded thereof.
Summary of the invention
The object of the invention is to fill up the blank of the clone of eggplant CHI family member, expression pattern analysis and eggplant CHI albumen and encoding gene thereof.The invention provides cDNA, gDNA and the aminoacid sequence of a kind of eggplant CHI; Further, the invention provides the Subcellular Localization of eggplant SmCHI gene, additionally provide the expression pattern of eggplant SmCHI gene under different tissues organ and low temperature.Present invention also offers promoter sequence and the component analysis thereof of SmCHI gene.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the invention provides a kind of protein with eggplant enzyme, namely chalcone isomerase activity, it comprises:
A protein that () is made up of the such as aminoacid sequence shown in SEQ ID NO.3; Or
B () aminoacid sequence in (a) passes through replacement, lacks or adds one or several amino acid and has the protein derivative by (a) of eggplant enzyme, namely chalcone isomerase activity.
Preferably, described protein comprises aminoacid sequence shown in SEQ ID NO.3 through 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, or within C-terminal and/or N-terminal add 1 ~ 20 amino acid and the aminoacid sequence that obtains.
Preferably, described protein for 1 ~ 10 amino acid in aminoacid sequence shown in SEQ ID NO.3 replace by the similar or close amino acid of character and the sequence formed.
Second aspect, the invention provides a kind of nucleotide sequence with the gene of the protein of eggplant enzyme, namely chalcone isomerase activity according to claim 1 of encoding, the gDNA sequence of described gene is as shown in SEQ ID NO.2.
Preferably, the cDNA sequence of described gene comprises:
(a) base sequence as shown in SEQ ID NO.1 1st ~ 705; Or
B the base sequence shown in () Yu SEQ ID NO.1 1st ~ 705 has the base sequence of the homology of at least 70%; Or
C () can carry out the base sequence of hybridizing with the base sequence shown in SEQ ID NO.1 1st ~ 705.
Preferably, described nucleotide sequence comprises the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQ ID NO.1 1st ~ 705, insertion and/or replacement, and 5 ' and/or 3 ' end interpolation 60 with inner nucleotide.
The third aspect, the invention provides a pair primer for the gDNA of the nucleotide sequence of the gene described in claim 4 that increases, and the sequence of described primer is as shown in SEQ ID NO.7 and SEQ ID NO.8.
Fourth aspect, the invention provides a pair primer for the cDNA of the nucleotide sequence of the gene described in claim 4 or 5 that increases, and the sequence of described primer is as shown in SEQ ID NO.5 and SEQ ID NO.6.
5th aspect, present invention also offers a kind of promotor with the nucleotide sequence of the gene of the protein of eggplant enzyme, namely chalcone isomerase activity, described promotor comprises the base sequence shown in SEQ ID NO.4.
In the present invention, " DNA of separation ", " DNA of purifying " refer to, the sequence that this DNA or fragment have been arranged in its both sides from native state is separated, also refer to that this DNA or fragment with under native state are separated with the component of nucleic acid, and separate with the protein accompanied in cell.
In the present invention, term " SmCHI albumen coded sequence " refers to encode the nucleotide sequence of the polypeptide with eggplant SmCHI protein-active, 1st ~ 705 nucleotide sequences as shown in SEQ ID NO.1 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in 1st ~ 705 Nucleotide shown in SEQ ID NO.1, have one or more codon replace by the degenerate codon of same amino acid of encoding after produce sequence.Due to the degeneracy of codon, the aminoacid sequence shown in SEQID NO.3 so the degenerate sequence being low to moderate about 70% with 1st ~ 705 nucleotide sequence homologies shown in SEQID NO.1 also can be encoded out.This term also comprises the nucleotide sequence with the homology at least 70% of the nucleotide sequence shown in SEQ ID NO.1.
This term also comprises encoding and has the variant form with sequence shown in the SEQ ID NO.1 of the albumen of natural eggplant SmCHI identical function.These variant forms comprise (but being not limited to): be generally the disappearance of 1 ~ 90 Nucleotide, insertion and/or replacement, and 5 ' and/or 3 ' end be added to 60 with inner nucleotide.
In the present invention, term " SmCHI albumen " refer to have eggplant SmCHI protein-active SEQ ID NO.3 shown in the polypeptide of sequence.This term also comprise have with eggplant SmCHI albumen identical function, the variant form of SEQ ID NO.3 sequence.These variant forms comprise (but being not limited to): be generally 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, and add one or amino acid within being 20 at C-terminal and/or N-terminal.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, add at C-terminal and/or N-terminal the function that or several amino acid also can not change protein usually.This term also comprises active fragments and the reactive derivative of eggplant SmCHI albumen.
The variant form of eggplant SmCHI albumen of the present invention comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, can the albumen coded by DNA of DNA hybridization relevant to eggplant SmCHI and the polypeptide utilizing the antiserum(antisera) of eggplant SmCHI albumen to obtain or albumen under high or low high stringency conditions.
In the present invention, " eggplant SmCHI conservative variation polypeptide " refers to compared with the aminoacid sequence shown in SEQ ID NO.3, have 10 amino acid at the most replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out replacing according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | Preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；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 | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also comprises the analogue of eggplant SmCHI albumen or polypeptide.The difference of these analogues and eggplant SmCHI related polypeptide can be the difference on aminoacid sequence, can be also the difference do not affected on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also by site-directed mutagenesis or the biological technology of other known moleculars.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide enumerated.
(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation, as carried out glycosylation modified and polypeptide that is that produce in those in the synthesis of polypeptide and processing or further procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) by being exposed to by polypeptide and completing.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the polypeptide of solubility property.
In the present invention, the expression pattern of the methods analyst eggplant SmCHI gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of the mRNA transcript namely analyzing eggplant SmCHI gene in cell.
In addition, according to eggplant SmCHI nucleotide sequence of the present invention and aminoacid sequence, can on the homology basis of nucleic acid homology or marking protein, screening eggplant SmCHI associated homologous gene or homologous protein.
In order to obtain the dot matrix with eggplant SmCHI genes involved, can screen eggplant cDNA library with DNA probe, these probes are under low high stringency conditions, use
32what P was correlated with to eggplant SmCHI all or part ofly does radioactivity mark and obtains.The cDNA library being suitable for screening is the library from eggplant.The method built from the cDNA library of interested cell or tissue is that biology field is well-known.In addition, many such cDNA libraries also can buy, such as, purchased from Clontech, Stratagene, Palo Alto, Cal..This screening method can identify the nucleotide sequence of the gene family relevant to eggplant SmCHI.
Eggplant SmCHI associated nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For 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 cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.
After obtaining relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.
In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.
Except producing with recombination method, the fragment also available solid phase technique of albumen of the present invention, is produced (people such as Stewart, (1969) Solid phase peptide synthssis, WH Freeman Co., San Francisco by direct peptide synthesis; MerrifieldJ. (1963) J.Am Chem.Soc 85:2149-2154).Synthetic protein can carry out by hand or automatically in vitro.Such as, can with the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems from dynamic synthetic peptide.Each fragment of chemosynthesis albumen of the present invention can be distinguished, then chemically connected the molecule producing total length.
Eggplant is the vegetable crop of extensively plantation, and the pericarp of purple eggplant contains abundant anthocyanidin.Result of study in recent years shows, it is best that the anti-oxidation health of purple eggplant acts in Vegetables crop.The present invention is directed to the present situation of current eggplant Research foundation weakness, clone obtains eggplant enzyme, namely chalcone isomerase gene SmCHI, for utilizing genetic engineering technique to improve plant quality from now on, obtaining and there is the medicine of high antioxidant or food provides theoretical foundation, there is very large using value.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is Homology search (GAP) result of eggplant CHI gene of the present invention and tomato CHI gene mRNA.
Fig. 2 is that eggplant CHI albumen of the present invention compares (FASTA) result with the amino acid sequence homologous of tomato CHI albumen, and wherein, identical amino acid marks with amino acid monocase between two sequences.
Fig. 3 is the Subcellular Localization of eggplant CHI gene of the present invention.
Fig. 4 is the expression of eggplant CHI gene of the present invention at different tissues.
The expression of Fig. 5 is eggplant CHI gene of the present invention different treatment time at low temperatures.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
The clone of embodiment 1, eggplant SmCHI gene
1. the acquisition of vegetable material
This tests vegetable material used is eggplant fine germplasm resources Lanshan County standing grain line eggplant.Lanshan County's standing grain line eggplant phenotype is purple stem, pale reddish brown, purple fruit.Experiment material is cultivated in the artificial plastic greenhouse of Pujiang town, Minhang District, Shanghai space-breeding land.Nursery under field conditions (factors), growth is also solid.Gather the pericarp of eggplant for extracting RNA and DNA.
The extraction of 2.RNA and DNA
TRIzol method is adopted to extract total serum IgE (TRIzol is purchased from Sangon Biotech (Shanghai) Co., Ltd.).By the integrity of denaturing formaldehyde gel electrophoresis qualification RNA, then in upper purity and the concentration measuring RNA of spectrophotometer (Thermo Scientific NANODROP1000Spectrophotometer).
Ezup pillar genome DNA extraction test kit is adopted to extract STb gene (Sangon Biotech (Shanghai) Co., Ltd.).
3. the full-length clone of gene
Based on the conserved sequence design pair of degenerate primers of CHI gene in Genbank.Extraction RNA is carried out reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited), with the first chain cDNA for template, utilize primer
F1(SEQ ID NO.5)：5′-ATGGCGBCYGTTACHAAATTGCAG-3′
R1(SEQ ID NO.6)：5′-TTACTGGAYTGWCGATAGCYCACTTC-3′
Carry out PCR, amplification is reclaimed after obtaining expection length and is connected on pMD-19T (precious biotechnology (Dalian) company limited) carrier, transformation of E. coli DH5 α, utilize α complementation and bacterium colony PCR screening positive clone, deliver to prompt base (Shanghai) trade Co., Ltd in the English Weihe River to check order, obtain cDNA sequence SEQ ID NO.1.
According to sequencing result design pair of primers
F2(SEQ ID NO.7)：5′-ATGGCGGCCGTTACTAAATTGCAG-3′
R2(SEQ ID NO.8)：5′-TTACTGGATTGTCGATAGCCCACTTC-3′
Take DNA as template, the gDNA total length of amplification SmCHI, reclaim after amplification obtains product and be connected on pMD-19T carrier, transformation of E. coli DH5 α, utilize α complementation and bacterium colony PCR screening positive clone, deliver to prompt base (Shanghai) trade Co., Ltd in the English Weihe River to check order, obtain gDNA sequence SEQ ID NO.2.
The sequence information of embodiment 2, eggplant CHI gene and homology analysis
The new eggplant CHI full length gene CDS open reading frame sequence of the present invention is 705bp, and detailed sequence is shown in SEQ IDNO.1.Derive the aminoacid sequence of eggplant CHI according to CDS opening code-reading frame sequence, totally 234 amino-acid residues, molecular weight is 25248.8 dalton, and theoretical iso-electric point (pI) is 5.37, and detailed sequence is shown in sequence shown in SEQ ID NO.3.The genomic dna of SmCHI comprises 4 exons and 3 introns, and detailed sequence is shown in sequence shown in SEQ ID NO.2.
The CDS open reading frame sequence of eggplant CHI and the aminoacid sequence blast program of proteins encoded thereof are carried out Nucleotide and protein homology search in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that it and tomato CHI gene (XM_004238946.2) have 86.81% consistence on nucleotide level, (the mRNA sequence of Query: eggplant CHI as shown in Figure 1; The mRNA sequence of Sbjct: tomato CHI); On amino acid levels, they between the two similarity be 85.47%, (Query: the aminoacid sequence of eggplant CHI as shown in Figure 2; Sbjct: the aminoacid sequence of tomato CHI).As can be seen here, all there is higher homology in eggplant CHI gene and tomato CHI gene from nucleic acid or protein level.
The promotor cloned and sequenced of embodiment 3, eggplant CHI gene
Adopt GenomeWalking Kit test kit (precious biotechnology (Dalian) company limited), design primer
SP1：5′-CCATTTCGCAGCTAGAAACGGAATTG-3′
SP2：5′-CTTCCTTCAATCTCCAAACCTCTACTCC-3′
SP3：5′-GGTATTGTTGGAACCAACAGGCTTCAC-3′
Take DNA as template, carry out three-wheel pcr amplification, reclaim after amplification obtains product and be connected on pMD-19T (precious biotechnology (Dalian) company limited) carrier, transformation of E. coli DH5 α, utilize α complementation and bacterium colony PCR screening positive clone, deliver to prompt base (Shanghai) trade Co., Ltd in the English Weihe River and check order.Sequencing result finds to obtain eggplant CHI gene promoter 1718bp.Sequence is submitted to PLACE and PlantCARE two cis element on-line prediction softwares and analyzes, and finds that it contains a lot of light response element, comprises Box 4, Box I, G-Box etc.; It also expresses relevant element containing some to hormone induction, such as, respond relevant ABRE to dormin, respond relevant CGTCA-motif and TGACG-motif to methyl jasmonate, and respond relevant AuxRR-core to growth hormone; Also comprise low temperature response element LTR and MYB binding site MBS.Detailed sequence is shown in sequence shown in SEQ ID NO.4.
The Subcellular Localization of embodiment 4, eggplant CHI gene
(1) structure of carrier
Design upstream and downstream primer
5′-GCCGCCATGGCGGCCGTTACTAAATTGCAG-3′
5′-GGACTAGTCTGGATTGTCGATAGCCCACTTC-3′
Increase.Plant expression vector Pcambia1302 has GFP albumen, can be used for observation of subcellular localization.Double digestion is carried out to the amplified production of restriction enzyme site and Pcambia1302 respectively with NcoI and SpeI, transformation of E. coli DH5 α after T4DNALigase (precious biotechnology (Dalian) company limited) connects, delivers to prompt base (Shanghai) trade Co., Ltd in the English Weihe River and checks order.Bacterium liquid correct for sequencing result is extracted plasmid, transformation Agrobacterium GV3101, after PCR checking, be stored in-80 DEG C of refrigerators, namely successfully build SmCHI-1302 carrier.
(2) plantation of tobacco
This formula tobacco seed is sprinkled in matrix, 25 DEG C of full suns.After growing about one week, individual plant proceeds in basin.About choose eugonic blade after two weeks and carry out instantaneous conversion.
(3) instantaneous conversion
The single bacterium colony of the Agrobacterium of picking SmCHI-1302 carrier, be inoculated in 10ml YEP liquid nutrient medium, 28 DEG C, 200rpm cultivates 24h, 4 DEG C, 5000rpm centrifugal 10min collection thalline, with the MS liquid nutrient medium suspension thalline of certain volume to OD
600value is about 0.6.Draw bacterium liquid with the aseptic needle tubing of 1ml and be injected in tobacco leaf lower surface, 12h in dark is put in after carrying out mark, dislocation is under light again, adopts laser confocal microscope (Leica) to observe after 2 days, finds that SmCHI expresses (Fig. 3) on tenuigenin and nucleus.
Embodiment 5, the differential expression of eggplant CHI gene in different tissues and expression pattern at low temperatures
1. the acquisition of vegetable material
Gather Fructus Solani melongenae root, stem, leaf, flower, pericarp, pulp respectively in the fructescence, drop at once in liquid nitrogen after sample is wrapped with aluminium platinum paper respectively, then proceed to stored for future use in-80 DEG C of Ultralow Temperature Freezers.
Sowing artificial climate incubator same period will be arranged in and the consistent eggplant seedling of growing way is placed in 4 DEG C, the blade of process material is got respectively at 0h, 1h, 3h, 6h, 12h, 24h and 48h, drop at once in liquid nitrogen after wrapping with aluminium platinum paper, then proceed to stored for future use in-80 DEG C of Ultralow Temperature Freezers.
The extraction of 2.RNA
TRIzol method is adopted to extract total serum IgE (TRIzol is purchased from Sangon Biotech (Shanghai) Co., Ltd.).With plain agar sugar gel electrophoresis (gum concentration 1.2%; 0.5 × TBE electrophoretic buffer; 150v, 15min) detect integrity.In electrophoretic band, maximum rRNA brightness should be the 1.5-2.0 of Article 2 rRNA brightness doubly, otherwise represents the degraded of rRNA sample.Purity good RNA, A
260/ A
280and A
260/ A
230be about about 2.0.Calculate rna content by spectrophotometric determination OD value.
The acquisition of 3.cDNA
With the total serum IgE of 500ng for template, according to precious biotech firm TaKaRa PrimeScript
tMit is for subsequent use that RT reagent KitPerfect Real Time test kit operation instructions carries out reverse transcription acquisition cDNA.
4. design Auele Specific Primer to carry out the expression amount of real-time fluorescence quantitative PCR analyzing gene in different tissues and under low temperature.According to the eggplant CHI gene order obtained, primer-design software is utilized to be designed for the Auele Specific Primer that in Real-time PCR, SmCHI gene quantification is analyzed,
CHI-F：5′-CCTTGACGGGTAAGCAATACTCTG-3′
CHI-R：5′-GATGGAGGCACCATGTGGGAAGGA-3′
Reference gene is Actin (GU984779.1), and its primer is
ACTIN-F：5′-GTCGGAATGGGACAGAAGGATG-3′
ACTIN-R：5′-GTGCCTCAGTCAGGAGAACAGGGT-3′
5. make the typical curve of goal gene and reference gene.With EASY Dilution (test kit provides), standard substance cDNA solution is carried out gradient dilution, then respectively with dilution after cDNA solution for template, carry out Real-time pcr amplification with the Auele Specific Primer of goal gene and reference gene, reaction terminates rear drafting solubility curve and typical curve.Analyze solubility curve, judge whether the solubility curve of goal gene and reference gene obtains simple spike, to judge to use this primer can obtain single pcr amplification product.By the appropriate dilutions multiple of typical curve determination template cDNA.
6. the Real time PCR of goal gene in testing sample.With the cDNA Article 1 chain of synthesis for template, carry out quantitative fluorescence analysis respectively by the primer amplified of goal gene and reference gene, Real-time PCR reaction is carried out on FTC-3000 real-time quantitative instrument, and reaction system is 20 μ L.Reaction adopts three-step approach, 95 DEG C of sex change 1min, then 40 circulations: 95 DEG C of 30s; 58 DEG C of 30s; 72 DEG C of 45s.Whether, after each amplification completes, all do solubility curve, be special generation to check amplified production.
7. adopt 2
-△ △ Ctmethod makes relative quantitative assay.Result shows that SmCHI gene has expression in the root of eggplant, stem, leaf, flower, pericarp, pulp, expression amount wherein in pericarp is the highest, next is leaf and stem, and expression amount in root is minimum, illustrates that the expression of SmCHI gene has obvious Spatial Difference (Fig. 4).Under low temperature stress, the expression amount of SmCHI gene constantly rises along with the prolongation of stress time, reaches maximum value at 48h, is untreated 3.65 times, shows its induction being subject to low temperature and express (Fig. 5).
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (9)
1. there is a protein for eggplant enzyme, namely chalcone isomerase activity, it is characterized in that, comprising:
A protein that () is made up of the such as aminoacid sequence shown in SEQ ID NO.3; Or
B () aminoacid sequence in (a) passes through replacement, lacks or adds one or several amino acid and has the protein derivative by (a) of eggplant enzyme, namely chalcone isomerase activity.
2. the protein with eggplant enzyme, namely chalcone isomerase activity according to claim 1, it is characterized in that, described protein comprises aminoacid sequence shown in SEQ ID NO.3 through 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, or within C-terminal and/or N-terminal add 1 ~ 20 amino acid and the aminoacid sequence that obtains.
3. the protein with eggplant enzyme, namely chalcone isomerase activity according to claim 1 and 2, it is characterized in that, described protein for 1 ~ 10 amino acid in aminoacid sequence shown in SEQ ID NO.3 replace by the similar or close amino acid of character and the sequence formed.
4. a coding nucleotide sequence with the gene of the protein of eggplant enzyme, namely chalcone isomerase activity according to claim 1, it is characterized in that, the gDNA sequence of described gene is as shown in SEQ ID NO.2.
5. have the nucleotide sequence of the gene of the protein of eggplant enzyme, namely chalcone isomerase activity described in coding according to claim 4, it is characterized in that, the cDNA sequence of described gene comprises:
(a) base sequence as shown in SEQ ID NO.1 1st ~ 705; Or
B the base sequence shown in () Yu SEQ ID NO.1 1st ~ 705 has the base sequence of the homology of at least 70%; Or
C () can carry out the base sequence of hybridizing with the base sequence shown in SEQ ID NO.1 1st ~ 705.
6. the nucleotide sequence with the gene of the protein of eggplant enzyme, namely chalcone isomerase activity according to claim 5, it is characterized in that, comprise the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQ ID NO.1 1st ~ 705, insertion and/or replacement, and at the base sequence that 5 ' and/or 3 ' hold interpolation 60 to be formed with inner nucleotide.
7. a pair primer for the gDNA of the nucleotide sequence of the gene described in claim 4 that increases, the sequence of described primer is as shown in SEQ ID NO.7 and SEQ ID NO.8.
8. a pair primer for the cDNA of the nucleotide sequence of the gene described in claim 4 or 5 that increases, the sequence of described primer is as shown in SEQ ID NO.5 and SEQ ID NO.6.
9. a promotor with the nucleotide sequence of the gene of the protein of eggplant enzyme, namely chalcone isomerase activity according to claim 4, is characterized in that, described promotor comprises the base sequence shown in SEQ ID NO.4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510112447.2A CN104745561B (en) | 2015-03-13 | 2015-03-13 | Eggplant enzyme, namely chalcone isomerase SmCHI albumen and its encoding gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510112447.2A CN104745561B (en) | 2015-03-13 | 2015-03-13 | Eggplant enzyme, namely chalcone isomerase SmCHI albumen and its encoding gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104745561A true CN104745561A (en) | 2015-07-01 |
| CN104745561B CN104745561B (en) | 2017-11-14 |
Family
ID=53585821
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510112447.2A Active CN104745561B (en) | 2015-03-13 | 2015-03-13 | Eggplant enzyme, namely chalcone isomerase SmCHI albumen and its encoding gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104745561B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN114574624A (en) * | 2022-04-15 | 2022-06-03 | 重庆市农业科学院 | DNA molecular marker for distinguishing eggplant pulp color and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1650017A (en) * | 2002-02-28 | 2005-08-03 | 荷兰联合利华有限公司 | Production of dadzein in transgenic plants |
-
2015
- 2015-03-13 CN CN201510112447.2A patent/CN104745561B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1650017A (en) * | 2002-02-28 | 2005-08-03 | 荷兰联合利华有限公司 | Production of dadzein in transgenic plants |
| US20050241014A1 (en) * | 2002-02-28 | 2005-10-27 | Colliver Steven P | Nutritionally enhanced plants |
Non-Patent Citations (5)
| Title |
|---|
| ACCESSION NO:XM_004238946: "PREDICTED: Solanum lycopersicum chalcone--flavanone isomerase (LOC1 01249265), mRNA", 《GENBANK DATABASE》 * |
| YANJIE ZHANG等: "Anthocyanin Accumulation and Molecular Analysis of Anthocyanin Biosynthesis-Associated Genes in Eggplant (Solanum melongena L.)", 《AGRIC. FOOD CHEM》 * |
| 周发俊等: "植物查尔酮异构酶分子生物学研究进展(综述)", 《河北科技师范学院学报》 * |
| 周莎莎: "基因克隆与表达研究进展", 《现代农业科技》 * |
| 李琳玲等: "植物查尔酮异构酶研究进展", 《生物技术通讯》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105950644B (en) * | 2016-06-23 | 2019-06-04 | 江西省农业科学院蔬菜花卉研究所 | The albumen and application of asparagus enzyme, namely chalcone isomerase gene and its coding |
| 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 |
| CN114574624A (en) * | 2022-04-15 | 2022-06-03 | 重庆市农业科学院 | DNA molecular marker for distinguishing eggplant pulp color and application |
| CN114574624B (en) * | 2022-04-15 | 2024-03-19 | 重庆市农业科学院 | DNA molecular marker for distinguishing eggplant pulp color and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104745561B (en) | 2017-11-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103333233B (en) | Agapanthus praecox auxin receptor protein TIR1 and coding gene and probe thereof | |
| CN107164391A (en) | A kind of strawberry floral genes FvbHLH78 and its application | |
| CN108603198A (en) | Manipulate the composition and method of development of plants | |
| CN104745560A (en) | Eggplant chalcone synthase SmCHS1 protein and coding gene thereof | |
| CN103614348B (en) | Turmeric flavanone-3-hydroxylase TfF3H albumen and encoding gene thereof | |
| CN109517812A (en) | Azalea chalcone synthase RsCHS albumen and its encoding gene | |
| CN104745561A (en) | Eggplant chalcone isomerase SmCHI protein and coding gene thereof | |
| CN105087508A (en) | Eggplant flavanone 3-hydroxylase SmF3H as well as gene and application thereof | |
| CN105440114A (en) | Eggplant cryptochrome gene SmCRY1 and application thereof | |
| CN105441396A (en) | Eggplant constitutively photomorphogenic SmCOP1 protein and coding gene thereof | |
| CN105440116A (en) | Eggplant cryptochrome gene SmCRY2 and application thereof | |
| CN105837670A (en) | African agapanthus auxin response factor ApARF2 and encoding gene and probe thereof | |
| CN103074307B (en) | Tulipa gesneriana TfbHLH1 protein, encoding gene thereof and probe | |
| CN103483437A (en) | Protein of key gene ApCO for photoperiod and flowering pathway of agapanthus praecox ssp.orientalis as well as coding gene and probe of gene | |
| CN102978194A (en) | Tulip chalcone isomerase TfCHI protein and coding gene thereof and probe | |
| CN102965349A (en) | Tulip flavanonol-3'- hydroxylase TfF3' H protein, and coding gene and probe thereof | |
| CN103756982B (en) | Tulipa fosteriana flavonol synthase TfFLS protein and encoding gene thereof | |
| CN105037514A (en) | Bermuda grass 'Tifway' dehydrin-L, encoding gene and probe thereof | |
| CN104017781B (en) | Agipanthus Plant hormones regulators,gibberellins synthesis dioxygenase APGA20ox albumen and encoding gene and probe | |
| CN104961814B (en) | Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 and its encoding gene and probe | |
| CN103589698B (en) | Tulip chalcone synthase TfCHS albumen and encoding gene thereof | |
| CN103695406A (en) | Tulip phenylalanine ammonialyase TfPAL protein and coding gene thereof | |
| CN104974992A (en) | Eggplant GDP-L-galactose phosphatase SmGGP protein and encoding gene | |
| CN103333232B (en) | Agapanthus praecox gibberellin acceptor APGID1a protein and coding gene and probe thereof | |
| CN105085642A (en) | Agapanthus africanus auxin signal transcriptional control egg white Aux/IAA2 and encoding gene and probe thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |