CN105440115B - Eggplant SmHY5 albumen and its encoding gene - Google Patents
Eggplant SmHY5 albumen and its encoding gene Download PDFInfo
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Abstract
The present invention discloses a kind of eggplant SmHY5 (Long Hypocotyl 5) gene and its functional analysis, and the cDNA of the gene has nucleotide sequence shown in SEQ ID NO.1.The amino acid sequence of the gene coding is as shown in SEQ ID NO.2.The gene has expression in root, stem, leaf, flower, pericarp, pulp, and the expression quantity in each tissue is without significant difference.The present invention provides theoretical foundation to improve plant quality using technique for gene engineering from now on, has very big application value.
Description
Technical field
The invention belongs to field of biotechnology, the key enzyme and its encoding gene being related in eggplant photo-signal channel, specifically
It is related to a kind of eggplant SmHY5 (Long Hypocotyl 5) albumen and its encoding gene.
Background technique
HY5 is the transcription factor for the positive regulation photomorphogenesis being found earliest, and the sequential analysis of protein of coding is shown,
HY5 belongs to basic leucine type zipper (b-ZIP) type transcription factor, it is positioned at nucleus, can regulate and control base with a variety of light
The promoter of cause binds directly to regulate and control the expression of these genes.To HY5 protein level the study found that HY5 protein accumulation by
To the regulation of light, the intracorporal HY5 albumen of Arabidopsis thaliana Seedlings for continuing to grow under dark condition is largely degraded, almost detects not
To the accumulation of HY5 albumen；And under the conditions of irradiation, HY5 albumen can accumulate rapidly；The degradation of this HY5 depends on 26S ubiquitin
Proteasome degradation pathway, and the E3 ubiquitin ligase for regulating and controlling HY5 degradation is exactly photomorphogenesis negative regulatory factor COP1.It is dark
Under the conditions of nucleus is located in due to COP1, degrade internal most HY5 albumen, and under illumination condition, COP1 is by thin
Karyon is transferred to cytoplasm, is released to the degradation of HY5 and is largely accumulated, and such HY5 can function.
Clone obtains HY5 gene, such as arabidopsis, grape, apple, corn from many plants at present.Eggplant is weight
The vegetable crop wanted, but correlative study relatively lags.Currently, not having any and eggplant HY5 gene and its encoding albumen
Pertinent literature report.
Summary of the invention
For the defects in the prior art, it is an object of the invention to fill up the blank of eggplant HY5 gene.The present invention provides
A kind of cDNA and amino acid sequence of eggplant HY5；Further, the present invention provides eggplant SmHY5 genes at different groups
Knit the expression pattern of organ.
The purpose of the present invention is what is be achieved through the following technical solutions:
In a first aspect, the present invention provides a kind of eggplant HY5 albumen, including amino acid sequence shown in SEQ ID No.2.
Second aspect, the present invention provide a kind of gene nucleic acid sequence for encoding eggplant HY5 albumen, the cDNA sequence of the gene
Column include:
(a) base sequence as shown in SEQ ID NO.1 the 1st~477；Or
(b) there is the base sequence of at least 70% homology with base sequence shown in SEQ ID NO.1 the 1st~477
Column；Or
(c) can with SEQ ID NO.1 the 1st~477 shown in the base sequence that is hybridized of base sequence.
Preferably, the cDNA sequence includes 1~90 core in nucleic acid sequence shown in SEQ ID NO.1 the 1st~477
Missing, insertion and/or the substitution of thuja acid, and in 5 ' and/or 3 ' end additions 60 with inner nucleotide.
The third aspect, the present invention provide a kind of for expanding the primer pair of the gene, the base sequence of the primer pair
As shown in SEQ ID NO.3, SEQ ID NO.4.
Fourth aspect, the present invention provide a kind of primer of the quantitative fluorescent PCR analysis of gene for encoding eggplant SmHY5 albumen
Right, the base sequence of the primer pair is as shown in SEQ ID NO.5, SEQ ID NO.6.
5th aspect, the present invention provide it is a kind of encode eggplant SmHY5 albumen gene in genetic engineering regulation plant in light
The purposes of lower upgrowth situation.
Preferably, the plant includes arabidopsis.
In the present invention, term " SmHY5 gene coded sequence " refers to the 1st~477 nucleotide shown in SEQ ID NO.1
Sequence and its degenerate sequence.The degenerate sequence refers to, is located in the 1st~477 nucleotide shown in SEQ ID NO.1, there is one
The sequence that a or multiple codons generate after being encoded replaced the degenerate codon of same amino acid.Due to the letter of codon
And property, so the degenerate sequence with the 1st~477 nucleotide sequence homology shown in SEQ ID NO.1 down to about 70%
Amino acid sequence shown in SEQ ID NO.2 can be encoded out.The term further includes and nucleotide sequence shown in SEQ ID NO.1
Homology at least 70% nucleotide sequence.
The term further includes the SEQ ID NO.1 institute that can be encoded Ju You with the albumen of natural eggplant SmHY5 identical function
Show the variant form of sequence.These variant forms include (but being not limited to): being usually missing, the insertion of 1~90 nucleotide
And/or replace, and be added to 60 at 5 ' and/or 3 ' ends with inner nucleotide.
In the present invention, the expression pattern of the method analysis eggplant SmHY5 gene product of real-time fluorescence quantitative PCR can be used,
Analyze presence or absence and quantity of the mRNA transcript of eggplant SmCOP1 gene in cell.
In addition, eggplant SmHY5 nucleotide sequence according to the present invention and amino acid sequence, it can be in nucleic acid homology or table
Up on the homology basis of protein, eggplant SmHY5 associated homologous gene or homologous protein are screened.
In order to obtain with the dot matrix of eggplant SmHY5 related gene, eggplant cDNA library can be screened with DNA probe, these
Probe is used under low high stringency conditions32P does obtained by radioactivity label the relevant all or part of eggplant SmHY5.It is suitble to
In the cDNA library of screening be the library from eggplant.The method for constructing the cDNA library from interested cell or tissue
It is that molecular biology field is well-known.In addition, many such cDNA libraries are also commercially available, such as purchased from
Clontech,Stratagene,Palo Alto,Cal..This screening technique can identify gene relevant to eggplant SmHY5
The nucleotide sequence of family.
Eggplant SmHY5 associated nucleotide full length sequence of the invention or its segment can usually use PCR amplification method, recombination method
Or artificial synthesized method obtains.For PCR amplification method, can disclosed related nucleotide sequence according to the present invention, especially
Open reading frame sequence carrys out design primer, and made with the commercially available library cDNA or by conventional method well known by persons skilled in the art
The standby library cDNA expands as template and obtains related sequence.When sequence is longer, it is often necessary to which PCR expands twice or repeatedly for progress
Increase, then the segment that each time amplifies is stitched together by proper order again.
After obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually by it
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
Optical signal can regulate and control many secondary metabolism approachs, such as the synthesis of anthocyanidin.Eggplant is that the vegetables planted extensively make
Object, the pericarp of purple eggplant anthocyanidin rich in.Result of study in recent years shows that the anti-oxidation health of purple eggplant is made
It is best in Vegetables crop.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, SmHY5 gene is expressed in arabidopsis mutant strain hy5, eggplant HY5 gene has and arabidopsis HY5 gene phase
As function.
2, the present invention is directed to the status of current eggplant Research foundation weakness, clones the key gene in photo-signal channel
SmHY5 gene, to improve plant quality using technique for gene engineering from now on, obtaining, there is the drug of high antioxidant or food to mention
For theoretical foundation, there is very big application value.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 be eggplant HY5 albumen of the invention compared with the amino acid sequence homologous of tomato HY5 albumen (FASTA) as a result,
Wherein, identical amino acid is marked between two sequences with amino acid monocase；
Fig. 2 is the expression of eggplant HY5 gene of the invention in different tissues；
Fig. 3 is the RT-PCR detection for turning SmHY5 gene plant；
Fig. 4 is the phenotype recovery situation for turning SmHY5 gene pairs arabidopsis mutant strain hy5.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc.
Molecular cloning: described in laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989)
Condition, or according to the normal condition proposed by manufacturer.
The clone of embodiment 1, eggplant SmHY5 gene
1. the acquisition of vegetable material
This experiment vegetable material used is eggplant fine germplasm resources Lanshan County standing grain line eggplant.Experimental material is cultivated in Shanghai City
In the artificial vinyl house of Minxing District Pujiang town space-breeding land.Nursery under field conditions (factors) is grown and solid.Acquire eggplant
Blade for extracting RNA.
The extraction of 2.RNA
Total serum IgE (TRIzol is purchased from Sangon Biotech (Shanghai) Co., Ltd.) is extracted using TRIzol method.Use first
Aldehyde is denaturalized the integrality of gel electrophoresis identification RNA, then in spectrophotometer (Thermo Scientific NANODROP 1000
Spectrophotometer the purity and concentration of RNA are measured on).
3. the full-length clone of gene
Conserved sequence based on HY5 gene in Genbank designs a pair of degenerate primers.RNA will be extracted and carry out reverse transcription
(Prime Script II 1st Strand cDNA Synthesis Kit: precious bioengineering (Dalian) Co., Ltd), with
One chain cDNA is template, utilizes primer
F1 (SEQ ID NO.3): 5 '-ATGCARGARCAAGCGACRAGYTC-3 '
R1 (SEQ ID NO.4): 5 '-YTACTTCCKCCCTTCCTGTGCAC-3 '
PCR is carried out, amplification recycles after obtaining expected length and is connected to pMD-19T (the limited public affairs of precious bioengineering (Dalian)
Department) on carrier, bacillus coli DH 5 alpha is converted, using α is complementary and bacterium colony PCR screening positive clone, send to Invitrogen (Shanghai)
Trade Co., Ltd is sequenced, and cDNA sequence SEQ ID NO.1 is obtained.
The sequence information and homology analysis of embodiment 2, eggplant HY5 gene
The new eggplant HY5 full length gene CDS open reading frame sequence of the present invention is 477bp, and detailed sequence is shown in SEQ ID
NO.1.The amino acid sequence of eggplant HY5 is derived according to CDS opening code-reading frame sequence, totally 158 amino acid residues, molecular weight
For 17453.4 dalton, theoretical isoelectric point (pI) is 9.64, and detailed sequence is shown in sequence shown in SEQ ID NO.2.
By the CDS open reading frame sequence of eggplant HY5 and its amino acid sequence blast program for encoding albumen in Non-
Redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+
Nucleotide and protein homology search are carried out in SwissProt+Superdate+PIR database, as a result, it has been found that it and tomato
HY5 gene (NM_001247891.1) has 92.02% consistency on nucleotide level；On amino acid levels, they two
Similitude is up to 93.04% between person, (Query: the amino acid sequence of eggplant HY5 as shown in Figure 1；Sbjct: the ammonia of tomato HY5
Base acid sequence).It can be seen that eggplant HY5 gene and tomato HY5 gene no matter all exist from nucleic acid or protein level it is higher
Homology.
The expression of embodiment 3, eggplant HY5 gene in different tissues
1. the acquisition of vegetable material
Eggplant root, stem, leaf, flower, pericarp, pulp are acquired respectively in fructescence, and sample is wrapped with aluminium platinum paper respectively
It puts into liquid nitrogen at once afterwards, is then transferred to stored for future use in -80 DEG C of ultra low temperature freezers.
2. the extraction of RNA
Total serum IgE (TRIzol is purchased from Sangon Biotech (Shanghai) Co., Ltd.) is extracted using TRIzol method.With general
Logical agarose gel electrophoresis (gum concentration 1.2%；0.5 × TBE electrophoretic buffer；150v, 15min) detection integrality.Electrophoresis strip
Maximum rRNA brightness should be 1.5-2.0 times of Article 2 rRNA brightness in band, otherwise indicate the degradation of rRNA sample.Purity is preferable
RNA, A260/A280And A260/A230About 2.0 or so.With spectrophotometric determination OD value and calculate rna content.
3. the acquisition of cDNA
Using the total serum IgE of 500ng as template, according to precious biotech firm TaKaRa PrimeScriptTM RT reagent Kit
It is spare that Perfect Real Time kit operating instruction carries out reverse transcription acquisition cDNA.
4. design specific primer is to carry out expression quantity of the real-time fluorescence quantitative PCR analysis gene in different tissues.Root
According to the eggplant HY5 gene order obtained, using primer-design software designed for SmHY5 gene in Real-time PCR
The specific primer of quantitative analysis,
HY5-F (SEQ ID NO.5): 5 '-GACAAGTTCCATTGCGGCTAGTTC-3 '
HY5-R (SEQ ID NO.6): 5 '-TCCATCTCTACCAGAAGCTGACGT-3 '
Reference gene is Actin (GU984779.1), and primer is
ACTIN-F (SEQ ID NO.7): 5 '-GTCGGAATGGGACAGAAGGATG-3 '
ACTIN-R (SEQ ID NO.8): 5 '-GTGCCTCAGTCAGGAGAACAGGGT-3 '
5. making the standard curve of target gene and reference gene.With EASYDilution (kit offer) by standard items
CDNA solution carries out gradient dilution, then respectively using the cDNA solution after diluting as template, with target gene and reference gene
Specific primer carries out Real-time PCR amplification, draws solubility curve and standard curve after reaction.Analysis dissolution is bent
Can line, judges whether the solubility curve of target gene and reference gene obtains simple spike, to judge obtain list using the primer
One pcr amplification product.The appropriate dilutions multiple of template cDNA is determined by standard curve.
6. the Real time PCR of target gene in sample to be tested.Using first chain of cDNA of synthesis as template, point
Quantitative fluorescence analysis is not carried out with the primer amplified of target gene and reference gene, Real-time PCR reaction exists
It is carried out on FTC-3000 real-time quantitative instrument, reaction system is 20 μ L.Reaction uses three-step approach, 95 DEG C of denaturation 1min, and then 40
Circulation: 95 DEG C of 30s；58℃30s；72℃45s.Every time amplification after the completion of, do solubility curve, with examine amplified production whether be
It is special to generate.
7. using 2-△△CtMethod makees relative quantitative assay.The result shows that SmHY5 gene is in the root of eggplant, stem, leaf, flower, fruit
There is expression in skin, pulp, and the expression quantity in each tissue illustrates that the expression of SmHY5 gene does not have without significant difference
Spatial Difference (Fig. 2).
Embodiment 4, eggplant HY5 gene turn the functional verification of arabidopsis
Conversion carrier used be pCAMBIA1302 (Cambia), arabidopsis material be HY5 mutant strain hy5 (Lu et al.,
2015.Red-light-dependent interaction of phyB with SPA1 promotes COP1–SPA1
dissociation and photomorphogenic development in Arabidopsis.Molecular
Plant.8:467–478.).Agrobacterium containing SmHY5-1302 is cultivated to OD 0.8~2.0,6000rpm and is centrifuged 5min,
Supernatant is abandoned, bacterial sediment is resuspended with MS liquid, adjusts OD600It is 0.8~1.2, infects 10~60sec of arabidopsis floral, dark culture
It is normally cultivated after 12h, collects mature seed.
The T of collection0For transgenic seed, it is layered on containing on 50mg/L hygromycin resistance 1/2MS plate, 4 DEG C of vernalization 3d, light
According to incubator culture 6-10d, the length of root long is chosen, and grows the healthy and strong seedling of true leaf, transplanting to native basin, culture.The T of collection1
Continue to be layered on for seed and choose resistance containing on 50mg/L hygromycin resistance 1/2MS plate: the not anti-strain than being 3:1 is collected
T2For seed.T2Generation survival rate on the 50mg/L hygromycin resistance plate is 100% to think to screen pure and mild resistant plant.It is right
The strain of acquisition detects the expression quantity (Fig. 3) of SmHY5 using Real-time PCR.Arabidopsis actin (NM_179953) used
Primer is as follows:
SEQ ID NO.9:5 '-GTCTGGATTGGAGGGTC-3 '
SEQ ID NO.10:5 '-TGAGAAATGGTCGGAAA-3 '
The same mutant strain of positive transformants that screening is obtained, wild type are placed under light together and cultivate.The results show that hy5 exists
Hypocotyl length under light is noticeably greater than wild type WT, and transgenic plant SmHY5/hy5 has restored this phenotype (Fig. 4).This
Illustrate that eggplant HY5 gene has function similar with arabidopsis HY5 gene.
The invention further relates to sequence it is as follows:
SEQ ID NO.1 ((SmHY5 gene cDNA sequence):
ATGCAAGAGCAAGCGACAAGTTCCATTGCGGCTAGTTCTATACCTTCAAGTAGTGAGAGATCTTCTAGT
TCAGCTCTCCATCTTGAACACAAAGAAGGTATGGAGAGTGATGATGAGATCAGAAGAGTGCCGGAGATAGGCGGAGA
AGCCACCGGAACCACGTCAGCTTCTGGTAGAGATGGAAGTTCCGCCACCCTTCAAGTTCAACCATTAACTGGGACTC
AAAAGAAGCGAGGAAGAAGCCCAGCTGACAAAGAAAACAAGAGGTTAAAAAGGTTGCTGAGAAATAGAGTGTCTGCA
CAGCAAGCAAGGGAGAGGAAGAAAGCATACTTGATAGATCTGGAAGCAAGGGTGAAGGAACTGGAAACAAAGAATGC
AGAACTTGAAGAGAGGTTGTCCACTTTGCAAAATGAGAACCAAATGCTTAGACATATACTGAAGAACACAACAGCAG
GTGCACAGGAAGGGCGGAAGTAA
SEQ ID NO.2 (SmHY5 gene amino acid sequence):
MQEQATSSIAASSIPSSSERSSSSALHLEHKEGMESDDEIRRVPEIGGEATGTTSASGRDGSSATLQVQ
PLTGTQKKRGRSPADKENKRLKRLLRNRVSAQQARERKKAYLIDLEARVKELETKNAELEERLSTLQNENQMLRHIL
KNTTAGAQEGRK
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (6)
1. a kind of eggplantSmHY5Albumen, which is characterized in that the amino acid sequence of the albumen is as shown in SEQ ID NO.2.
2. eggplant described in a kind of coding claim 1SmHY5The gene of albumen, which is characterized in that the cDNA sequence of the gene
The base sequence as shown in SEQ ID NO.1 the 1st~477.
3. one kind is for expanding eggplant described in claim 2SmHY5The primer pair of the gene of albumen, which is characterized in that described to draw
The base sequence of object pair is as shown in SEQ ID NO.3, SEQ ID NO.4.
4. one kind is for encoding eggplant described in claim 2SmHY5The primer pair of the quantitative fluorescent PCR analysis of the gene of albumen,
It is characterized in that, the base sequence of the primer pair is as shown in SEQ ID NO.5, SEQ ID NO.6.
5. a kind of coding eggplant as claimed in claim 2SmHY5The gene of albumen is grown under light in genetic engineering regulation plant
The purposes of situation.
6. encoding eggplant according to claim 5SmHY5The purposes of the gene of albumen, which is characterized in that the plant is quasi-
Southern mustard.
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| CN109810181B (en) * | 2019-01-04 | 2022-04-01 | 南京农业大学 | Pear transcription factor PyHY5, and recombinant expression vector and application thereof |
| CN111778276B (en) * | 2020-07-24 | 2022-07-12 | 上海交通大学 | Eggplant SmBIC2 gene and application of protein |
| CN111944772B (en) * | 2020-07-31 | 2022-03-04 | 上海交通大学 | Eggplant cryptochrome blue light inhibitor SmBIC1 protein and coding gene |
| CN113789334B (en) * | 2021-09-28 | 2022-08-02 | 浙江大学 | Application of HY5 gene in regulation and control of plant resistance to pest and disease damage |
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| CN103215275A (en) * | 2013-05-20 | 2013-07-24 | 上海交通大学 | Gene sequence for synthesizing relative transcription factor SmMYB by eggplant anthocyanidin |
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