CN103215275A - Gene sequence for synthesizing relative transcription factor SmMYB by eggplant anthocyanidin - Google Patents
Gene sequence for synthesizing relative transcription factor SmMYB by eggplant anthocyanidin Download PDFInfo
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- CN103215275A CN103215275A CN2012104365547A CN201210436554A CN103215275A CN 103215275 A CN103215275 A CN 103215275A CN 2012104365547 A CN2012104365547 A CN 2012104365547A CN 201210436554 A CN201210436554 A CN 201210436554A CN 103215275 A CN103215275 A CN 103215275A
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Abstract
The invention discloses a gene sequence for synthesizing a relative transcription factor SmMYB by eggplant anthocyanidin, comprising a nucleotide sequence shown in 1st-1035th nucleotides in SEQ ID NO.1, wherein a polypeptide coded by the gene sequence comprises an amino acid sequence is shown in SEQ ID NO.2. The gene sequence in the invention is a key regulation and control gene for synthesizing eggplant anthocyanidin, the relative transcription factor SmMYB is synthesized by eggplant anthocyanidin. A downstream gene which is regulated and controlled by the gene sequence and is closely related to anthocyanidin synthesis, and other transcription factor genes manually acting with the gene sequence, are screened. The gene sequence in the invention is applied to obtain an eggplant kind with high anthocyanidin content and excellent resistance quality.
Description
Technical field
What the present invention relates to is a kind of gene order of biological technical field, specifically is the gene order of the synthetic associated transcription factor SmMYB of a kind of eggplant anthocyanidin.
Background technology
Anthocyanidin extensively exists at nature, belongs to the flavonoid class material, usually with carbohydrate formation glucosides such as glucose, is called anthocyanogen.Because that anthocyanidin has is anti-oxidant, remove biochemical pharmacology effect such as free radical, since the eighties, the whole world is studied increasingly extensive deep to it.Eggplant is to originate from the vegetable crop that extensively plant in the whole world in the Asia, and its different varieties is widely different on shape and color, and purple eggplant is a kind the most general on the market.Purple solanberry skin is rich in delphinidin-3-rutinoside, is the very strong anthocyanogen of a kind of stability, and nutritive value is abundant.Nowadays, improve the important direction that interior flavonoid (anthocyanidin) the class material of plant materials has become plant breeding.
Paz-Ares etc. have reported the gene C 1 of the coding myb transcription factor that identifies for the first time from corn in " The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators " that " EMBO J " 1987 the 6th volumes 3553-3558 page or leaf is delivered.Multidigit investigator such as Espley, Takahashi, Dal Cin isolates the synthetic relevant myb gene of a plurality of and anthocyanidin in succession from plants such as Arabidopis thaliana, apple, petunia afterwards.Albert etc. are in " Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning " literary composition that " Plant J " 2011 the 65th volumes 771-784 page or leaf is delivered; propose MYB albumen, bHLH albumen, the plain synthetic pattern of WD40 albumen coordinated regulation petunia cyanine, provide fundamental basis for people probe into the synthetic regulatory mechanism of plant anthocyanidin.
Research to solanaceous crops anthocyanidin biosynthetic controlling at present mainly concentrates in petunia, potato, the tomato, research to eggplant anthocyanidin also concentrates on oxidation-resistance evaluation and key gene screening, and gene clone and regulatory mechanism research of transcription factor are not appeared in the newspapers as yet.The synthetic relevant myb transcription factor gene SmMYB of separating clone eggplant anthocyanidin of the present invention, its spatial and temporal expression characteristic is analyzed, provide fundamental basis for probing into eggplant anthocyanidin biosynthetic controlling mechanism, also the further exploitation for eggplant fruit look breeding and eggplant health-care effect provides condition.
By prior art documents, do not find the technical theme identical or similar as yet with the present invention.
Summary of the invention
The gene order that the purpose of this invention is to provide the synthetic associated transcription factor SmMYB of a kind of eggplant anthocyanidin.This gene order cDNA total length 1035bp, 278 amino acid are derived in the position, coding region between 69bp-905bp, and its coding and deduced amino acid are:
1 M N T A T V A K S L G V R K G A W T E E
1 ATGAATACTGCTACTGTTGCTAAGTCATTGGGAGTGAGGAAAGGTGCATGGACTGAAGAA
21 E D L L L R K C M D K Y G E G K W H L V
61 GAAGATTTACTATTGAGGAAATGTATGGACAAGTATGGTGAAGGCAAGTGGCATCTTGTT
41 P T R S G L N R C R K S C R L R W L N Y
121 CCCACTAGATCAGGTCTAAATAGATGCCGAAAGAGTTGTAGACTAAGATGGTTGAATTAT
61 L R P H I K R G D F A P D E I D L I L R
181 TTAAGACCACATATCAAAAGAGGTGACTTTGCTCCGGACGAAATAGATCTCATTTTGAGA
81 L H K L L G N R W S L I A G R F P E R T
241 CTTCATAAGCTTCTAGGCAACAGATGGTCACTTATTGCTGGAAGATTTCCGGAAAGAACA
101 A N D V K N Y W N T H I Q K K L T N S R
301 GCAAACGATGTGAAAAACTATTGGAACACACACATACAAAAAAAGTTAACAAATTCGCGG
121 P Q M Q E R K H N N A L K I T K N T I L
361 CCTCAGATGCAAGAGAGAAAGCACAATAATGCCCTCAAGATCACCAAAAACACCATACTA
141 R P Q P R P P P P P P P P P P P P P R T
421 AGACCTCAACCTCGACCTCCACCTCCACCTCCACCTCCACCTCCACCTCCACCTCGGACC
161 F S S A K N V S W C T N K N M N I T N T
481 TTCTCAAGTGCAAAGAATGTTTCTTGGTGCACCAATAAAAATATGAATATCACAAACACA
181 L D K D N E R H K E I G V N T C E K P K
541 TTAGATAAAGATAACGAACGCCACAAAGAAATTGGAGTAAATACATGTGAGAAGCCAAAA
201 G D A T S S S I D D D G V Q W W T S L L
601 GGTGATGCAACATCGTCGTCTATAGACGATGATGGAGTTCAATGGTGGACAAGTTTGCTG
221 E N C N E I E E E A T A V L S F E E E N
661 GAAAATTGCAATGAAATTGAGGAAGAAGCAACAGCAGTACTGAGCTTTGAGGAAGAAAAT
241 K F L P N L L H E E N N S P P M Q Q G Q
721 AAGTTTTTACCAAATTTGTTGCATGAAGAAAATAATTCACCACCCATGCAACAAGGACAA
261 N D G W D D F S V D I D L W N L F N *
781 AATGATGGTTGGGATGACTTTTCAGTGGATATTGACCTATGGAATCTATTTAATTAG
The present invention is achieved by the following technical solutions:
The gene order of the synthetic associated transcription factor SmMYB of a kind of eggplant anthocyanidin, it has among the SEQ ID NO.1 from the nucleotide sequence shown in the Nucleotide 1-1035 position.
Described gene order encoded polypeptides has the aminoacid sequence shown in the SEQ ID NO.2.
The application of the synthetic associated transcription factor SmMYB gene of described eggplant anthocyanidin in obtaining high anthocyanidin content and the colory eggplant kind of resistance.
The present invention is according to the Genebank database, and utilization information biology means are relatively designed degenerated primer by homologous sequence, and the method with molecular cloning clones MYB(SmMYB in the purple eggplant) gene; Through sequential analysis, determine it is the eggplant myb gene; The tissue expression analysis finds that the synthetic associated transcription factor SmMYB gene of eggplant anthocyanidin expression amount in purple solanberry skin is respectively organized apparently higher than other tissue of purple eggplant and white eggplant; After the bagging shading treatment, it is similar to this SmMYB gene expression amount variation tendency that the fluorescence half-quantitative detection finds that eggplant pericarp anthocyanidin content changes, and illustrates that this gene is synthetic relevant with eggplant anthocyanidin.
The bacillus coli DH 5 alpha that the present invention relates to, BL-21 bacterial strain " Sa nurse Brooker J, Russell DW. molecular cloning experiment guide [M]. Huang Peitang, Wang Jiaxi, Zhu Houchu waits and translates. the 3rd edition. Beijing: Science Press, 2002 " in open; Bacillus coli DH 5 alpha, BL-21 can obtain by disclosing commercially available commercial channel, as sky, Shanghai root biotech firm, CompanyAddress: Room 606, No. 1 building, No. 27 boats star commercial affairs building, Shanghai City Cao Xilu 258 lanes.
Myb transcription factor gene of the present invention is an eggplant anthocyanidin synthetic key controlling gene, and this gene synthesizes thereby regulate and control anthocyanidin by regulate the expression of downstream gene in conjunction with cis element.
The present invention has following beneficial effect: the gene that utilizes the synthetic associated transcription factor SmMYB of eggplant anthocyanidin of the present invention, can filter out be subjected to its regulation and control with the synthetic closely-related downstream gene of anthocyanidin, and other transcription factor genes interactional, thereby probe into eggplant anthocyanidin synthesis path regulatory mechanism with it.Utilize the present invention can obtain high anthocyanidin content and the colory eggplant kind of resistance.
Description of drawings
Fig. 1 is the SmMYB expression analysis figure of gene organization of the present invention.
Fig. 2 is shading treatment consequence peel anthocyanidin content figure.
Fig. 3 is SmMYB gene expression analysis figure in the pericarp after the shading treatment.
Embodiment
The invention will be further described below in conjunction with specific embodiment and accompanying drawing, and these embodiment only are used to the present invention is described and can not limit the scope of protection of present invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, molecular cloning laboratory manual (the New York:Cold Spring Harbor Laboratory Press that writes of Sambrook etc. (1989) for example, 1989) condition described in, or the condition of advising according to manufacturer.Test used PrimeScript
TM1st Strand cDNA Synthesis Kit, the Taq enzyme of high-fidelity, pMD19-T carrier are the products of Dalian TaKaRa company.
1, experiment material is drawn materials---
The good eggplant germ plasm resource YZ14 and the YZ3 that preserve with the applicant are experiment material.The YZ14 phenotype is stingless, purple stem, pale reddish brown, purple fruit, below all is called purple eggplant; The YZ3 phenotype is spinosity, green stem, spend in vain, gingko, has more tangible wild eggplant proterties, below all is called white eggplant.Experiment material is all planted in the artificial plastic greenhouse in space breeding base, Minhang District Pujiang town, Shanghai City, and it is also solid to grow seedlings under the natural condition, grow.Get root, purple stem, purple blade, purple petal, the purple fruit of purple eggplant, root, green stem, green blade, white petal, the white fruit of white eggplant.
2, extract purple eggplant petal RNA---
A) take by weighing 0.5g left and right sides purple petal, the adding liquid nitrogen fills the back branch and is milled into meal, before the liquid nitrogen volatilization, in sample transfer to a RNase-free 1.5ml centrifuge tube, the concuss mixing, the centrifugal 5min of 12000rpm draws in supernatant to the new RNase-free 1.5ml centrifuge tube;
B) add the abundant mixing of 1/2 volume dehydrated alcohol in lysate sample adsorption column is put into collection tube, with pipettor solution is all added in the adsorption column, leave standstill 2min, the centrifugal 1min of 8000rpm outwells waste liquid in the collection tube;
C) adsorption column is put back in the collection tube, added 500 μ l RW Soultion, leave standstill 1min, the centrifugal 1min of 10000rpm outwells waste liquid in the collection tube;
D) adsorption column is put back in the collection tube, added 500 μ l RPE Soultion, leave standstill 2min, the centrifugal 1min of 10000rpm outwells waste liquid in the collection tube;
E) repeating step d) once;
F) adsorption column is put back in the collection tube the centrifugal 2min of 10000rpm;
G) adsorption column is put into the 1.5ml centrifuge tube of RNase-free, added 30-50 μ l DEPC-treatedddH in adsorption film central authorities
2O leaves standstill 5min, the centrifugal 2min of 12000rpm, with resulting RNA solution in-70 ℃ of preservations;
H) identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
3, SmDFR gene cDNA full-length clone---
According to myb gene DNA in the plant in conjunction with territory conserved regions design pair of degenerate primers (tabulating 2 as follows), flower with purple long eggplant carries out RT-PCR and PCR as the total RNA of material extraction, reverse transcription is carried out with reference to giving birth to worker AMV ThermoScript II specification sheets, and the PCR reaction conditions is: 94 ℃ of 4min; 94 ℃ of 30s, 55 ℃ of 45s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 8min.The PCR product that reclaims is connected on the pMD19-T carrier, and transformed into escherichia coli DH-5 α utilizes α complementation and bacterium colony PCR screening positive clone, delivers to the big gene of Shanghai China and checks order.
According to myb gene intermediate segment nucleotide sequence design 3 ' RACE amplifying specific primer that has obtained and nido check primer (tabulating 1 as follows), carry out the terminal amplification of 3 ' cDNA with 3 ' universal primer UPM and NUP respectively.Design 5 ' RACE amplifying specific primer and nido check primer (tabulating 1 as follows) carry out the terminal amplification of 5 ' cDNA with SMARTer5 ' Primer and SMARTer5 ' Nested Primer respectively.Operation steps is with reference to SMARTer
TMRACE cDNAAmplification Kit test kit (Takara) specification sheets is finished.Cloning process is the same.
According to RACE splicing sequences Design full length gene amplifying specific primer (tabulating 1 as follows), the cDNA that obtains with reverse transcription is as pcr template, amplification eggplant SmMYB gene cDNA full length sequence, reverse transcription is carried out with reference to giving birth to worker AMV ThermoScript II specification sheets, and the PCR reaction conditions is 98 ℃ of 3min; 98 ℃ of 10s, 55 ℃ of 15s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 8min.Cloning process is the same.
Table 1 the primer of the present invention
Table 2 shading treatment experimental design of the present invention
"-" expression shading, light is seen in "+" expression;
Clone and isolate the cDNA sequence of the SmDFR gene of purple eggplant, its total length is 1503bp, and protein-coding region is total to 837bp from 69-905,278 amino acid of coding of deriving.SmMYB sequence total length 1035bp, open reading frame is totally 837 Nucleotide from 69bp to 905bp, 278 amino acid of deriving, molecular weight is 32123.2, iso-electric point is 8.47; InterPro database qualification result shows that SmMYB has two MYB-DNA-binding structural domains; The Subcellular Localization prediction is positioned SmMYB albumen in the nucleus; PredictProtein predicts that this amino acid contains the helix turn helix structure.SmMYB deduced amino acid and other plant R2R3-MYB transcription factor aminoacid sequence are compared, and structure evolutionary tree, find SmMYB and capsicum CaMYB(CAE75745.1) evolutionary distance is nearest, homology is up to 71%, with plants of Solanaceae such as petunia, tobacco, potatos higher sequence similarity degree is arranged all, with Arabidopis thaliana AtMYB113(NP_176811.1) evolutionary relationship relative far away, homology is 47%.Have one section proline(Pro) (P) enrichment region, acidic amino acid enrichment region at SmMYB gene C-end, prompting SmMYB may have transcriptional activation function.Prove that thus the anthocyanidin transcription activating protein of SmMYB gene coded protein and other species belongs to same protein family, has similar function.
CDNA coding region complete sequence and derivation protein sequence are seen SEQ ID.NO.1 and SEQ ID.NO.2.
Extract root, stem epidermis, leaf, petal, the pericarp RNA of purple eggplant, white eggplant, with internal control gene SmActin in contrast, adopt SmMYB-RT-F/SmMYB-RT-R and SmActin-F/SmActin-R primer (table 1) to be carried out the fluorescence sxemiquantitative pcr analysis of SmMYB gene.
Typical curve adopts the standard cDNA sample of concentration known to draw, and 5 standard models are set, and is respectively 10 times of cDNA stoste and dilutions, and 100 times, the cDNA sample of 1000 times and 10000 times.SYBRPrimeScriptRT-PCR Kit(Takara is adopted in the operation of quantitative PCR), experimental procedure is carried out according to the test kit specification sheets.Fluorescence sxemiquantitative PCR is reflected at Mastercyclerep realplex(Eppendorf) carry out on the PCR instrument.The reaction conditions program is 95 ℃ of pre-sex change 30s, 95 ℃ of sex change 5s, and 60 ℃ of annealing 30s, 72 ℃ are extended 20s, totally 40 circulations.
Purple eggplant of two calibration curve method analyses and white eggplant respectively organize myb gene fluorescence half-quantitative detection result to show, referring to Fig. 1, the synthetic associated transcription factor SmMYB gene expression amount in purple solanberry skin of eggplant anthocyanidin is the highest, all has low scale to reach in other tissue of purple eggplant and white eggplant.
1, purple eggplant bagging shading treatment experimental design.
Purple eggplant YZ14 needs 20 days approximately from flowering period to the commodity ripening stage, and the flower of choosing open 90 ° of petal carries out the bagging shading treatment.Processing is divided into A, B, C, D, E, F, negative control CK-and positive control CK+ totally 8 kinds (table 2), and every kind of processing was carried out fruit harvesting in the 8th day, 10 days, 15 days, 20 days in the back of blooming respectively, cut and got the eggplant pericarp as experiment material.
2, anthocyanidin content is measured.
Bloom back 8 days, 10 days, 15 days, the pericarp of gathering in 20 days of treatment group and control group are carried out anthocyanidin content and are measured.Measuring method is: get the 1g tissue, add hydrochloric acid-ethanol 4mL of 2% in the 10mL centrifuge tube, place 32 ℃ of thermostat containers, dipping 2h then takes out centrifuge tube 8000 * g (RCF under 4 ℃, relative centrifugal force) centrifugal 10min, collect supernatant liquor, add hydrochloric acid-ethanol 3mL of 2% again, repeat to extract 3 times, merge supernatant liquor, constant volume is to 10mL.The anthocyanogen extracting solution respectively with 10 times of pH 1.0 buffered soln and pH 4.5 buffered soln dilutions, stablize behind the 60min respectively at 525nm and 700nm place mensuration light absorption value.Hydrochloric acid-ethanolic soln with 2% is as blank.The anthocyanidin content calculation formula is as follows:
Anthocyanin content (mg/g)
Wherein Δ A is a light absorption value; F is an extension rate; M is the relative molecular mass 449.2 of Cyanidin-3-glucoside; ε is the molar extinction coefficient 26900 of Cyanidin-3-glucoside; M is sample quality (g).
3, the synthetic associated transcription factor SmMYB genetic expression component analysis of eggplant anthocyanidin.
The analysis of SmMYB expression amount is carried out in bloom back 8 days, 10 days, 15 days, the pericarp of gathering in 20 days of treatment group and control group, primer is SmMYB-RT-F/SmMYB-RT-R and SmActin-F/SmActin-R(table 1), fluorescence sxemiquantitative PCR is reflected at Mastercyclerep realplex(Eppendorf) carry out on the PCR instrument.The reaction conditions program is 95 ℃ of pre-sex change 30s, 95 ℃ of sex change 5s, and 60 ℃ of annealing 30s, 72 ℃ are extended 20s, totally 40 circulations; Melt curve analysis is determined as from 65 ℃ to 95 ℃.Two calibration curve method analyzing gene expressions, the result shows that the low light level obviously suppresses SmMYB genetic expression, referring to Fig. 2, and anthocyanidin content obviously reduces referring to Fig. 3.Comparison diagram 2 and Fig. 3 can find that the synthetic associated transcription factor SmMYB gene expression amount variation tendency of eggplant anthocyanidin is similar to the anthocyanidin content variation tendency, illustrate that the synthetic associated transcription factor SmMYB gene of eggplant anthocyanidin is synthetic relevant with eggplant anthocyanidin.
Claims (3)
1. the gene order of the synthetic associated transcription factor SmMYB of eggplant anthocyanidin is characterized in that this gene order has among the SEQ ID NO.1 from the nucleotide sequence shown in the Nucleotide 1-1035 position.
2. the gene order of the synthetic associated transcription factor SmMYB of eggplant anthocyanidin according to claim 1 is characterized in that described gene order encoded polypeptides has the aminoacid sequence shown in the SEQ ID NO.2.
3. the application of the synthetic associated transcription factor SmMYB gene of eggplant anthocyanidin according to claim 2 in obtaining high anthocyanidin content and the colory eggplant kind of resistance.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059926A (en) * | 2014-06-24 | 2014-09-24 | 上海交通大学 | Related genes for synthesis of eggplant anthocyanin and coding protein of related genes |
| CN105440115A (en) * | 2015-12-28 | 2016-03-30 | 上海交通大学 | Eggplant SmHY5 protein and coding gene thereof |
| CN107176982A (en) * | 2017-06-27 | 2017-09-19 | 中国热带农业科学院橡胶研究所 | Regulate and control transcription factor and its encoding gene and the application of rubber tree anthocyanidin synthesis |
| CN108503699A (en) * | 2018-06-12 | 2018-09-07 | 中国科学院西北高原生物研究所 | Matrimony vine gene and its coding protein, recombinant vector, and application thereof |
| CN108676805A (en) * | 2018-06-25 | 2018-10-19 | 河南省农业科学院 | Petunia ASRs genomes and its application |
| CN109748959A (en) * | 2017-11-08 | 2019-05-14 | 中国科学院遗传与发育生物学研究所 | Anthocyanidin synthesis associated protein SlANT1L and its encoding gene and application |
| CN111778276A (en) * | 2020-07-24 | 2020-10-16 | 上海交通大学 | Eggplant SmBIC2 gene and application of protein |
| CN111996198A (en) * | 2020-08-26 | 2020-11-27 | 山东农业大学 | Anthocyanin regulatory gene SmbHLH1 in eggplant stem and application thereof |
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| CN104059926A (en) * | 2014-06-24 | 2014-09-24 | 上海交通大学 | Related genes for synthesis of eggplant anthocyanin and coding protein of related genes |
| CN105440115A (en) * | 2015-12-28 | 2016-03-30 | 上海交通大学 | Eggplant SmHY5 protein and coding gene thereof |
| CN105440115B (en) * | 2015-12-28 | 2019-02-22 | 上海交通大学 | Eggplant SmHY5 albumen and its encoding gene |
| CN107176982B (en) * | 2017-06-27 | 2019-12-03 | 中国热带农业科学院橡胶研究所 | Regulate and control the transcription factor and its encoding gene and application that rubber tree anthocyanidin synthesizes |
| CN107176982A (en) * | 2017-06-27 | 2017-09-19 | 中国热带农业科学院橡胶研究所 | Regulate and control transcription factor and its encoding gene and the application of rubber tree anthocyanidin synthesis |
| CN109748959B (en) * | 2017-11-08 | 2021-03-23 | 中国科学院遗传与发育生物学研究所 | Anthocyanin synthesis related protein SlANT1L, and coding gene and application thereof |
| CN109748959A (en) * | 2017-11-08 | 2019-05-14 | 中国科学院遗传与发育生物学研究所 | Anthocyanidin synthesis associated protein SlANT1L and its encoding gene and application |
| CN108503699A (en) * | 2018-06-12 | 2018-09-07 | 中国科学院西北高原生物研究所 | Matrimony vine gene and its coding protein, recombinant vector, and application thereof |
| CN108676805A (en) * | 2018-06-25 | 2018-10-19 | 河南省农业科学院 | Petunia ASRs genomes and its application |
| CN108676805B (en) * | 2018-06-25 | 2021-09-24 | 河南省农业科学院 | Petunia ASRs genome and application thereof |
| CN111778276A (en) * | 2020-07-24 | 2020-10-16 | 上海交通大学 | Eggplant SmBIC2 gene and application of protein |
| CN111996198A (en) * | 2020-08-26 | 2020-11-27 | 山东农业大学 | Anthocyanin regulatory gene SmbHLH1 in eggplant stem and application thereof |
| CN111996198B (en) * | 2020-08-26 | 2022-06-24 | 山东农业大学 | Anthocyanin regulatory gene SmbHLH1 in eggplant stem and application thereof |
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Application publication date: 20130724 |