CN103205438A - OVATE gene sequence related to fruit shape control of eggplant - Google Patents
OVATE gene sequence related to fruit shape control of eggplant Download PDFInfo
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Abstract
The invention discloses an OVATE gene sequence related to fruit shape control of eggplant. The gene sequence comprises nucleotide sequences which are shown as the first position to the 1077th position of nucleotide in SEQ ID NO.1 or SEQ ID NO.2. By using the OVATE gene sequence related to fruit shape control of eggplant, ornamental materials of different fruit shapes of the eggplant and the eggplant variety for enlarging the fruits to increase production can be obtained by methods and means such as mutation breeding and transgenosis.
Description
Technical field
What the present invention relates to is a kind of gene order of biological technical field, is specifically related to a kind of OVATE gene order relevant with eggplant control fruit shape.
Background technology
The shape of fruit is varied, and it is subjected to the attention of biologist, geneticist and breeding man because of having inseparable relation with fruit weight.In the species to the fruit shape research, tomato is favored owing to worldwide being eaten by everybody, thereby studies as the model plant of Solanaceae.The reported first of present gene SUN, OVATE about the fruit shape, LC and the FAS gene of separating clone is all from tomato.Tomato OVATE gene is undergone mutation at this gene coded sequence as a kind of regulatory gene, when terminator extracts, produces the fruit of pyriform, and such fruit type is unexistent in hybrid strain.On capsicum, it is the comparison common phenomena that grafting causes the elongated of fruit, and by the analysis to capsicum OVATE genetic expression, finds that the difference of oblate and microscler capsicum is on the OVATE expression of gene amount height.To the interference of capsicum OVATE gene, make oblate capsicum elongation to become microscler capsicum that great variation has taken place the fruit type.
Though eggplant has than the more fruit shape of tomato, correlative study relatively lags behind, and the sequence of fruit shape genes involved and research do not appear in the newspapers as yet.On the basis based on research such as tomato and capsicum, both feasible to the research of the relevant OVATE gene of eggplant fruit type, can enrich the associated viscera of OVATE gene again from another angle.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the gene order of the relevant OVATE of a kind of eggplant control fruit shape is provided, in order to by to operations such as the regulation and control of this genetic expression and interference, realize the change to eggplant fruit shape.
Technical scheme of the present invention is:
The relevant OVATE gene order of a kind of eggplant control fruit shape, it has among the SEQ ID NO.1 from the nucleotide sequence shown in the Nucleotide 1-1077 position.
Described gene order encoded polypeptides has the aminoacid sequence shown in the SEQ ID NO.3.
Another technical scheme of the present invention is:
The relevant OVATE gene order of a kind of eggplant control fruit shape, it has among the SEQ ID NO.2 from the nucleotide sequence shown in the Nucleotide 1-1077 position.
Described gene order encoded polypeptides has the aminoacid sequence shown in the SEQ ID NO.3.
The present invention adopts the mode of homologous clone to obtain eggplant OVATE gene, the cDNA total length 1192bp of this eggplant OVATE gene, genes encoding head of district 1077bp according to the ovate sequence of Solanaceae model plant tomato.The long 1659bp of genome sequence, it comprises 1 intron and 2 exons, the long 689bp of first exon, second long 388bp of exon, the middle long 467bp of intron.The non-coding region of 48bp is arranged in OVATE gene start codon upstream, and there is the non-coding region of 67bp in the terminator downstream.358 amino acid of OVATE genes encoding, this aminoacid sequence has the structural domain of OVATE supergene family.OVATE gene and deduced amino acid thereof are:
CGATTAAGCAGTGGTATCAACGCAGAGTACATGGGGGAGACAAAAAGAATGGGAAAAAGCTTCAAGCTTCGGTTCTCCAGAGTTATGGCT
M G K S F K L R F S R V M A
TCTTTCAACACCTGCCGTTCCAAGGAGCCTTCTTCTCTTCCCCAAAATCCTAATTTCTTCCCGCATAGTAACAATAAGCTCAGTTGTACA
S F N T C R S K E P S S L P Q N P N F F P H S N N K L S C T
AAACTCATCTCCTCCGATTTCCATCTTATCGATCAAAATCAAAATCCCCGTAATTATAATTACGTGCCGGAATCCATGATGATCTCCGTT
K L I S S D F H L I D Q N Q N P R N Y N Y V P E S M M I S V
GGCTGTAGGCCAGAATTCAAGTGGGAGAAAGAAGAGAAGTTTCACGTGGTTTCCAGCTCGTTCGTCTCCGAAGAAGAAGAAGAGGAGGAG
G C R P E F K W E K E E K F H V V S S S F V S E E E E E E E
ATCAAGTTGGCCTTACGACCTCCTGTTACTCCTCCGCGCTTCAGTACCTCTGAGAAGAAGAAGAAACGGCGAGTTAAAAAGTCGAAAACA
I K L A L R P P V T P P R F S T S E K K K K R R V K K S K T
AAGAGTAGAATGATCAGAATGAGCACTTCGTCAGCTGAGGAGTACAGCGGGATTTTAAGCGGTACGGATCAAACTGTGGATAATTATAAC
K S R M I R M S T S S A E E Y S G I L S G T D Q T V D N Y N
GAAGAAGAGGAAACTGAAACTTTAGTTTCATCTTCCAAAAGTTGTTTTGATTTCTCAACTGATGACTCATCCACTGATTTCAACCCTCAG
E E E E T E T L V S S S K S C F D F S T D D S S T D F N P Q
TTGGAAACCATATGTGAGACCGCTACAATGAGGCGTCGTCACAAGAAGAACGCCACCACCAAGAGGAGACTCAAGCATTCTAGACCAAGT
L E T I C E T A T M R R R H K K N A T T K R R L K H S R P S
TTTTCCTCTTCAAAAGGAAGAAGATCATCAGTTTCTACGTCATCGGACAGTGAGTTACCAGCGAGGTTATCAGTGTTCAAAAAGCTGATA
F S S S K G R R S S V S T S S D S E L P A R L S V F K K L I
CCGTGTAGCGTCGATGGAAAAGTGAAGGAGAGTTTCGCTATCGTGAAGAAATCTCAGGACCCATACGAAGATTTCAAGAGGTCAATGATG
P C S V D G K V K E S F A I V K K S Q D P Y E D F K R S M M
GAGATGATTTTGGAGAAGGAAATGTTTGAGAAGAATGAGCTTGAGCAGCTTCTACAGTGTTTTCTGTCGTTGAATGGAAAGCATTATCAT
E M I L E K E M F E K N E L E Q L L Q C F L S L N G K H Y H
GGAGTGATAGTTGAGGCTTTCTCCGATATTTGGGAGACTTTGTTTTTAGGTAATAATGATAATGATAGAGTAAGGAGGATGTCAAGTCAT
G V I V E A F S D I W E T L F L G N N D N D R V R R M S S H
GATCCCCACCCACCTACTGTAGGAAGAAGAAGAACGTCCCATTGATTACTCCATGTCGGCTAATCATGATTTAGTCGTTAATTAATAAAT
D P H P P T V G R R R T S H -
TTAATTTTTCTTTTATTTTGTT
Test kits such as the RNA extraction that the present invention relates to, PCR purifying, plasmid recovery obtain by disclosing commercially available commercial channel, purchase in giving birth to worker's biotechnology (Shanghai) limited-liability company, address: No. 698, Shanghai Songjiang Qu Xiang Min Lu.Reverse transcription,
Max DNA Polymerase enzyme, clone's pMD19-T carrier and intestinal bacteria JM19 bacterial strain etc. are purchased in precious biotechnology (Dalian) company limited CompanyAddress: No. 19, Dongbei 2ed Street ,Economic Development Zone ,Dalian City ,Liaoning Province.
The present invention has following beneficial effect: described OVATE gene is the negative regulator gene of eggplant control fruit shape, utilize this OVATE gene relevant with eggplant control fruit shape, can be by to operations such as the regulation and control of this genetic expression and interference, also can be by this gene be carried out site mutation, change the sequence of OVATE gene in the eggplant, thereby eggplant fruit shape is changed, reach and improve its ornamental value and output, realize higher economic worth.
Description of drawings
Fig. 1 is the RACE electrophorogram of cDNA total length amplification of the present invention.
Among the figure, article one swimming lane is 5 ' RACE, and the second swimming lane is 3 ' RACE, the 3rd the negative contrast of swimming lane, the 4th the positive contrast of swimming lane.
Fig. 2 is eggplant OVATE albumen conserved domain of the present invention.
Among the figure, the OVATE structural domain is positioned at amino acid whose 270 to 330, shown in the Ovate label character.
Fig. 3 is OVATE gene structure of the present invention.
Among the figure, first dark frame is first exon, and second dark frame is second exon, and the line between the dark frame is intron, and the line at two ends is non-coding region.
Fig. 4 is the difference of purple long eggplant and circle eggplant different times OVATE genetic expression.
Among the figure, it is microscler eggplant that white is filled histogram, and it is oblate eggplant that black is filled histogram, and expression is respectively and spends back 5 days period, commodity phase fruit and three periods of mature fruit.
Embodiment
The present invention uses the information biology means according to the Genebank database, designs gene specific primer by the homologous sequence comparison, and the method with RACE equimolecular clone clones OVATE gene in the eggplant; Through sequential analysis, determine it is eggplant OVATE gene; The tissue expression analysis revealed, this OVATE gene is in the ovary tissue of blooming back 5 days, the expression amount of oblate eggplant is apparently higher than the expression amount of microscler eggplant, the effect that influences fruit length with OVATE negative gene regulation and control in tomato and the capsicum is consistent, illustrates that this gene is relevant with eggplant fruit shape.
Below in conjunction with specific embodiment and accompanying drawing, further set forth the specific embodiment of the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the 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 RT-PCR Kit, high-fidelity
Max archaeal dna polymerase, pMD19-T carrier are precious biological (Dalian) Engineering Co., Ltd (TaKaRa) products.
Embodiment: clone and the expression analysis of the relevant OVATE gene of eggplant fruit shape
1, the eggplant experiment material is drawn materials
Eggplant strain purple long eggplant and the Bai Yuan eggplant of collecting and preserving with the applicant are material.Purple long eggplant is representative purple eggplant strain, and by the laboratory plantation for many years, self propagated is reserved seed for planting, and has stingless, purple stem, pale reddish brown, long strip shape fruit phenotype that purple eggplant Cultivar has.The fruit in commodity ripening stage, black purple, the fruit type is long and big, so the name purple long eggplant.White circle eggplant is the eggplant strain that seminar preserves, and has stingless, pale reddish brown, white pericarp, oblate fruit phenotype, the commodity ripening stage.Experiment material is cultivated in the artificial plastic greenhouse in space breeding base, Minhang District Pujiang town, Shanghai City.Grow seedlings under field conditions (factors), growth is also solid.Experiment is drawn materials and need be used the purple long eggplant back five days ovary of blooming, green blade, immature phase fruit pulp, commodity ripening stage fruit pulp.
2, the total RNA of the total RNA extracting of UNIQ-10 pillar eggplant
1) takes by weighing 0.1g left and right sides eggplant flesh tissue, in liquid nitrogen, be ground to powder, transfer to fast in the centrifuge tube of 1.5ml sterilization; Add 450 μ l RLT Buffer at once, with the abundant mixing of homogenizer homogenate 30sec; The centrifugal 5min of 12000rpm draws supernatant liquor in new centrifuge tube.
2) add 1/2 volume dehydrated alcohol in the supernatant liquor, abundant mixing is transferred in the adsorption column and leaves standstill 2mim, and the centrifugal 1mim of 8000rpm outwells the waste liquid in the collection tube.
3). adsorption column is put back in the collection tube, added 500 μ l RW Solution, leave standstill 1min, the centrifugal 1min of 10000rpm outwells the waste liquid in the collection tube.
4) adsorption column is put back in the collection tube, added 500 μ l RPE Solution, leave standstill 2min, the centrifugal 1min of 10000rpm outwells the waste liquid in the collection tube.
5) repeating step 4).
6) adsorption column is put back in the collection tube the centrifugal 2min of 10000rpm.
7) adsorption column is put into new 1.5ml centrifuge tube, central authorities add the distilled water that 30 μ l DEPC handle at the adsorption column film, leave standstill 5min, and the centrifugal 2min of 12000rpm places resulting RNA solution-80 ° of C preservations or is used for follow-up test.
3, the RACE of eggplant OVATE gene cDNA total length clone
Be query based on tomato OVATE gene order among the Genbank, retrieval eggplant est database splices with ContigExpress software the est sequence that obtains, and designs eggplant OVATE gene specific primer.OVATE gene RACE special primer (tabulating 1 as follows), 5 ' end of two sequences of amplification and 3 ' end full length sequence.SMARTer is used in the RACE experiment
TMRACEcDNA Amplification Kit test kit (Clontech) is operated, and the cDNA that obtains is template, carries out first run PCR with OVATE gene specific primer GSP1, GSP2, carries out nest-type PRC with NGSP1, NGSP2.5 ' RACE nest-type PRC result detects through 2% agarose gel electrophoresis, cuts glue and reclaims target fragment.The glue recycling step reclaims test kit with reference to giving birth to worker SanPrep pillar DNA glue.Pcr amplification product purifying rear clone to pMD-19T carrier (Takara), is imported intestinal bacteria JM19 bacterial strain, detect through α complementation and M13 universal primer, the positive transformant that fragment is correct send China big gene sequencing.By 3 ' RACE and 5 ' RACE experiment, cloned the cDNA full length sequence of the OVATE gene of purple long eggplant and Bai Yuanqie.CDNA length is 1192bp, and protein-coding region is from the 49th to the 1125th of cDNA 1077bp altogether, 358 amino acid of coding of deriving, and cDNA coding region sequence and derivation protein sequence are seen SEQ ID.NO.1 and SEQ ID.NO.2.
Table 1 is the used RACE primer of the present invention
The nucleotide sequence that order-checking is obtained is compared with listed OVATE gene order among the BLASTn method of NCBI and the Genbank, and is the highest with the homology degree of the coding region sequence of the OVATE gene of tomato, specifically sees Table 2.Aminoacid sequence BLASTp retrieval in ncbi database with eggplant OVATE genes encoding detects the structural domain that it has the OVATE protein family, as shown in Figure 2.The aminoacid sequence of eggplant OVATE genes encoding and tomato OVATE protein sequence carry out sequence alignment, see Table 3.
The sequence alignment of table 2 tomato and eggplant OVATE gene nucleotide
Tomato: tomato OVATE gene nucleotide series (the NCBI accession number is NM_001247292)
Eggplant: eggplant OVATE gene nucleotide series
The aminoacid sequence comparison of table 3 tomato and eggplant OVATE genes encoding
Tomato: tomato OVATE gene coding amino acid sequence (the NCBI accession number is NP_001234221)
Eggplant: eggplant OVATE gene coding amino acid sequence
4, the analysis that relatively reaches gene structure of eggplant OVATE gene coding region
1) the CTAB method is extracted eggplant leaf DNA
Take by weighing eggplant young tender leaf agreement that contracts a film or TV play to an actor or actress 2g and put into the mortar that freezes, add an amount of liquid nitrogen grinding to white powder, after collecting with the PE of 1.5ml pipe, adds 65 ℃ of warm good CTAB and extract damping fluid 600ul and fully shake and shake up.65 ℃ of water-bath 45min, during soft vibration 3-5 time.Add isopyknic saturated phenol/chloroform/Virahol (25:24:1) mixed solution after being chilled to room temperature, softly put upside down mixing, the centrifugal 10min of 13000rpm under 4 ℃ of cold condition.Draw supernatant liquor to new PE pipe, add isopyknic chloroform/Virahol (24:1) mixed solution, softly put upside down mixing, the centrifugal 10min of 13000rpm under 4 ℃ of cold condition.After drawing supernatant liquor 2 NaAC of dropping (PH4.8-5.2) and adding and the isopyknic cooling Virahol of supernatant liquor, place 30min deposit D NA for-20 ℃.In 4 ℃ of centrifugal 15min of following 12000rpm, remove supernatant liquor, the ethanol with 75% cleans DNA three times.Being deposited on the aseptic technique platform room temperature dries.With the band TE of RNAase or distilled water dissolving, detect the DNA quality with NANO DROP1000, and it is standby to be stored in-20 ℃ of refrigerators.
2) compare of analysis of purple long eggplant and Bai Yuan eggplant OVATE gene coded sequence
Be template with purple long eggplant and the common reverse transcription cDNA of Bai Yuan eggplant and DNA, initiator codon and terminator codon Position Design primer (forward primer: ccg CTC GAG ATG GGA AAA AGC TTC AAG CTT CGG reverse primer: aaaaCTG CAG ATG GGA CGT TCT TCT TCT TCC TAC), amplification eggplant OVATE gene coded sequence and genomic full length sequence.Pcr amplification adopts high-fidelity
Max archaeal dna polymerase, system are with reference to specification sheets, and annealing temperature is made as 55 ℃.The PCR product adds the A end reaction through giving birth to worker PCR product purification test kit purifying, is connected to the pMD19-T carrier, and transformed into escherichia coli JM19 send the order-checking of bacterium liquid.PCR product purification and add A tail experimental implementation step with reference to SanPrep pillar PCR product purification and DNA A-Tailing Kit test kit specification sheets.
The sequence of purple long eggplant and Bai Yuan eggplant cDNA amplification is compared with ClustalX2, at the 468th and the 804th the synonym replacement has taken place, but to not influence of amino acid, sequence alignment result such as table 4.
Table 4 purple long eggplant and the comparison of Bai Yuan eggplant OVATE gene coded sequence
Zichang: eggplant purple long eggplant OVATE gene coded sequence
Baiyuan: eggplant is round eggplant OVATE gene coded sequence in vain
3) gene structure analysis
Be the OVATE genome sequence that obtains of template with eggplant cDNA with eggplant DNA be that the OVATE gene order of template acquisition is carried out the analysis of gene structure.Gene structure display server is adopted in the gene structure analysis, uses with reference to website (gsds.cbi.pku.edu.cn) explanation.This gene has 2 exons and an intron, the long 689bp of first exon, and second long 388bp of exon, intron length is 467bp.The eggplant intron zone of different fruit types does not have the site variation.The gene structure that obtains is seen accompanying drawing 3.
5, eggplant OVATE expression of gene is analyzed
Get the total RNA of tissue extraction of purple long eggplant and Bai Yuan eggplant different times, with
RT reagent Kit With gDNA Eraser (Perfect Real Time) reverse transcription generates cDNA, and quantitative PCR kit adopts
Premix Ex Taq
TM(Tli RNaseH Plus).Reaction system reference reagent box specification sheets, the PCR program is as follows:
The used quantification PCR primer of table 5 the present invention
The step2 that quantitative fluorescent PCR is arranged on stage2 carries out signals collecting.The method of quantitative PCR adopts the relative quantification method, by with the difference of the difference calculating OVATE genetic expression of the Ct value of internal control gene ACTIN, namely 2
-△ △ CtStandard measure.The quantification PCR primer sequence sees Table 5.
Quantitative PCR is the result show, the relevant OVATE gene of eggplant control fruit type expression amount in the eggplant of two kinds of different fruit types has significant difference.The expression amount of microscler eggplant purple long eggplant is all lower at different times, and oblate white round eggplant expression amount spend back 5 days very high, commodity ripening stage afterwards and aging fruit expression amount reduce.Report on this and tomato and the capsicum expression more consistent, illustrate that eggplant OVATE gene also is that pattern by negative regulation is to the fruit type generation effect of eggplant.
Claims (3)
1. an eggplant is controlled the relevant OVATE gene order of fruit shape, and it is characterized in that: described gene order has among the SEQ ID NO.1 from the nucleotide sequence shown in the Nucleotide 1-1077 position.
2. an eggplant is controlled the relevant OVATE gene order of fruit shape, and it is characterized in that: described gene order has among the SEQ ID NO.2 from the nucleotide sequence shown in the Nucleotide 1-1077 position.
3. eggplant according to claim 1 and 2 is controlled the relevant OVATE gene order of fruit shape, and it is characterized in that: described gene order encoded polypeptides has the aminoacid sequence shown in the SEQ ID NO.3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175054A (en) * | 2019-07-02 | 2021-01-05 | 中国科学院遗传与发育生物学研究所 | Method for regulating and controlling plant type of rice |
| CN112457380A (en) * | 2019-09-09 | 2021-03-09 | 中国科学院遗传与发育生物学研究所 | Protein for regulating and controlling content of fruit shape and/or fruit juice of plant, related biological material and application thereof |
-
2013
- 2013-04-18 CN CN2013101358527A patent/CN103205438A/en active Pending
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175054A (en) * | 2019-07-02 | 2021-01-05 | 中国科学院遗传与发育生物学研究所 | Method for regulating and controlling plant type of rice |
| CN112175054B (en) * | 2019-07-02 | 2022-07-26 | 中国科学院遗传与发育生物学研究所 | Method for regulating rice plant type |
| CN112457380A (en) * | 2019-09-09 | 2021-03-09 | 中国科学院遗传与发育生物学研究所 | Protein for regulating and controlling content of fruit shape and/or fruit juice of plant, related biological material and application thereof |
| CN112457380B (en) * | 2019-09-09 | 2022-07-05 | 中国科学院遗传与发育生物学研究所 | Protein for regulating and controlling content of plant fruit shape and/or fruit juice, related biological material and application thereof |
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Application publication date: 20130717 |