CN1757732A - CDNA sequence of coding sweet potato phytoene synthetase - Google Patents

Abstract

A cDNA nucleotide sequence for coding sweet potato's phytoene synthetase is disclosed. It is obtained through separating it from the tuber of sweet potato and cloning it. Its cDNA length is 1500 bp. Its open reading frame is 1314 bp. It can catalyze the condensation of two GGPPs to generate phytoene, so it it's a key enzyme for synthesizing the beta-carotene of sweet potato.

Description

The cDNA sequence of coding sweet potato phytoene synthetase

Affiliated field

The present invention relates to the cDNA sequence of the coding phytoene synthetase (Ipomoeabatatas phytoene synthase) of expression in the sweet potato (Ipomoea batatas).

Technical background

(biosynthesizing of β-Carotene) mainly betides in higher plant, algae, fungi and the bacterial body β-Hu Luobusu, can not the de novo synthesis β-Hu Luobusu in the animal body.β-Hu Luobusu is the precursor of vitamin A (Vitamin A), can be converted into vitamin A as required in human body.It has nutrition, painted dual function, is the outstanding nourishing food additive of category-A (foodadditives) that the Food and Argriculture OrganizationFAO (FAO) and the foodstuff additive joint specialist council of The World Health Organization (WHO) are assert.In recent years, increasing medical research shows that β-Hu Luobusu can resist multiple cancer, particularly can reduce the lung cancer morbidity rate.β-Hu Luobusu strengthens body immunity at the cancellation free radical, and protection human health aspects such as preventing cardiovascular disease play an important role.

Natural beta-carotin cis-isomeride ratio height, at present chemical synthesis also can't be synthesized the β-Hu Luobusu cis-isomeride, it is anticancer, anti-cardiovascular disease and nourishing function are higher than the alltrans isomer far away and cis-isomeride is by clinical proof.

Carotenoid is synthetic to be a very huge secondary metabolism approach, synthesizing of β-Hu Luobusu from geranyl geranyl tetra-sodium (GGPP), successively through the catalysis of phytoene synthetase, phytoene dehydrogenase, sigma carotene dehydrogenase and lycopene beta cyclase, final synthetic β-Hu Luobusu, phytoene synthetase is a β-Hu Luobusu synthetic key enzyme.Before the present invention comes forth, any cDNA sequence that discloses or reported the coding sweet potato phytoene synthetase of mentioning in the present patent application is not arranged as yet.

Summary of the invention

The cDNA sequence that the purpose of this invention is to provide coding sweet potato phytoene synthetase.

In the present invention, " isolating " cDNA is meant, this DNA or fragment have been arranged in the sequence of its both sides under native state separates, and refers to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separates with follow its protein in cell.

In the present invention, various carrier known in the art be can select for use,, plasmid, clay etc. comprised as commercially available carrier.When producing the nucleotide sequence of coding sweet potato phytoene synthetase of the present invention, the nucleotide sequence of coding sweet potato phytoene synthetase operationally can be connected in expression regulation sequence, thereby form the sweet potato phytoene synthetase expression carrier.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

The cDNA sequence of coding sweet potato phytoene synthetase of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

After obtaining relevant sequence, obtain relevant sequence in large quantity with recombination method.Normally it is cloned into carrier, changes cell again over to, from the host cell after the propagation, separate obtaining relevant sequence then by ordinary method.

In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the nucleotide sequence of code book invention sweet potato phytoene synthetase.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.

Table 2 has been listed the homology comparison diagram of the cDNA sequence and capsicum phytoene synthetase cDNA (the GenBank Accession No.X68017) sequence of coding sweet potato phytoene synthetase of the present invention.

Below in conjunction with concrete steps, further set forth the present invention.Should be understood that these steps only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following step, usually according to normal condition, " molecular cloning ", " laboratory manual " (New York:Cold Spring Harbor Laboratory Press such as Sambrook for example, 1989) condition described in, or the condition of advising according to manufacturer.

Step 1

The clone of the cDNA sequence of coding sweet potato phytoene synthetase

1. separate tissue (isolation)

Red heart sweet potato variety Kingsoft 72 derives from crop institute of University Of Agriculture and Forestry In Fujian potato class research department, field planting 80 days, gets tender root of children, uses liquid nitrogen flash freezer.

2. the separation (total RNA isolation) of total RNA and detection

Get root, grind, add the 50ml pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to 50ml after the homogenate and newly manage, and extracted total RNA (TRIzol Reagents, Invitrogen, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, electrophoretogram presents 3 clear differentiable bands, 28SRNA wherein: 18SRNA ratio is about 2: 1, shows total RNA degraded basically, can be used for the clone of the cDNA sequence of coding sweet potato phytoene synthetase.

3. the clone of full length cDNA sequence (Cloning of Full-length cDNA)

Carry out the full length cDNA sequence clone, divide four-stage to carry out:

(1)RT-PCR

According to the nucleic acid conserved sequence of the coding phytoene synthetase of daffodil, corn, tomato, Radix Dauci Sativae, capsicum, the over-designed primer, the forward and the reverse primer of conserved sequence are respectively: SEQ ID NO.1 and SEQ ID NO.2.Adopt the method (TaKaRa test kit) of RT-PCR, obtain conserved sequence psy con (422bp), be connected on the T-easy carrier, with SP6 or T7 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of the phytoene synthase gene of conserved sequence that obtains and known tomato (Lycopersicon esculentum), capsicum (C.annuum), citrus (Citrus unshiu) is respectively 83%, 83%, 82%, thinks that tentatively it is the fragment of a coding sweet potato phytoene synthetase cDNA.

(2)3’-RACE

The total RNA of reverse transcription obtains the first chain cDNA.

First round PCR (primer is GR3 (SEQ ID NO.9)+SEQ ID NO.3)

Second takes turns PCR (GR3N (SEQ ID NO.10)+SEQ ID NO.4) obtains other process of psy3 (551bp) as shown in (1).

The existing database of sequencing result (Genebank+EMBL) search, the homology of nucleotide sequence that obtains the coding phytoene synthetase of its nucleotide sequence and known tomato (Lycopersiconesculentum) and capsicum (C.annuum), citrus (Citrus unshiu) is respectively 83%, 83%, 82%, is the fragment of a coding sweet potato phytoene synthetase cDNA so can further confirm it.

(3)5’-RACE

First round PCR (GR5 (SEQ ID NO.11)+SEQ ID NO.5)

Second takes turns PCR (GR5N (SEQ ID NO.12)+SEQ ID NO.6) obtains other process of psy5 (615bp) as shown in (1).

(4) coding region of pcr amplification sweet potato phytoene synthetase cDNA

By being used in combination aforesaid method, the full length nucleotide sequence of splicing candidate's coding sweet potato phytoene synthetase, on the basis that obtains this sequence (comprising complete open reading frame at least) information, further design forward primer psyF (SEQ IDNO.7) and reverse primer psyR (SEQ ID NO.8), is template with total RNA through Oligo (dT) reverse transcription, Pyrobest enzyme with high-fidelity, carry out pcr amplification, the PCR condition is 94 ℃ of 5min, thereupon with 94 ℃ of 30sec, 60 ℃ of 30sec and 72 ℃ of 2min carry out 30 circulations, extend 10min with 72 ℃ at last.The electrophoresis detection pcr amplification product, obtaining expanding fragment length is 1,363bp.

Therefore, make up above-mentioned 4 kinds of results that method obtains, obtained the full length cDNA sequence (table 1) of coding sweet potato phytoene synthetase.

The gene that result's proof of BLAST newly obtains from sweet potato really is phytoene synthetase cDNA.Because known homology phytoene synthetase can synthesize phytoene by catalysis GGPP, infer that this cDNA has identical functions.

Step 2

The sequence information of sweet potato phytoene synthetase cDNA and homology analysis:

The length of the sweet potato phytoene synthetase full-length cDNA that the present invention is new is 1,500bp, and open reading frame is positioned at 55-1368 position Nucleotide, and detailed sequence sees Table 1.The full length cDNA sequence of sweet potato phytoene synthetase is carried out the nucleotide homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, the phytoene synthetase cDNA's of sweet potato phytoene synthetase cDNA and capsicum (Capsicum annuum) (GenBank Accession No.X68017) has a homogeny of 76% on nucleotide level as a result, can think that both have very high similarity on function.

Step 3

The functional analysis of sweet potato phytoene synthetase cDNA:

In this step, the cDNA sequence construct of coding sweet potato phytoene synthetase is gone among the plant expression vector, and transformation of tobacco, to identify its function.

1. the structure of plant expression vector

According to sweet potato phytoene synthetase full length cDNA sequence (table 1), design amplifies the primer of entire reading frame, and introduce restriction endonuclease sites respectively on positive anti-primer: positive anti-primer is introduced XbaI and KpnI restriction enzyme site respectively.Amplified production with acquisition in the step 1 is a template, behind pcr amplification, guarantees that the reading frame of sweet potato phytoene synthetase is entirely true.Carry out 37 ℃ of enzymes with restriction endonuclease XbaI and KpnI respectively and cut 3h, reclaim purpose fragment 1,363bp; Carrier p2300 cuts 3h with XbaI and 37 ℃ of enzymes of KpnI restriction endonuclease, reclaims the purpose fragment respectively.The purpose fragment of the sweet potato phytoene synthetase cDNA that will cut through enzyme is connected with the p2300 carrier of cutting through corresponding restriction endonuclease, and transformed into escherichia coli DH5 α cultivates 20h for 37 ℃, and the PCR that carries out recon identifies and enzyme is cut evaluation.

2. plant expression vector transforms Agrobacterium

(1) gets a competence Agrobacterium EHA105, add about 1 μ g, the mixing gently of plant expression vector that contains sweet potato phytoene synthetase cDNA;

(2) quick-frozen 2 minutes in liquid nitrogen, 37 ℃ of incubations 5 minutes;

(3) add 500 μ l YEB liquid nutrient mediums, 28 ℃ jog 2-4 hour, eliminate competence;

(4) get 50-200 μ l bacterium liquid respectively, separate application is selected on the flat board in containing suitable antibiotic YEB, is inverted for 28 ℃ and cultivates two days.

(5) identify the single bacterium colony of the Agrobacterium EHA105 that is positive, be inoculated into and contain the 50mg/L Rifampin, in the 20ml liquid YEB substratum of 100mg/L kantlex, to logarithmic phase, get an amount of Agrobacterium doubly with liquid MS medium dilution 20-30 in 28 ℃ of constant temperature shaking table shaking culture 30 hours.

3. genetic transformation of tobacco and regeneration

(1) gets aseptic tobacco leaf, excision blade edge and Zhong Mai, 28 ℃ of pre-cultivations 2 days in Ms+1.0mg/L NAA solid medium;

(2) retrieve material, put into the Agrobacterium that has goal gene that aseptic MS liquid nutrient medium diluted, soaked 15 minutes, low speed shook 15 minutes in 28 ℃ of shaking tables are arranged then;

(3) take out vanelets, inhale with aseptic filter paper and remove unnecessary bacterium liquid, 28 ℃ of dark cultivations two days in the MS solid medium;

(4),, after aseptic filter paper is inhaled and removed unnecessary bacterium liquid, change over to and contain 100mg/LKm and 500mg/L Cb division culture medium M with adding the aseptic washing twice of 500mg/L Cb vanelets ₁In, be cultured under 28 ℃ of light and differentiate callus, until growing bud, per therebetween 15 days subcultures are once;

(5) bud that will grow to 3-5cm changes root media 1/2Ms over to and goes up root induction.

(6) after being accredited as positive transgene tobacco, continue to cultivate into seedling.

4. the extraction of transgene tobacco blade carotenoid

(1) get each transgene tobacco blade, lyophilized powder is made in lyophilize rapidly, puts in the vacuum drying oven and keeps in Dark Place, and measure as early as possible.

(2) accurately take by weighing lyophilized powder 1.000g in tool plug triangular flask, and the adding extracting solution (acetone: 45mL ethanol=3: 2), shake up, cover bottle stopper, use ultrasonic extraction 20 minutes, filter in the 50mL volumetric flask and constant volume.Getting 20mL filtrate puts in the separating funnel, add 10mL sherwood oil mixing, adding 10mL distilled water distributes, getting sherwood oil puts in the rotary evaporation bottle mutually, surplus water adds the 10mL sherwood oil reallocates, and merges 2 sherwood oils and puts on the Rotary Evaporators 40-45 ℃ of evaporate to dryness mutually, with the dissolving of 2.0mL Virahol, filter the back and go up machine mensuration.

5. high effective liquid chromatography for measuring transgene tobacco blade carotenoid

(1) preparation of β-Hu Luobusu standard specimen: β-Hu Luobusu mark Huaihe River product (Sigma company product) 1.0mg, with a small amount of trichloromethane dissolving, use petroleum ether dissolution again, solution changes in the 25.0ml volumetric flask, use the sherwood oil constant volume, concentration is 0.04mg/ml, and refrigerator is preserved.Face the time spent, draw 1.0ml in the 10.0ml volumetric flask, add moving phase to scale, this moment, concentration was 0.004mg/ml.

(2) preparation of Lyeopene standard specimen: Lyeopene mark Huaihe River product (Sigma company product) 1.0mg, with a small amount of trichloromethane dissolving, use petroleum ether dissolution again, solution changes in the 25.0ml volumetric flask, uses the sherwood oil constant volume, and concentration is 0.04mg/ml, and refrigerator is preserved.Face the time spent, draw 1.0ml in the 10.0ml volumetric flask, add moving phase to scale, this moment, concentration was 0.004mg/ml.

(3) measuring method: moving phase: acetonitrile: trichloromethane (92: 8).Detect wavelength: dual wavelength is measured, 470nm (0-min) and 450nm (8-13min).Flow velocity: 1.0mL/min.Column temperature: 35 ℃.Observing samples has the size variation of absorption peak at 470nm and 450nm place.

Mark that the present invention relates to and sequence apportion are as follows:

(1) information of SEQ ID NO.1

(i) sequence signature:

(A) length: 20bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.1

TGGTGT/cAGGC/aGG/aACAGATGA

(2) information of SEQ ID NO.2

(i) sequence signature:

(A) length: 23bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.2

CTTCCTCTTCTA/g/c/t?GCATCTTCT/gCC

(3) information of SEQ ID NO.3

(i) sequence signature:

(A) length: 24bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.3

CATTCTCAGAGACGTAGGCGAAGA

(4) information of SEQ ID NO.4

(i) sequence signature:

(A) length: 24bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.4

CTAGACGGGGGAGGGTCTATTTAC

(5) information of SEQ ID NO.5

(i) sequence signature:

(A) length: 24bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.5

AGGGCGGTTGGAGTTATATGCGAT

(6) information of SEQ ID NO.6

(i) sequence signature:

(A) length: 23bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.6

AGTCTCGTACCAGTCTCCGTCAT

(7) information of SEQ ID NO.7

(i) sequence signature:

(A) length: 28bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.7

GCTCTAGACACCTCAGCTCAAGAATGTC

(8) information of SEQ ID NO.8

(i) sequence signature:

(A) length: 26bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.8

CAGAATGCTTTCAGCTTCTTCAGTAC

(9) information of SEQ ID NO.9

(i) sequence signature:

(A) length: 25bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.9

GCTGTCAACGATACGCTACGTAACG

(10) information of SEQ ID NO.10

(i) sequence signature:

(A) length: 23bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.10

CGCTACGTAACGGCATGACAGTG

(11) information of SEQ ID NO.11

(i) sequence signature:

(A) length: 23bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.11

CGACTGGAGCACGAGGACACTGA

(12) information of SEQ ID NO.12

(i) sequence signature:

(A) length: 26bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.12

GGACACTGACATGGACTGAAGGAGTA

The cDNA sequence of table 1 coding sweet potato phytoene synthetase

＜110〉University Of Agriculture and Forestry In Fujian

＜120〉the cDNA sequence of coding sweet potato phytoene synthetase

<160>1

<170>Patent?In?Version?2.1

<210>1

<211>1500

<212>DNA

＜213〉sweet potato (Ipomoea batatas)

<221>gene

<222>(1)…(1500)

<400>Full?length?sequence?of?phytoene?synthase?gene?from?Ipomoea?batatas

1 GAAAACAAAA?TCTTGGAAGT?AGTTATATAG?AAACTATTTC?ACCTCAGCTC?AAGA ATGTCT

61 AGTGCCTTGC?TGTGGGCTGT?TTCTCCCTCT?TCTGAGCTAT?CGAATGGCAC?TGGAATCTTT

121 TATTCGGTGA?GGGAGGGAAT?CCGGATTGTG?GATTCGTCGA?GGTTCCTTGG?CAGGAACAGG

181 AGTTTGGTGT?ACAAAGGCAG?GGCTAAGAAG?GGTAAGAAAC?AAAGATGCAC?TTTGGCATCT

241 TTCAATGCAG?ACTCGAGGTA?TCTTTGCTCG?GGAGGGTCGA?GCTTGAAGAA?TGGAGGGAAA

301 TCCTCTGTGC?TTTCGAATGC?GGTGGTTAGC?CCAGCCGGGG?AAATGGCGAT?GTCATCTGAG

361 CAAAAGGTGT?ACGATGTAGT?GTTGAAGCAG?GCTGCTTTGG?TGAATAGGAG?GTTGAGATCC

421 ATAGATAATT?TGGAGGTGAA?GCCCGATATA?GCCCTTCCGG?GCGATTTGGG?CGTGTTGAGT

481 GAAGCTTATG?ATCGATGCGG?TGAAGTATGT?GCAGAGTATG?CTAAGACGTT?TTATTTGGGA

541 ACCATGCTAA?TGACACCTGA?GAGAAGAAGA?GCTATCTGGG?CGATATATGT?GTGGTGTAGG

601 CGGACAGATG?AGCTCGTTGA?CGGGCCTAAT?GCATCGCATA?TAACTCCAAC?CGCCCTGGAC

661 AGATGGGAGG?CTCGGCTGGA?AGACGTATTC?AGAGGGCGCC?CGTTTGATAT?GCTCGACGCT

721 GCACTATCAG?ATACAGTATC?CAGGTTTCCA?GTTGATATTC?AGCCCTTTAG?GGATATGATT

781 GAAGGAATGC?GAATGGACCT?CTGGAAATCG?AGATACGATA?ACTTTGATGA?GCTATACCTG

841 TACTGTTATT?ACGTTGCTGG?TACAGTTGGT?TTGATGAGTG?TCCCGGTTAT?GGGCATTGCG

901 CCCGAATCAA?AGGCAACTAC?AGAGAGTGTC?TATAATGCCG?CTTTGGCTTT?AGGCATCGCT

961 AATCAACTAA?CCAACATTCT?CAGAGACGTA?GGCGAAGATG?CTAGACGGGG?GAGGGTCTAT

1021?TTACCTCAAG?ATGAATTAGC?TCAAGCGGGG?CTCTCTGATG?AGGATATATA?TGCTGGAAAA

1081?GTTACTGATA?AGTGGAGGAA?CTTCATGAAG?AAGCAAATCA?AGAGAGCAAG?GAAGTTCTTC

1141?GACGAGGCCG?AGAGAGGCGT?GACTGAACTT?AGCTCCGCTA?GTCGATGGCC?AGTGTGGGCG

1201?TCGCTGCTGT?TGTACCGCAA?GATTCTCGAC?GAGATCGAAG?CCAACGACTA?CAACAACTTC

1261?ACAAGGAGAG?CCTATGTAAG?CAAGCCAAAG?AAACTGCTTG?CATTGCCTAT?TGCATATGCA

1321?AAAGCTGTGA?TTCGACCATC?AACAACTGCT?TCCCCTCTGG?CAAAAGCTTG?AACACAAATT

1381?ATTATACTGT?GTAATACTGT?ACTGAAGAAG?CTGAAAGCAT?TCTGTACATT?ACAACTTTGT

1441?AATATTGCAA?TGTAAAATCA?ACAGTAGATA?GTGAATTCAG?TTCCTCAAAA?AAAAAAAAAA

Table 2 Percent Identity:75%in nt overlap

1 GAAAACAAAATCTTGGAAGTAGTTATATAGAAACTATTTCACCTCAGCTCAAGAATGTCT

||||||

1 ......................................................ATGTCT

61 AGTGCCTTGCTGTGGGCTGTTTCTCCCTCTTCTGAGCTATCGAATGGCACTGGAATCTTT

|||||||?|?||||?|||||||| |||?||| |?||?||?||?||?|||?||||

7 GTTGCCTTGTTATGGGTTGTTTCTC...CTTGTGACGTCTCAAACGGGACAGGATTCTTG

121 TATTCGGTGAGGGAGGGAATCCGGATTGTGGATTCGTCGAGGTTCCTTGGCAGGAACAGG

||?|| |?|||||||?|||||||?|?|||||||||?||

64 GTATCCGTTCGTGAGGGAAACCGGATTTTTGATTCGTCGGGGCGT...............

181 AGTTTGGTGTACAAAGGCAGGGCTAAGAAGGGTAAGAAACAAAGATGCACTTTGGCATCT

|||?| | || |||| |||?|

109 ..............AGGAATTTGGCGTGCAATGAGAGAATCAAGAGAGGAGGTGGAAAA.

241 TTCAATGCAGACTCGAGGTATCTTTGCTCGGGAGGGTCGAGCTTGAAGAATGGAGGGAAA

|||?| | | |||| |?|||||| | |?||||?|||||

154 ..CAAAGGTGGAGTTTTGGTTCTTACTT.GGGAGGAGCACAAACTGGAAGTGGACGGAAA

301 TCCTCTGTGCTTTCGAATGCGGTGGTTAGCCCAGCCGGGGAAATGGCGATGTCATCTGAG

| |||||?|?||| | ||||?|| ||?||?||?||||||?||||||||||?||

211 TTTTCTGTACGTTCTGCTATCGTGGCTACTCCGGCTGGAGAAATGACGATGTCATCAGAA

361 CAAAAGGTGTACGATGTAGTGTTGAAGCAGGCTGCTTTGGTGAATAGGAGGTTGAGATCC

| |?|||?||?|||||?||?||||?||||||?||?||||||||?|| |?|||||||

271 CGGATGGTATATGATGTGGTTTTGAGGCAGGCAGCCTTGGTGAAGAGACAGCTGAGATCG

421 ATAGATAATTTGGAGGTGAAGCCCGATATAGCCCTTCCGGGCGATTTGGGCGTGTTGAGT

| |||?|?||?||?|||||| ||||||?| ||||||| |||||||?||||||||

331 ACCGATGAGTTAGATGTGAAGAAGGATATACCTATTCCGGGGACTTTGGGCTTGTTGAGT

481 GAAGCTTATGATCGATGCGGTGAAGTATGTGCAGAGTATGCTAAGACGTTTTATTTGGGA

|||||?||||||?|?|| |||||||||||||||||||?||?|||||||||||?||?|||

391 GAAGCATATGATAGGTGTAGTGAAGTATGTGCAGAGTACGCAAAGACGTTTTACTTAGGA

541 ACCATGCTAATGACACCTGAGAGAAGAAGAGCTATCTGGGCGATATATGTGTGGTGTAGG

||?|||||||||||?||?|||||||||| ||||||||||| ||||?||?|||||?|||

451 ACGATGCTAATGACTCCGGAGAGAAGAAAGGCTATCTGGGCAATATACGTATGGTGCAGG

601 CGGACAGATGAGCTCGTTGACGGGCCTAATGCATCGCATATAACTCCAACCGCCCTGGAC

|?|||||?||?||?|||||?||?||?||||||||?||?||?||||| |?|||?|?||

511 AGAACAGACGAACTTGTTGATGGTCCGAATGCATCACACATTACTCCGGCGGCCTTAGAT

661 AGATGGGAGGCTCGGCTGGAAGACGTATTCAGAGGGCGCCCGTTTGATATGCTCGACGCT

||?|||||?| ||||?|||||?||?|||||?||?||?||?|||||?||||||||?|||

571 AGGTGGGAAGACAGGCTAGAAGATGTTTTCAGTGGACGGCCATTTGACATGCTCGATGCT

721 GCACTATCAGATACAGTATCCAGGTTTCCAGTTGATATTCAGCCCTTTAGGGATATGATT

|| |?||?||?|||||?|||| ||||||||||||||||||||?||?||||||||||||

631 GCTTTGTCCGACACAGTTTCCAAATTTCCAGTTGATATTCAGCCATTCAGAGATATGATT

781 GAAGGAATGCGAATGGACCTCTGGAAATCGAGATACGATAACTTTGATGAGCTATACCTG

|||||||||||?||||||?| ||||?||?|||||| ||||||||?||?||||||||

691 GAAGGAATGCGTATGGACTTGAGGAAGTCAAGATACAGAAACTTTGACGAACTATACCTA

841 TACTGTTATTACGTTGCTGGTACAGTTGGTTTGATGAGTGTCCCGGTTATGGGCATTGCG

||?||||||||||||||||||||?|||||?|||||||||||?|| ||||||||||?||

751 TATTGTTATTACGTTGCTGGTACGGTTGGGTTGATGAGTGTTCCAATTATGGGCATCGCA

901 CCCGAATCAAAGGCAACTACAGAGAGTGTCTATAATGCCGCTTTGGCTTTAGGCATCGCT

||?||||||||||||||?||?|||||?||?||||||||?|||||||||||?||?|||||

811 CCTGAATCAAAGGCAACAACGGAGAGCGTATATAATGCTGCTTTGGCTTTGGGGATCGCA

961 AATCAACTAACCAACATTCTCAGAGACGTAGGCGAAGATGCTAGACGGGGGAGGGTCTAT

|||||?||?||||||||?||?|||||?||?||?||||||||?|||?|?||?||?||||||

871 AATCAGCTGACCAACATACTTAGAGATGTTGGAGAAGATGCCAGAAGAGGAAGAGTCTAT

1021 TTACCTCAAGATGAATTAGCTCAAGCGGGGCTCTCTGATGAGGATATATATGCTGGAAAA

||?|||||||||||||||||?||?||?||?||?||?||?||?||?||||?||||||||?|

931 TTGCCTCAAGATGAATTAGCACAGGCAGGTCTATCCGACGAAGACATATTTGCTGGAAGA

1081?GTTACTGATAAGTGGAGGAACTTCATGAAGAAGCAAATCAAGAGAGCAAGGAAGTTCTTC

||?||?|||||?|||||?|?||||||||||||?||||| ||||?|||||?||||||||

991 GTGACCGATAAATGGAGAATCTTCATGAAGAAACAAATTCAGAGGGCAAGAAAGTTCTTT

1141?GACGAGGCCGAGAGAGGCGTGACTGAACTTAGCTCCGCTAGTCGATGGCCAGTGTGGGCG

||||||||?||||?|||?|||||?|||?|?|||?|?||||||?|||||||?||||?|||

1051?GACGAGGCAGAGAAAGGAGTGACCGAATTGAGCGCAGCTAGTAGATGGCCTGTGTTGGCA

1201?TCGCTGCTGTTGTACCGCAAGATTCTCGACGAGATCGAAGCCAACGACTACAACAACTTC

||?||||||||||||||||?|||?||?|||||||||||||||||?|||||||||||||||

1111?TCTCTGCTGTTGTACCGCAGGATACTGGACGAGATCGAAGCCAATGACTACAACAACTTC

1261?ACAAGGAGAGCCTATGTAAGCAAGCCAAAGAAACTGCTTGCATTGCCTATTGCATATGCA

||||?||||||?|||||?|||||?|||||||| ||?|||||||?|||||||||||||||

1171?ACAAAGAGAGCTTATGTGAGCAAACCAAAGAAGTTGATTGCATTACCTATTGCATATGCA

1321?AAAGCTGTGATTCGACCATCAACAACTGCTTCCCCTCTGGCAAAAGCTTGAACACAAATT

|||?||?| | ||| |||?|?|?|||| || |

1231?AAATCTCTTGTG.....................CCTTCTACAAGAACATGAAATCAGGAT

1381?ATTATACTGTGTAATACTGTACTGAAGAAGCTGAAAGCATTCTGTACATTACAACTTTGT

||||| || | |||||

1270?TTTATATAAATCAAGGCCAA..TGAAGC

1441?AATATTGCAATGTAAAATCAACAGTAGATAGTGAATTCAGTTCCTCAAAAAAAAAAAAAA

Above-listed: the cDNA sequence of coding sweet potato phytoene synthetase

Following: the cDNA sequence of capsicum phytoene synthetase (GenBank Accession No.X68017)

Claims (1)

1, the cDNA sequence of coding sweet potato phytoene synthetase is characterized in that getting from red heart sweet potato root tuber separating clone, and the length of its full-length cDNA nucleotide sequence is 1,500bp, and wherein open reading frame is positioned at 55-1368 position Nucleotide, and is specific as follows:

1 GAAAACAAAA?TCTTGGAAGT?AGTTATATAG?AAACTATTTC?ACCTCAGCTC?AAGA ATGTCT

61 AGTGCCTTGC?TGTGGGCTGT?TTCTCCCTCT?TCTGAGCTAT?CGAATGGCAC?TGGAATCTTT

121 TATTCGGTGA?GGGAGGGAAT?CCGGATTGTG?GATTCGTCGA?GGTTCCTTGG?CAGGAACAGG

181 AGTTTGGTGT?ACAAAGGCAG?GGCTAAGAAG?GGTAAGAAAC?AAAGATGCAC?TTTGGCATCT

241 TTCAATGCAG?ACTCGAGGTA?TCTTTGCTCG?GGAGGGTCGA?GCTTGAAGAA?TGGAGGGAAA

301 TCCTCTGTGC?TTTCGAATGC?GGTGGTTAGC?CCAGCCGGGG?AAATGGCGAT?GTCATCTGAG

361 CAAAAGGTGT?ACGATGTAGT?GTTGAAGCAG?GCTGCTTTGG?TGAATAGGAG?GTTGAGATCC

421 ATAGATAATT?TGGAGGTGAA?GCCCGATATA?GCCCTTCCGG?GCGATTTGGG?CGTGTTGAGT

481 GAAGCTTATG?ATCGATGCGG?TGAAGTATGT?GCAGAGTATG?CTAAGACGTT?TTATTTGGGA

541 ACCATGCTAA?TGACACCTGA?GAGAAGAAGA?GCTATCTGGG?CGATATATGT?GTGGTGTAGG

601 CGGACAGATG?AGCTCGTTGA?CGGGCCTAAT?GCATCGCATA?TAACTCCAAC?CGCCCTGGAC

661 AGATGGGAGG?CTCGGCTGGA?AGACGTATTC?AGAGGGCGCC?CGTTTGATAT?GCTCGACGCT

721 GCACTATCAG?ATACAGTATC?CAGGTTTCCA?GTTGATATTC?AGCCCTTTAG?GGATATGATT

781 GAAGGAATGC?GAATGGACCT?CTGGAAATCG?AGATACGATA?ACTTTGATGA?GCTATACCTG

841 TACTGTTATT?ACGTTGCTGG?TACAGTTGGT?TTGATGAGTG?TCCCGGTTAT?GGGCATTGCG

901 CCCGAATCAA?AGGCAACTAC?AGAGAGTGTC?TATAATGCCG?CTTTGGCTTT?AGGCATCGCT

961 AATCAACTAA?CCAACATTCT?CAGAGACGTA?GGCGAAGATG?CTAGACGGGG?GAGGGTCTAT

1021 TTACCTCAAG?ATGAATTAGC?TCAAGCGGGG?CTCTCTGATG?AGGATATATA?TGCTGGAAAA

1081 GTTACTGATA?AGTGGAGGAA?CTTCATGAAG?AAGCAAATCA?AGAGAGCAAG?GAAGTTCTTC

1141 GACGAGGCCG?AGAGAGGCGT?GACTGAACTT?AGCTCCGCTA?GTCGATGGCC?AGTGTGGGCG

1201 TCGCTGCTGT?TGTACCGCAA?GATTCTCGAC?GAGATCGAAG?CCAACGACTA?CAACAACTTC

1261 ACAAGGAGAG?CCTATGTAAG?CAAGCCAAAG?AAACTGCTTG?CATTGCCTAT?TGCATATGCA

1321 AAAGCTGTGA?TTCGACCATC?AACAACTGCT?TCCCCTCTGG?CAAAAGCT TG?AACACAAATT

1381 ATTATACTGT?GTAATACTGT?ACTGAAGAAG?CTGAAAGCAT?TCTGTACATT?ACAACTTTGT

1441 AATATTGCAA?TGTAAAATCA?ACAGTAGATA?GTGAATTCAG?TTCCTCAAAA?AAAAAAAAAA