CN1757735A - CDNA sequence of coding sweet potato phytoene dehydrogenase - Google Patents

Abstract

A cDNA nucleotide sequence for coding sweet potato's phytoene dehydrogenase is disclosed. It is obtained through separating it from the tuber of sweet potato and cloning it. Its cDNA length is 2181 bp. Its open reading frame is 1685 bp. It can catalyze the dehydrogenation of phytoene to sequentially generate phytofluene and zeta-carotene, so it is a key enzyme for synthesizing beta-carotene of sweet potato.

Description

The cDNA sequence of coding sweet potato phytoene dehydrogenase

Affiliated field

The present invention relates to the cDNA sequence of the coding phytoene dehydrogenase (Ipomoeabatatas phytoene desaturase) 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 β-Hu Luobusu cyclase, final synthetic beta carotene, phytoene dehydrogenase is one of beta carotene synthetic key enzyme.Before the present invention comes forth, any cDNA sequence that discloses or reported the coding sweet potato phytoene dehydrogenase 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 dehydrogenase.

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 dehydrogenase operationally can be connected in expression regulation sequence, thereby form the sweet potato phytoene dehydrogenase 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 dehydrogenase 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 phytoene dehydrogenase gene (the GenBank Accession No.X59948) sequence of coding sweet potato phytoene dehydrogenase gene of the present invention and tomato.

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 dehydrogenase

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 dehydrogenase.

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 dehydrogenase 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 pas con (531bp), 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 dehydrogenase gene of conserved sequence that obtains and known tomato (Lycopersicon esculentum), capsicum (C.annuum), potato (Solanum tuberosum) is 86%, thinks that tentatively it is the fragment of a coding sweet potato phytoene dehydrogenase 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 pds3 (1060bp) as shown in (1).

The existing database of sequencing result (Genebank+EMBL) search, the homology that obtains the phytoene dehydrogenase gene of its nucleotide sequence and known tomato (Lycopersiconesculentum) and capsicum (C.annuum), potato (Solanum tuberosum) is respectively 86%, 86%, 84%, is the fragment of a phytoene dehydrogenase 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) obtains other process of pds5 (610bp) as shown in (1).

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

By being used in combination aforesaid method, the full length cDNA sequence of splicing candidate's coding sweet potato phytoene dehydrogenase, on the basis that obtains this sequence (comprising complete open reading frame at least) information, further design forward primer pdsF (SEQ ID NO.7) and reverse primer pdsR (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 1,845bp.

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 dehydrogenase.

The gene that result's proof of BLAST newly obtains from sweet potato really is phytoene dehydrogenase cDNA.Because known phytoene dehydrogenase can form phytofluene and sigma carotene by the catalysis phytoene, infer that this cDNA has identical functions.

Step 2

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

The length of the sweet potato phytoene dehydrogenase full length gene cDNA that the present invention is new is 2,181bp, and open reading frame is positioned at 184-1969 position Nucleotide, and detailed sequence sees Table 1.The full length cDNA sequence of sweet potato phytoene dehydrogenase gene is carried out the nucleotide homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, the phytoene synthetase cDNA's (GenBankAccession No.X68017) of potato phytoene synthetase cDNA and capsicum (Capsicum annuum) 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 dehydrogenase cDNA:

In this step, the cDNA sequence construct of coding sweet potato phytoene dehydrogenase 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 dehydrogenase 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 SmaI and SacI 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 enzyme is entirely true.Carry out 37 ℃ of enzymes with restriction endonuclease SmaI and SacI respectively and cut 3h, reclaim purpose fragment 1,845bp; Carrier p2300 cuts 3h with SmaI and 37 ℃ of enzymes of Sacl restriction endonuclease, reclaims the purpose fragment respectively.The purpose fragment of the sweet potato phytoene dehydrogenase 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 dehydrogenase 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: 23bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.1

GGTGGAAAGGTAGCTGCT/aTGGAA

(2) information of SEQ ID NO.2

(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.2

GGCATGCAA/tAGT/cCTC/tTCAGG

(3) information of SEQ ID NO.3

(i) sequence signature:

(A) length: 22bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.3

CCGGATGAACTTTCGATGCAGT

(4) information of SEQ ID NO.4

(i) sequence signature:

(A) length: 22bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.4

ACGGAAATCCGCCTGAGAGACT

(5) information of SEQ ID NO.5

(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.5

AGTCTCGTACCAGTCTCCGTCAT

(6) information of SEQ ID NO.6

(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.6

ACCCAGCACATATCTCGTGCCTCCA

(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

TCCCCCGGGTGTGTGAGCAATGTTGAAT

(8) information of SEQ ID NO.8

(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.8

CGAGCTCGCGATTCTAGTGCACATT

(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

GCTGTCAACGAIACGCTACGTAACG

(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

Table 1 sweet potato phytoene dehydrogenase full length cDNA sequence table

＜110〉University Of Agriculture and Forestry In Fujian

＜120〉sweet potato phytoene dehydrogenase full length cDNA sequence table

<160>1

<170>Patent?In?Version?2.1

<210>1

<211>2181

<212>DNA

＜213〉sweet potato (Ipomoea batatas)

<221>gene

<222>(1)…(2181)

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

1 CGAAAAAAAA?TTTGACAACG?ATTAGCTTCA?ATTCAAATAT?CCCATATGCA?GTTTATAGAA

61 AATTTTCCCC?TGCTGTTCTT?TCCCTCAAAA?AGGATTAGTG?GTGTGTGAGC?AATGTTGAAT

121 TGATACTCTT?GAAGAATTAT?AGTTTCCCAA?AGTGATCGCG?TTTTCAGTAT?AGGATTTGGC

181 AAA ATGTCCC?ACATTGGACA?TGTTTCTGCT?GTTAGCTTGC?CGTCTTCCTC?TAGAGTTGGT

241 TGTCGTGTTG?GTTCAGCAGA?AGTAAATGCG?CTATCATTTG?GATCGAGCTA?CTCAATGGGC

301 TCTAAATTGA?GGATTCCTAC?TAGCAATGGT?GTTATGCCCA?AATTAGGGAG?GGGGTACCGT

361 CCTTTGAAGG?TCGTTTGCAT?GGACTATCCG?AGGCCAGAAC?TTGAGAATAC?TGTTAACTAT

421 ATCGAAGCTG?CTTACCTGTC?CTCTTCATTT?CGTTCTGCTC?CGCGTCCAAG?TAAACCATTG

481 GAAGTTGTTA?TTGCTGGTGC?CGGTTTGGCT?GGGTTGTCTA?CTGCAAAATA?TTTAGCAGAT

541 GCGGGCCACA?AACCAATACT?GCTGGAGGCA?CGAGATGTGC?TGGGAGGAAA?GGTAGCTGCA

601 TGGAAAGATG?ATGACGGGGA?CTGGTATGAG?ACTGGCTTAC?ACATATTCTT?TGGGGCATAC

661 CCGAATATAC?AGAACCTGTT?TGGAGAACTA?GGCATCAATG?ATCGCTTGCA?GTGGAAGGAA

721 CATTCTATGA?TATTTGCAAT?GCCAAACAAA?CCAGGAGAGT?TCAGCCGATT?TGATTTCCCT

781 GAAGTTTTAC?CATCTCCATT?TAACGGGGTG?TGGGCCATCC?TAAGGAATAA?TGAGATGCTT

841 ACGTGGCCGG?AGAAAGTGAA?GTTTGCTATT?GGTCTGTTGC?CAGCAATGCT?TGGTGGACAG

901 TCTTATGTTG?AAGCTCAAGA?TGGGATATCT?GTTAAGGACT?GGATGAGAAA?GCAGGGTATA

961 CCAGACAGGG?TGACTGATGA?GGTGTTCATT?GCCATGTCAA?AGGCTCTGAA?TTTCATTAAT

1021?CCGGATGAAC?TTTCGATGCA?GTGCATATTG?ATTGCTTTGA?ACAGATCTCT?TCAGGAGAAA

1081?CATGGTTCAA?AAATGGCCTT?TTTAGACGGA?AATCCGCCAG?AAAGGCTTTG?CTTGCCGATT

1141?GTTGAACATA?TCAAGTCACG?AGGCGGTAAA?GTCAAACTTA?ACTCACGTAT?AAATAAAATT

1201?GAGCTGAATG?AAGATGGAAG?TGTCAAGAGT?TTTCTTCTAA?GTAATGGAAC?TGTTATTAAA

1261?GGAGATGCTT?TTGTGTTCGC?TGGGCCTGTT?GATATCCTGA?AGCTTCTTTT?GCCCGAGGAC

1321?TGGAAAGAGA?TCCCATATTT?CAAAAAGCTG?GAGAAATTAG?TTGGAGTTCC?CGTAATAAAT

1381?GTTCATATAT?GGTTTGACAG?AAAACTGAAG?AACACATACG?ATCATCTCCT?CTTTAGCAGA

1441?AGCTCACTTC?TGAGTGTGTA?TGCTGACATG?TCGGTAACAT?GCAAGGAATA?TTACAACCCC

1501?AATCAGTCTA?TGTTGGAGTT?GGTGTTCGCG?CCTGCAGAAG?AATGGGTGTC?AAAAAGCGAC

1561?TCAGAAATTA?TAGAAGCTAC?GATGAAGGAA?CTAGCCAAAT?TGTTTCCCGA?CGAAATTTCT

1621?GCTGATCAGA?GCAAAGCAAA?AATACTGAAG?TACCATGTTG?TCAAAACTCC?AAGGTCTGTG

1681?TACAAAACCG?TGCCAGGATG?TGAGCCATGT?CGCCCCTTGC?AAAAATCCCC?CATAGACGGA

1741?TTCTATTTAG?CAGGCGACTA?CACGAAACAG?AAGTACTTGG?CTTCAATGGA?AGGCGCTGTT

1801?CTATCCGGGA?AGCTTTGTGC?ACAGGCGATT?TTAAAGGATT?ACGAGTCACT?CCTCGCTCGA

1861?CAGCA GAAAA?TGCTGGCCGA?GGCAAGCGCA?AACATCAGTT?GAAAACGAAC?TTAACCTGTT

1921?CTAATGTGCA?CTAGAATCGC?GAGGTCGTTG?AACCAAGTTA?TTTAGGTAGA?TTGTGTACAG

1981?TACTGCATAT?ACCAAGAACA?CACCAAAATT?GAGAAGAAGA?TGCAATGTGT?AACCTGGTAA

2041?AAGGACAAGC?TAGGAAACAT?CTGAATTCAT?GTAAAGCTGA?TATACCATGT?TTGTTGATGC

2101?TGTTAAGAAT?TTTGAAGGTC?CATAGCCTTC?TTTACTAAGT?AGCAATCCAG?CCTGTTTACA

2161?TCTGTTTTAG?CAAAAAAAAA?A

Table 2 Percent Identity:70%in nt overlap

1 CTTTTACTAGTTATAGCATTCGGTATCTTTTTCTGGGTAACTGCCAAACCACCACAAATT

61 ACAAGTTTCCATTTAACTCTTCAACTTCAACCCAACCAAATTTATTTCCTTAATTGTGCA

121 GAACCACTCCCTATATCTTCTAGGTGCTTTCATTCGTTCCGAGGTAAGAAAAGATTTTTG

181 TTTCTTTGAATGCTTTATGCCACTCGTTTAACTTCTGAGGTTTGTGGATCTTTTAGGCGA

1 ..............CGAAAAAAAATTTGACAACGATTAGCTTCAATTCAAATATCCCATA

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

241 CTTTTTTTTTTTTTGTATGTAAAATTTGTTTCATAAATGCTTC...TCAACATAAATCTT

47 TGCAGTTTATAGAAAATTTTCCCCTGCTGTTCTTTCCCTCAAAAAGGATTAGTGGTGTGT

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

298 GACAAAGAGAAGGAATTTTACCAAGTATTTAGGTTCAGAAATGGATAATTTTCTTACTGT

107 GAGCAATGTTGAATTGATACTCTTGAAGAATTATAGTTTCC.......CAAAGTGATCGC

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

358 GAAATATCCTTATGGCAGGTTTTACTGTTATTTTTCAGTAAAATGCCTCAAATTGGACTT

160 GTTTTCAGTATAGGATTTGGCAAAATGTCCCACATTGGACATGTTTCTGCTGTTAGCTTG

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

418 GTTTCTGCTGTTAACTTGAGAGTCCAAGGTAGTTCAGCTTATCTTTGGAGCTCGAGGT..

220 CCGTCTTCCTCTAGAGTTGGTTGTCGTGTTGGTTCAGCAGAAGTAAATGCGCTATCATTT

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

476 .CGTCTTCTTTGGGAACTGAAAGTCGAGATGGTTGCTTGCAAAGGAATTCGTTATGTTTT

280 GGATCGAGCTACTCAATGGGCTCTAAATTGAGGATTCCTACTAGCAATGGTGTTATGCCC

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

535 GCTGGTAGCGAATCAATGGGTCATAAGTTAAAGATTCGTACTCCCCATGCCACGACCAGA

340 AAATTAGGGAGGGGGTACCGTCCTTTGAAGGTCGTTTGCATGGACTATCCGAGGCCAGAA

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

595 AGATTGGTTAAGGACTTGGGGCCTTTAAAGGTCGTATGCATTGATTATCCAAGACCAGAG

400 CTTGAGAATACTGTTAACTATATCGAAGCTGCTTACCTGTCCTCTTCATTTCGTTCTGCT

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

655 CTGGACAATACAGTTAACTATTTGGAGGCTGCATTTTTATCATCAACGTTCCGTGCTTCT

460 CCGCGTCCAAGTAAACCATTGGAAGTTGTTATTGCTGGTGCCGGTTTGGCTGGGTTGTCT

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

715 CCGCGCCCAACTAAACCATTGGAGATTGTTATTGCTGGTGCAGGTTTGGGTGGTTTGTCT

520 ACTGCAAAATATTTAGCAGATGCGGGCCAGAAACCAATACTGCTGGAGGCACGAGATGTG

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

775 ACAGCAAAATATTTGGCAGATGCTGGTCACAAACCGATACTGCTGGAGGCAAGGGATGTT

580 CTGGGAGGAAAGGTAGCTGCATGGAAAGATGATGACGGGGACTGGTATGAGACTGGCTTA

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

835 CTAGGTGGAAAGGTAGCTGCATGGAAAGATGATGATGGAGATTGGTACGAGACTGGTTTG

640 CACATATTCTTTGGGGCATACCCGAATATACAGAACCTGTTTGGAGAACTAGGCATCAAT

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

895 CATATATTCTTTGGGGCTTACCCAAATATTCAGAACCTGTTTGGAGAATTAGGGATTAAC

700 GATCGCTTGCAGTGGAAGGAACATTCTATGATATTTGCAATGCCAAACAAACCAGGAGAG

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

955 GATCGATTGCAATGGAAGGAACATTCAATGATATTTGCAATGCCAAGCAAGCCAGGAGAA

760 TTCAGCCGATTTGATTTCCCTGAAGTTTTACCATCTCCATTTAACGGGGTGTGGGCCATC

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

1015?TTCAGCCGCTTTGATTTCTCCGAAGCTTTACCCGCTCCTTTAAATGGAATTTTAGCCATC

820 CTAAGGAATAATGAGATGCTTACGTGGCCGGAGAAAGTGAAGTTTGCTATTGGTCTGTTG

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

1075?TTAAAGAATAACGAAATGCTTACATGGCCAGAGAAAGTCAAATTTGCAATTGGACTCTTG

880 CCAGCAATGCTTGGTGGACAGTCTTATGTTGAAGCTCAAGATGGGATATCTGTTAAGGAC

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

1135?CCAGCAATGCTTGGAGGGCAATCTTATGTTGAAGCTCAAGATGGGATAAGTGTTAAGGAC

940 TGGATGAGAAAGCAGGGTATACCAGACAGGGTGACTGATGAGGTGTTCATTGCCATGTCA

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

1195?TGGATGAGAAAGCAAGGTGTGCCGGACAGGGTGACAGATGAGGTGTTCATTGCTATGTCA

1000?AAGGCTCTGAATTTCATTAATCCGGATGAACTTTCGATGCAGTGCATATTGATTGCTTTG

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

1255?AAGGCACTCAACTTTATAAACCCTGACGAACTTTCAATGCAGTGCATTTTGATCGCATTG

1060?AACAGATCTCTTCAGGAGAAACATGGTTCAAAAATGGCCTTTTTAGACGGAAATCCGCCA

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

1315?AACAGGTTTCTTCAGGAGAAACATGGTTCAAAAATGGCCTTTTTAGATGGTAATCCTCCT

1120?GAAAGGCTTTGCTTGCCGATTGTTGAACATATCAAGTCACGAGGCGGTAAAGTCAAACTT

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

1375?GAGAGACTTTGCATGCCGATTGTTGAACACATTGAGTCAAAAGGTGGCCAAGTCAGACTG

1180?AACTCACGTATAAATAAAATTGAGCTGAATGAAGATGGAAGTGTCAAGAGTTTTCTTCTA

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

1435 AACTCACGAATAAAAAAGATTGAGCTGAATGAGGATGGAAGTGTCAAGAGTTTTATACTG

1240 AGTAATGGAACTGTTATTAAAGGAGATGCTTTTGTGTTCGCTGGGCCTGTTGATATCCTG

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

1495 AGTGACGGTAGTGCAATCGAGGGAGATGCTTTTGTGTTTGCCGCTCCAGTGGATATTTTC

1300 AAGCTTCTTTTGCCCGAGGACTGGAAAGAGATCCCATATTTCAAAAAGCTGGAGAAATTA

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

1555 AAGCTTCTATTGCCTGAAGACTGGAAAGAGATTCCATATTTCCAAAAGTTGGAGAAGTTA

1360 GTTGGAGTTCCCGTAATAAATGTTCATATATGGTTTGACAGAAAACTGAAGAACACATAC

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

1615 GTCGGAGTACCTGTGATAAATGTACATATATGGTTTGACAGAAAACTGAAGAACACATAT

1420 GATCATCTCCTCTTTAGCAGAAGCTCACTTCTGAGTGTGTATGCTGACATGTCGGTAACA

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

1675 GATCATTTGCTCTTCAGCAGAAGCTCACTGCTCAGTGTGTATGCTGACATGTCTGTTACA

1480 TGCAAGGAATATTACAACCCCAATCAGTCTATGTTGGAGTTGGTGTTCGCGCCTGCAGAA

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

1735 TGTAAGGAATATTACAACCCCAATCAGTCTATGTTGGAATTGGTTTTTGCACCTGCAGAA

1540 GAATGGGTGTCAAAAAGCGACTCAGAAATTATAGAAGCTACGATGAAGGAACTAGCCAAA

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

1795 GAGTGGATATCTCGCAGCGACTCAGAAATTATTGATGCAACGATGAAGGAACTAGCAACG

1600 TTGTTTCCCGACGAAATTTCTGCTGATCAGAGCAAAGCAAAAATACTGAAGTACCATGTT

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

1855 CTTTTTCCTGATGAAATTTCAGCAGATCAAAGCAAAGCAAAAATATTGAAGTACCATGTT

1660 GTCAAAACTCCAAGGTCTGTGTACAAAACCGTGCCAGGATGTGAGCCATGTCGCCCCTTG

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

1915 GTCAAAACTCCGAGGTCTGTTTATAAAACTGTGCCAGGTTGTGAACCCTGTCGGCCTTTA

1720 CAAAAATCCCCCATAGACGGATTCTATTTAGCAGGCGACTACACGAAACAGAAGTACTTG

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

1975 CAAAGATCCCCAATAGAGGGGTTTTATTTAGCCGGTGACTACACGAAACAGAAATACTTG

1780 GCTTCAATGGAAGGCGCTGTTCTATCCGGGAAGCTTTGTGCACAGGCGATTTTAAAGGAT

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

2035 GCTTCAATGGAAGGCGCTGTCTTATCAGGAAAGCTTTGTGCTCAAGCTATTGTACAGGAT

1840 TACGAGTCACTCCTCGCTCGACAGCAGAAAATGCTGGCCGAGGCAAGCGCAAACATCAGT

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

2095 TATGAGTTACTTGTTGGACGTAGCCAAAAGAAGTTGTCGGAAGCAAGCGTAGTTTAGCTT

1900?TGAAAACGAACTTAACCTGTTCTAATGTGCACTAGAATCGCGAGGTCGTTGAACCAAGTT

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

2155?TGTGGTTATTATTTAGCTTCTGTACACTAAATTTATGATGCAAGAAGCGTTGTACACAAC

1960?ATTTAGGTAGATTGTGTACAGTACTGCATATACCAAGAACACACCAAAATTGAGAAGAAG

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

2215?ATATAGAAGAAGAGTGCGAGGTGAAGCAAGTAGGAGAAATGTTAGGAAAGCTCCTATACA

2020?ATGCAATGTGTAACCTGGTAAAAGGACAAGCTAGGAAACATCTGAATTCATGTAAAGCTG

| |||

2275?AAAGGATGGCATG...............................................

2080?ATATACCATGTTTGTTGATGCTGTTAAGAATTTTGAAGGTCCATAGCCTTCTTTACT...

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

2288?................................TTGAAGAT...TAGCATCTTTTTAATCC

2137?.AAGTAGCAATCCAGCCTGTTTACATCTGTTTTAGCAAAAAAAAAA

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

2313?CAAGTTTAAATATAAAGCATATTTTATGGAATTC

Above-listed: the cDNA sequence of sweet potato phytoene dehydrogenase

Following: the nucleotide sequence of tomato phytoene dehydrogenase (GenBank Accession No.X59948)

Claims (1)

1, the cDNA sequence of coding sweet potato phytoene dehydrogenase 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 2,181bp, and wherein open reading frame is positioned at 184-1868 position Nucleotide, and is specific as follows:

1 CGAAAAAAAA?TTTGACAACG?ATTAGCTTCA?ATTCAAATAT?CCCATATGCA?GTTTATAGAA

61 AATTTTCCCC?TGCTGTTCTT?TCCCTCAAAA?AGGATTAGTG?GTGTGTGAGC?AATGTTGAAT

121 TGATACTCTT?GAAGAATTAT?AGTTTCCCAA?AGTGATCGCG?TTTTCAGTAT?AGGATTTGGC

181 AAA ATGTCCC?ACATTGGACA?TGTTTCTGCT?GTTAGCTTGC?CGTCTTCCTC?TAGAGTTGGT

241 TGTCGTGTTG?GTTCAGCAGA?AGTAAATGCG?CTATCATTTG?GATCGAGCTA?CTCAATGGGC

301 TCTAAATTGA?GGATTCCTAC?TAGCAATGGT?GTTATGCCCA?AATTAGGGAG?GGGGTACCGT

361 CCTTTGAAGG?TCGTTTGCAT?GGACTATCCG?AGGCCAGAAC?TTGAGAATAC?TGTTAACTAT

421 ATCGAAGCTG?CTTACCTGTC?CTCTTCATTT?CGTTCTGCTC?CGCGTCCAAG?TAAACCATTG

481 GAAGTTGTTA?TTGCTGGTGC?CGGTTTGGCT?GGGTTGTCTA?CTGCAAAATA?TTTAGCAGAT

541 GCGGGCCACA?AACCAATACT?GCTGGAGGCA?CGAGATGTGC?TGGGAGGAAA?GGTAGCTGCA

601 TGGAAAGATG?ATGACGGGGA?CTGGTATGAG?ACTGGCTTAC?ACATATTCTT?TGGGGCATAC

661 CCGAATATAC?AGAACCTGTT?TGGAGAACTA?GGCATCAATG?ATCGCTTGCA?GTGGAAGGAA

721 CATTCTATGA?TATTTGCAAT?GCCAAACAAA?CCAGGAGAGT?TCAGCCGATT?TGATTTCCCT

781 GAAGTTTTAC?CATCTCCATT?TAACGGGGTG?TGGGCCATCC?TAAGGAATAA?TGAGATGCTT

841 ACGTGGCCGG?AGAAAGTGAA?GTTTGCTATT?GGTCTGTTGC?CAGCAATGCT?TGGTGGACAG

901 TCTTATGTTG?AAGCTCAAGA?TGGGATATCT?GTTAAGGACT?GGATGAGAAA?GCAGGGTATA

961 CCAGACAGGG?TGACTGATGA?GGTGTTCATT?GCCATGTCAA?AGGCTCTGAA?TTTCATTAAT

1021 CCGGATGAAC?TTTCGATGCA?GTGCATATTG?ATTGCTTTGA?ACAGATCTCT?TCAGGAGAAA

1081 CATGGTTCAA?AAATGGCCTT?TTTAGACGGA?AATCCGCCAG?AAAGGCTTTG?CTTGCCGATT

1141 GTTGAACATA?TCAAGTCACG?AGGCGGTAAA?GTCAAACTTA?ACTCACGTAT?AAATAAAATT

1201 GAGCTGAATG?AAGATGGAAG?TGTCAAGAGT?TTTCTTCTAA?GTAATGGAAC?TGTTATTAAA

1261 GGAGATGCTT?TTGTGTTCGC?TGGGCCTGTT?GATATCCTGA?AGCTTCTTTT?GCCCGAGGAC

1321 TGGAAAGAGA?TCCCATATTT?CAAAAAGCTG?GAGAAATTAG?TTGGAGTTCC?CGTAATAAAT

1381 GTTCATATAT?GGTTTGACAG?AAAACTGAAG?AACACATACG?ATCATCTCCT?CTTTAGCAGA

1441 AGCTCACTTC?TGAGTGTGTA?TGCTGACATG?TCGGTAACAT?GCAAGGAATA?TTACAACCCC

1501 AATCAGTCTA?TGTTGGAGTT?GGTGTTCGCG?CCTGCAGAAG?AATGGGTGTC?AAAAAGCGAC

1561 TCAGAAATTA?TAGAAGCTAC?GATGAAGGAA?CTAGCCAAAT?TGTTTCCCGA?CGAAATTTCT

1621 GCTGATCAGA?GCAAAGCAAA?AATACTGAAG?TACCATGTTG?TCAAAACTCC?AAGGTCTGTG

1681 TACAAAACCG?TGCCAGGATG?TGAGCCATGT?CGCCCCTTGC?AAAAATCCCC?CATAGACGGA

1741 TTCTATTTAG?CAGGCGACTA?CACGAAACAG?AAGTACTTGG?CTTCAATGGA?AGGCGCTGTT

1801 CTATCCGGGA?AGCTTTGTGC?ACAGGCGATT?TTAAAGGATT?ACGAGTCACT?CCTCGCTCGA

1861 CAGCA GAAAA?TGCTGGCCGA?GGCAAGCGCA?AACATCAGTT?GAAAACGAAC?TTAACCTGTT

1921 CTAATGTGCA?CTAGAATCGC?GAGGTCGTTG?AACCAAGTTA?TTTAGGTAGA?TTGTGTACAG

1981 TACTGCATAT?ACCAAGAACA?CACCAAAATT?GAGAAGAAGA?TGCAATGTGT?AACCTGGTAA

2041 AAGGACAAGC?TAGGAAACAT?CTGAATTCAT?GTAAAGCTGA?TATACCATGT?TTGTTGATGC

2101 TGTTAAGAAT?TTTGAAGGTC?CATAGCCTTC?TTTACTAAGT?AGCAATCCAG?CCTGTTTACA

2161 TCTGTTTTAG?CAAAAAAAAA?A