CN103740744A - Zeaxanthin synthetic gene recombinant plasmid and preparation method and use thereof - Google Patents

Abstract

The invention discloses a zeaxanthin synthetic gene recombinant expression plasmid pET-Zea, wherein a base sequence is shown in SEQ ID NO:1. Meanwhile, the invention also discloses a preparation method of the pET-Zea, and application and an application method thereof in detection of the activity of a target receptor zeaxanthin synthetic gene. By adopting the zeaxanthin synthetic gene recombinant expression plasmid pET-Zea provided by the invention, accumulation of zeaxanthin in an escherichia coli cell body is successfully achieved after an escherichia coli BL21 strain is led. Therefore, a zeaxanthin production bacterial strain can be accurately and rapidly built. The method for preparing the zeaxanthin synthetic gene recombinant expression plasmid pET-Zea provided by the invention is simple and convenient, easy to operate and low in cost. By adopting the method disclosed by the invention, a related gene sequence in recombinant expression plasmid pET-Zea can be replaced by a to-be-detected gene sequence in the target receptor by digestion and connection, and then the to-be-detected gene function in the target receptor can be simply and effectively detected and validated by detecting whether the zeaxanthin is accumulated in the host cell body.

Description

A kind of zeaxanthin synthetic gene recombinant plasmid and its production and use

Technical field

The present invention relates to recombinant plasmid and its production and use, specifically a kind of zeaxanthin synthetic gene recombinant plasmid and its production and use.

Background technology

Zeaxanthin, also claims zeaxanthin (Zeaxanthin, 3,3-dihydroxyl-β-carotene), and fat-soluble cpds belongs to xenthophylls class carotenoid.Pharmacology in recent years and Physiologic Studies find that zeaxanthin has extremely strong oxidation-resistance, its due to the macular degeneration of prevention eyes blind and senile cataract, prevent Partial tumors generation and development, reduce aspect the danger generation of some cardiovascular diseases and there is remarkable effect.As the corn of one of three large staple food crops, zeaxanthin rich content in its seed, by genetic engineering breeding, cultivates the fresh edible maize that is rich in zeaxanthin, can further bring into play its healthcare applications and be worth, and has larger development prospect.At present; corn mass-producing transgenosis is mainly take callus as acceptor, and its induction and cultivation are subject to maize genotype very large with the restriction of drawing materials season, and the cycle of cultivating, transform, screening and regenerating is longer; the scale of transgeneic procedure is generally less, is difficult to guarantee to transform successfully.Agriculture bacillus mediated converted in-situ method take shoot tip meristem as acceptor, does not rely on tissue culture, is subject to genotype restriction little, simple to operate, and experimental period is short, can from most materials, obtain transfer-gen plant.But transformation efficiency need to improve, and the transformant that obtains is mostly mosaic, offspring's isolation identification is comparatively difficult, so also need further Improvement.Recently by building the synthetic zeaxanthin of zeaxanthin biological metabolism engineering, be subject to gradually researchist's favor, but because the synthesis step of zeaxanthin in microbe is many and complicated, the carrier that therefore development can the multiple synthetic enzyme of high efficient expression zeaxanthin becomes focus and the difficult point of people's research.In addition, to the detection of the function of Major Enzymes system in target recipient zeaxanthin metabolic pathway of synthesizing in R&D process, also there are a lot of puzzlements in researchist, as the biochemical reaction by vitro or the method for utilizing transgenation strain to have complementary functions detect, its operation steps is more loaded down with trivial details, and efficiency is not high yet.Therefore a kind of method that how to obtain efficient detection target recipient zeaxanthin synthetic gene activity is also the problem that this area researchist pays close attention to.

Summary of the invention

One of object of the present invention is just to provide the recombinant plasmid of the multiple synthetic gene of a kind of high efficient expression zeaxanthin, to can be accurately, rapid build goes out zeaxanthin and produces bacterial strain; Two of object of the present invention is just to provide a kind of preparation method of this recombinant plasmid; Three of object of the present invention is just to provide a kind of purposes of this recombinant plasmid, be the application of described recombinant plasmid in the function that detects the synthetic main phase correlation gene of zeaxanthin, more specifically say that described recombinant plasmid pET-Zea is in the application detecting in target recipient zeaxanthin synthetic gene activity.

The object of the invention is to realize by following technical scheme:

Zeaxanthin synthetic gene recombinant expression plasmid pET-Zea provided by the present invention, it carries crtE gene, crtB gene, crtI gene, crtY gene, crtZ gene, and the structure of this recombinant plasmid is as shown in Figure 1;

Described recombinant expression plasmid is on expression vector pET-28a, to be connected with crtE gene in turn, crtB gene, crtI gene, crtY gene, crtZ gene, wherein between expression vector pET-28a and crtE gene, there is Nde I restriction enzyme site, between crtE gene and crtB gene, there is Hpa I restriction enzyme site, between crtB gene and crtI gene, there is Mfe I restriction enzyme site, between crtI gene and crtY gene, there is Nhe I restriction enzyme site, between crtY gene and crtZ gene, there is Aha III restriction enzyme site, between crtZ gene and expression vector pET-28a, there is Not I restriction enzyme site.

Described zeaxanthin synthetic gene recombinant expression plasmid pET-Zea, its base sequence is as shown in SEQ ID NO:1.

Wherein said crtE gene, crtY gene, crtI gene, crtB gene, crtZ gene are respectively crtE gene, lycopene cyclase gene, Phytoene dehydrogenase gene, phytoene synthase gene and the Cartoene hydroxylase genes of pantoea agglomerans Pantoea agglomerans.

Zeaxanthin synthetic gene recombinant expression plasmid pET-Zea provided by the present invention, importing after e. coli strain bl21, has successfully realized the accumulation of zeaxanthin in Bacillus coli cells body, thus can be accurately, rapid build zeaxanthin production bacterial strain.

The preparation method of zeaxanthin synthetic gene recombinant expression plasmid pET-Zea provided by the present invention, comprises the steps:

I) gene amplification:

Extract the genomic dna of Pantoea agglomerans ACCC10495, adopt pcr amplification crtE, crtB, crtI, crtY, crtZ gene coding region, corresponding primer is respectively P1/P2, P3/P4, P5/P6, P7/P8, P9/P10:

The base sequence of described P1 is 5 '-CAGCATATGATGGTGAGTGGCAGT-3 '

The base sequence of described P2 is 5 '-TTAATTGTTAACTCAGGCGATTTTCAT-3 '

The base sequence of described P3 is 5 '-TTAATTGTTAACATGAGCCAACCGCCG-3 '

The base sequence of described P4 is 5 '-GGGCCCCAATTGCTAAACGGGACGCTG-3 '

The base sequence of described P5 is 5 '-GGGCCCCAATTGATGAAAAAAACCGTT-3 '

The base sequence of described P6 is 5 '-GGAATTCGCTAGCGAATTTCAGGCTGGCGGTGG-3 '

The base sequence of described P7 is 5 '-GGAATTCGCTAGCGTGAGGGATCTGATT-3 '

The base sequence of described P8 is 5 '-CCCTTTAAAGGGTCATCCTTTATCTCG-3 '

The base sequence of described P9 is 5 '-ATTGCGGCCGCTTATTCGGGCGAAGA-3 '

The base sequence of described P10 is 5 '-CCCTTTAAAGGGATGCTAGTAAATAGT-3 ';

II) structure cloned plasmids:

The amplified production of crtE, crtB, crtI, crtY, crtZ is carried out respectively to 1.5% agarose gel electrophoresis, then reclaim respectively the band at 924bp, 930bp, 1459bp, 1161bp, 531bp place, be connected, transform bacillus coli DH 5 alpha with cloning vector pMD18-T respectively, filter out positive colony, after fermentation, extract and obtain pMD18-crtE, pMD18-crtB, pMD18-crtI, pMD18-crtY and pMD18-crtZ cloned plasmids;

III) structure recombinant expression plasmid pET-Zea:

Prepared cloned plasmids is carried out respectively to double digestion, the direction that checking gene order is connected with carrier: pMD18-crtE is with Hpa I and EcoR I double digestion; PMD18-crtB is with Hpa I and EcoR I double digestion; PMD18-crtI is with Mfe I and EcoR I double digestion; PMD18-crtY is with Nhe I and EcoR I double digestion; PMD18-crtZ, with Aha III and EcoR I double digestion, chooses the cloned plasmids of gene order and pMD18-T Opposite direction connection;

The plasmid pMD18-crtE that screening is obtained and pMD18-crtB through Hpa I with EcoR I double digestion, be connected and obtain pMD18-crtEB; By pMD18-crtEB and pMD18-crtI through Mfe I with EcoR I double digestion, be connected and obtain pMD18-crtEBI; By pMD18-crtEBI and pMD18-crtY through Nhe I with EcoR I double digestion, be connected and obtain pMD18-crtEBIY; By pMD18-crtEBIY and pMD18-crtZ through Aha III with EcoR I double digestion, be connected and obtain plasmid pMD18-crtEBIYZ;

Expression vector pET-28a is cut through Sna I and Bgl II enzyme, otch is filled, connects and to obtain carrier pET-28a-SB;

By plasmid pMD18-crtEBIYZ and carrier pET-28a-SB, respectively through Nde I and Not I double digestion, enzyme is cut product and is connected and obtain expression plasmid pET-Zea.

The method of preparing zeaxanthin synthetic gene recombinant expression plasmid pET-Zea provided by the present invention is easy, easy to operate, cost is low.

Recombinant plasmid of the present invention can be used for detecting the function of the synthetic main phase correlation gene of zeaxanthin.More specifically address says the activity that can be used for detecting target recipient zeaxanthin synthetic enzyme.The synthetic main phase correlation gene of wherein said zeaxanthin refers to crtE gene, crtB gene, crtI gene, crtY gene, crtZ gene.

The method of detection target recipient zeaxanthin synthetic gene activity provided by the present invention is:

By zeaxanthin synthetic gene amplification to be detected in target recipient; Amplified production and recombinant expression plasmid pET-Zea compare according to following manner;

(a) amplified production and recombinant expression plasmid pET-Zea are respectively through Nde I and Hpa I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtE gene in target recipient, it has crtE activity;

(b) amplified production and recombinant expression plasmid pET-Zea are respectively through Hpa I and Mfe I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtB gene in target recipient, it has phytoene synthetase activity;

(c) amplified production and recombinant expression plasmid pET-Zea are respectively through Mfe I and Nhe I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtI gene in target recipient, it has phytoene dehydrogenase activity;

(d) amplified production and recombinant expression plasmid pET-Zea are respectively through through Nhe I and Aha III double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtY gene in target recipient, it has lycopene cyclase activity

(e) amplified production and recombinant expression plasmid pET-Zea are respectively through Aha III and Not I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, the absorption peak that zeaxanthin detected shows to carry crtZ gene in target recipient, has Cartoene hydroxylase activity.

The inventive method can be cut by enzyme, be connected, gene order to be detected in target recipient is replaced to the genes involved sequence in recombinant expression plasmid pET-Zea, then by detect in host cell body, whether accumulate zeaxanthin can be simply, effectively testing gene function in target recipient is detected and is verified.

Accompanying drawing explanation

Fig. 1 is recombinant expression plasmid pET-Zea collection of illustrative plates.

Fig. 2 is crtE, crtI, crtY, crtZ, crtB gene amplification product agarose gel electrophoretogram.

In Fig. 2: M1:DM5000marker; M2:DM2000marker; 1:crtE; 2:crtB; 3:crtY; 4:crtI; 5:crtZ.

Fig. 3 is that recombinant expression plasmid pET-Zea double digestion detects agarose gel electrophoretogram.

In Fig. 3: M1:DM5000marker; M2:DM2000marker1:Aha III and Not I double digestion; 2:Hpa I and Mfe I double digestion; 3:Nde I and Hpa I double digestion; 4:Mfe I and Nhe I double digestion; 5:Nhe I and Aha III double digestion.

Fig. 4 is the liquid chromatogram of zeaxanthin in pET-Zea/BL21 Recombinant E. coli Fermentation Broth.

Fig. 5 is the liquid chromatogram of zeaxanthin standard solution.

Fig. 6 recombinates the crtE gene of Pantoea ananatis MCCC1F01131 and plasmid pET-Zea and proceeds to the liquid chromatogram of zeaxanthin in intestinal bacteria secondary fermentation liquid.

Fig. 7 recombinates the crtB gene of Pantoea ananatis MCCC1F01131 and plasmid pET-Zea and proceeds to the liquid chromatogram of zeaxanthin in intestinal bacteria secondary fermentation liquid.

Fig. 8 recombinates the crtI gene of Pantoea ananatis MCCC1F01131 and plasmid pET-Zea and proceeds to the liquid chromatogram of zeaxanthin in intestinal bacteria secondary fermentation liquid.

Fig. 9 recombinates the crtY gene of Pantoea ananatis MCCC1F01131 and plasmid pET-Zea and proceeds to the liquid chromatogram of zeaxanthin in intestinal bacteria secondary fermentation liquid.

Figure 10 recombinates the crtZ gene of Pantoea ananatis MCCC1F01131 and plasmid pET-Zea and proceeds to the liquid chromatogram of zeaxanthin in intestinal bacteria secondary fermentation liquid.

Embodiment

Embodiment 1: the pET-Zea of recombinant expression plasmid builds

(1) gene amplification

Adopt the imitative extraction process of phenol to extract Pantoea agglomerans ACCC10495(purchased from Chinese agriculture microbial strains preservation administrative center) genomic dna as pcr amplification template, with ExTaq archaeal dna polymerase (purchased from Takara), corresponding primer (Beijing three rich polygala root synthetic) is in Table 1, increase respectively crtE, crtB, crtI, crtY, crtZ gene coding region.

Amplified production is in 1.5% agarose gel electrophoresis, and result is as Fig. 2, and the band that then reclaims respectively 924bp, 930bp, 1459bp, 1161bp, 531bp place must be expected amplified production.Expection amplified production respectively with cloning vector pMD18-T(purchased from Takara) be connected, transform bacillus coli DH 5 alpha.Picking list bacterium colony, take M13 Primer RV and M13 Primer M4 as primer pair (purchased from Takara), carry out positive colony detection, filter out positive colony plasmid, and by Beijing three rich polygala root company sequencing analysis gene order, the aminoacid sequence of each gene is shown in SEQ ID NO:2～SEQ ID NO:6.

Table 1: amplification crtE, crtI, crtY, crtZ, the required primer sequence information of crtB gene

Note: in table 1, double underline part is restriction endonuclease recognition sequence, single underscore part is restriction endonuclease protectiveness base.The primer sequence of above P1～P10 designs according to GenBank accession number M87280.

(2) build recombinant expression plasmid:

Positive colony plasmid carries out respectively double digestion, the direction that checking gene order is connected with carrier: pMD18-crtE is with Hpa I and EcoR I double digestion; PMD18-crtB is with Hpa I and EcoR I double digestion; PMD18-crtI is with Mfe I and EcoR I double digestion; PMD18-crtY is with Nhe I and EcoR I double digestion; PMD18-crtZ, with Aha III and EcoR I double digestion, chooses the cloned plasmids of gene order and pMD18-T Opposite direction connection.

The plasmid pMD18-crtE that screening is obtained and pMD18-crtB through Hpa I with EcoR I double digestion, be connected and obtain pMD18-crtEB; By pMD18-crtEB and pMD18-crtI through Mfe I with EcoR I double digestion, be connected and obtain pMD18-crtEBI; By pMD18-crtEBI and pMD18-crtY through Nhe I with EcoR I double digestion, be connected and obtain pMD18-crtEBIY; By pMD18-crtEBIY and pMD18-crtZ through Aha III with EcoR I double digestion, be connected and obtain plasmid pMD18-crtEBIYZ.

Expression vector pET-28a is cut through Sna I and Bgl II enzyme, otch is filled, connects and to obtain pET-28a-SB.

Plasmid pMD18-crtEBIYZ and expression vector pET-28a-SB, respectively through Nde I and Not I double digestion, connection, are obtained to expression plasmid pET-Zea, and sequencing result is shown in sequence table and SEQ ID NO:1, and plasmid map is as Fig. 1.

Embodiment 2: the enzyme of recombinant expression plasmid is cut checking

The recombinant expression plasmid pET-Zea that embodiment 1 is built, through Nde I and Hpa I double digestion, verifies the connection (Fig. 3, swimming lane 3) of crtE; Through Hpa I and Mfe I double digestion, the connection (Fig. 3, swimming lane 2) of checking crtB; Through Mfe I and Nhe I double digestion, the connection (Fig. 3, swimming lane 4) of checking crtI; Through Nhe I and Aha III double digestion, the connection (Fig. 3, swimming lane 5) of checking crtY; Through Aha III and Not I double digestion, the connection (Fig. 3, swimming lane 1) of checking crtZ.

Embodiment 3: the fermentation of recombinant bacterial strain detects

Recombinant expression plasmid pET-Zea is imported to coli strain BL21(coli strain BL21 purchased from Takara company) express, and measure the main ingredient of tunning by high performance liquid chromatography (HPLC), detect the recombinant expression plasmid building and whether accumulate zeaxanthin.

The actual conditions that carries out high performance liquid chromatography in the present embodiment is: chromatographic column: Hypersil ODS25um, 250 × 4.6mm; Moving phase: A is acetonitrile, and B is tetrahydrofuran (THF); Ultraviolet detection wavelength: 450nm; Flow velocity: 1.0mL/min; Sample size: 100 μ L.

(1) precision takes zeaxanthin standard substance 1.4mg, puts in 20mL volumetric flask, adds acetone solution, constant volume, makes the solution of concentration 0.07mg/mL, standby as standard solution;

According to a conventional method recombinant expression plasmid pET-Zea is imported to e. coli strain bl21 and obtain recombination bacillus coli, fermented liquid after 37 ℃ of liquid fermenting 48h, then by fermented liquid lyophilize, precision takes the thalline 0.1413g after lyophilize, be put in triangular flask, add acetone: the mixed solvent 20mL of hexanaphthene (1:1, v/v) composition, lucifuge supersound extraction 3 times, each 15min.After the extracting solution of 3 times is centrifugal under 4 ℃ of conditions, merge supernatant liquor, supernatant liquor is dried up with nitrogen, then add 20mL acetone solution, after 0.45 μ m filtering with microporous membrane, as test sample solution for standby.

(2) identify

Get in the standard solution 100 μ L injection liquid chromatographies that prepare and measure, result as shown in Figure 5;

Get in test sample solution 100 μ L injection liquid chromatographies and measure, result as shown in Figure 4.

Interpretation of result: known with Fig. 4 by comparison diagram 5, in constructed recombinant Bacillus coli cells body, there is the accumulation of zeaxanthin.

Embodiment 4: testing gene Function detection

Using crtE, the crtB of known sequence in Pantoea ananatis MCCC1F01131 and function, crtI, crtY, crtZ gene as the gene for the treatment of brake, with detection system of the present invention (being the constructed recombinant expression plasmid pET-Zea of embodiment 1), carry out the detection of gene function.

(1) testing gene amplification

Adopt genomic dna that the imitative extraction process of phenol extracts Pantoea ananatis MCCC1F01131 as pcr amplification template, with ExTaq archaeal dna polymerase, corresponding primer (table 2) increase respectively its crtE, crtB, crtI, crtY, crtZ gene coding region.Expection amplified production is connected respectively conversion bacillus coli DH 5 alpha with cloning vector pMD18-T.The positive bacterium colony of picking, by Beijing three rich polygala root company sequencing analysis gene order.

Table 2: amplification crtE, crtB, crtI, crtY, the required primer sequence information of crtZ gene

Note: in table 2, double underline part is restriction endonuclease recognition sequence, single underscore part is restriction endonuclease protectiveness base.Above P1'～P10' primer sequence designs according to GenBank accession number D90087.2.

(2) testing gene active function detects

(2.1) crtE active function detects

By expression plasmid pET-Zea and Pantoea ananatis MCCC1F01131crtE amplified production to be measured, respectively through Nde I and Hpa I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, and connects product and transforms e. coli bl21.After e. coli strain bl21 fermentation containing recombinant plasmid, carry out HPLC analysis, detect that zeaxanthin absorption peak is as Fig. 6.Show that Pantoea ananatis MCCC1F01131crtE gene to be measured has crtE activity.

(2.2) phytoene synthetase active function detects

By expression plasmid pET-Zea and Pantoea ananatis MCCC1F01131crtB amplified production to be measured, respectively through restriction endonuclease Hpa I and Mfe I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, and connects product and transforms e. coli bl21.After e. coli strain bl21 fermentation containing recombinant plasmid, carry out HPLC analysis, detect that zeaxanthin absorption peak is as Fig. 7.Show that Pantoea ananatis MCCC1F01131crtB gene to be measured has phytoene synthetase activity.

(2.3) phytoene dehydrogenase active function detects

By expression plasmid pET-Zea and Pantoea ananatis MCCC1F01131crtI amplified production to be measured, respectively through Mfe I and Nhe I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, and connects product and transforms e. coli bl21.After e. coli strain bl21 fermentation containing recombinant plasmid, carry out HPLC analysis, detect that zeaxanthin absorption peak is as Fig. 8.Show that Pantoea ananatis MCCC1F01131crtI gene to be measured has phytoene dehydrogenase activity.

(2.4) lycopene cyclase active function detects

By expression plasmid pET-Zea and Pantoea ananatis MCCC1F01131crtY amplified production to be measured, respectively through Nhe I and Aha III double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, and connects product and transforms e. coli bl21.After e. coli strain bl21 fermentation containing recombinant plasmid, carry out HPLC analysis, detect that zeaxanthin absorption peak is as Fig. 9.Show that Pantoea ananatis MCCC1F01131crtY gene to be measured has lycopene cyclase activity.

(2.5) Cartoene hydroxylase active function detects

By expression plasmid pET-Zea and Pantoea ananatis MCCC1F01131crtZ amplified production to be measured, respectively through Aha III and Not I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, and connects product and transforms e. coli bl21.After e. coli strain bl21 fermentation containing recombinant plasmid, carry out HPLC analysis, detect that zeaxanthin absorption peak is as Figure 10.Show that Pantoea ananatis MCCC1F01131crtZ gene to be measured has Cartoene hydroxylase activity.

The actual conditions that in above step (2.1)～(2.5), HPLC analyzes is consistent with the HPLC analysis condition of embodiment 3.

Sequence table

<110> University Of Hebei

<120> zeaxanthin synthetic gene recombinant plasmid and its production and use

<130>

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<170>PatentIn version3.3

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<213> zeaxanthin synthetic gene recombinant expression plasmid pET-Zea

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tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtagatctc gatcccgcga aattaatacg 2400

actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa 2460

ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc 2520

tggtgccgcg cggcagccat atggtgagtg gcagtaaagc gggcgtttcg cctcatcgcg 2580

aaatagaagt aatgagacaa tccattgacg atcacctggc tggcctgtta cctgaaaccg 2640

acagccagga tatcgtcagc cttgcgatgc gtgaaggcgt catggcaccc ggtaaacgga 2700

tccgtccgct gctgatgctg ctggccgccc gcgacctccg ctaccagggc agtatgccta 2760

cgctgctcga tctcgcctgc gccgttgaac tgacccatac cgcgtcgctg atgctcgacg 2820

acatgccctg catggacaac gccgagctgc gccgcggtca gcccactacc cacaaaaaat 2880

ttggtgagag cgtggcgatc cttgcctccg ttgggctgct ctctaaagcc tttggtctga 2940

tcgccgccac cggcgatctg ccgggggaga ggcgtgccca ggcggtcaac gagctctcta 3000

ccgccgtggg cgtgcagggc ctggtactgg ggcagtttcg cgatcttaac gatgccgccc 3060

tcgaccgtac ccctgacgct atcctcagca ccaaccacct caagaccggc attctgttca 3120

gcgcgatgct gcagatcgtc gccattgctt ccgcctcgtc gccgagcacg cgagagacgc 3180

tgcacgcctt cgccctcgac ttcggccagg cgtttcaact gctggacgat ctgcgtgacg 3240

atcacccgga aaccggtaaa gatcgcaata aggacgcggg aaaatcgacg ctggtcaacc 3300

ggctgggcgc agacgcggcc cggcaaaagc tgcgcgagca tattgattcc gccgacaaac 3360

acctcacttt tgcctgtccg cagggcggcg ccatccgaca gtttatgcat ctgtggtttg 3420

gccatcacct tgccgactgg tcaccggtca tgaaaatcgc ctgagttaac atgagccaac 3480

cgccgctgct tgaccacgcc acgcagacca tggccaacgg ctcgaaaagt tttgccaccg 3540

ctgcgaagct gttcgacccg gccacccgcc gtagcgtgct gatgctctac acctggtgcc 3600

gccactgcga tgacgtcatt gacgaccaga cccacggctt cgccagcgag gccgcggcgg 3660

aggaggaggc cacccagcgc ctggcccggc tgcgcacgct gaccctggcg gcgtttgaag 3720

gggccgagat gcaggatccg gccttcgctg cctttcagga ggtggcgctg acccacggta 3780

ttacgccccg catggcgctc gatcacctcg acggctttgc gatggacgtg gctcagaccc 3840

gctatgtcac ctttgaggat acgctgcgct actgctatca cgtggcgggc gtggtgggtc 3900

tgatgatggc cagggtgatg ggcgtgcggg atgagcgggt gctggatcgc gcctgcgatc 3960

tggggctggc cttccagctg acgaatatcg cccgggatat tattgacgat gcggctattg 4020

accgctgcta tctgcccgcc gagtggctgc aggatgccgg gctgaccccg gagaactatg 4080

ccgcgcggga gaatcgggcc gcgctggcgc gggtggcgga gcggcttatt gatgccgcag 4140

agccgtacta catctcctcc caggccgggc tacacgatct gccgccgcgc tgcgcctggg 4200

cgatcgccac cgcccgcagc gtctaccggg agatcggtat taaggtaaaa gcggcgggag 4260

gcagcgcctg ggatcgccgc cagcacacca gcaaaggtga aaaaattgcc atgctgatgg 4320

cggcaccggg gcaggttatt cgggcgaaga cgacgagggt gacgccgcgt ccggccggtc 4380

tttggcagcg tcccgtttag caattgatga aaaaaaccgt tgtgattggc gcaggctttg 4440

gtggcctggc gctggcgatt cgcctgcagg cggcagggat cccaaccgta ctgctggagc 4500

agcgggacaa gcccggcggt cgggcctacg tctggcatga ccagggcttt acctttgacg 4560

ccgggccgac ggtgatcacc gatcctaccg cgcttgaggc gctgttcacc ctggccggca 4620

ggcgcatgga ggattacgtc aggctgctgc cggtaaaacc cttctaccga ctctgctggg 4680

agtccgggaa gaccctcgac tatgctaacg acagcgccga gcttgaggcg cagattaccc 4740

agttcaaccc ccgcgacgtc gagggctacc ggcgctttct ggcttactcc caggcggtat 4800

tccaggaggg atatttgcgc ctcggcagcg tgccgttcct ctcttttcgc gacatgctgc 4860

gcgccgggcc gcagctgctt aagctccagg cgtggcagag cgtctaccag tcggtttcgc 4920

gctttattga ggatgagcat ctgcggcagg ccttctcgtt ccactccctg ctggtaggcg 4980

gcaacccctt caccacctcg tccatctaca ccctgatcca cgcccttgag cgggagtggg 5040

gggtctggtt ccctgagggc ggcaccgggg cgctggtgaa cggcatggtg aagctgttta 5100

ccgatctggg cggggagatc gaactcaacg cccgggtcga agagctggtg gtggccgata 5160

accgcgtaag ccaggtccgg ctggcggatg gtcggatctt tgacaccgac gccgtagcct 5220

cgaacgctga cgtggtgaac acctataaaa agctgctcgg ccaccatccg gtggggcaga 5280

agcgggcggc agcgctggag cgcaagagca tgagcaactc gctgtttgtg ctctacttcg 5340

gcctgaacca gcctcattcc cagctggcgc accataccat ctgttttggt ccccgctacc 5400

gggagctgat cgacgagatc tttaccggca gcgcgctggc ggatgacttc tcgctctacc 5460

tgcactcgcc ctgcgtgacc gatccctcgc tcgcgcctcc cggctgcgcc agcttctacg 5520

tgctggcccc ggtgccgcat cttggcaacg cgccgctgga ctgggcgcag gaggggccga 5580

agctgcgcga ccgcatcttt gactaccttg aagagcgcta tatgcccggc ctgcgtagcc 5640

agctggtgac ccagcggatc tttaccccgg cagacttcca cgacacgctg gatgcgcatc 5700

tgggatcggc cttctccatc gagccgctgc tgacccaaag cgcctggttc cgcccgcaca 5760

accgcgacag cgacattgcc aacctctacc tggtgggcgc aggtactcac cctggggcgg 5820

gcattcctgg cgtagtggcc tcggcgaaag ccaccgccag cctgagctag cgtgagggat 5880

ctgattttag tcggcggcgg cctggccaac gggctgatcg cctggcgtct gcgccagcgc 5940

tacccgcagc ttaacctgct gctgatcgag gccggggagc agcccggcgg gaaccatacc 6000

tggtcattcc atgaagacga tctgactccc gggcagcacg cctggctggc cccgctggtg 6060

gcccacgcct ggccgggcta tgaggtgcag tttcccgatc ttcgccgtcg cctcgcgcgc 6120

ggctactact ccattacctc agagcgcttt gccgaggccc tgcatcaggc gctgggggag 6180

aacatctggc taaactgttc ggtgagcgag gtgttaccca atagcgtgcg ccttgccaac 6240

ggtgaggcgc tgcttgccgg agcggtgatt gacggacgcg gcgtgaccgc cagttcggcg 6300

atgcaaaccg gctatcagct ctttcttggt cagcagtggc ggctgacaca gccccacggc 6360

ctgaccgtac cgatcctgat ggatgccacg gtggcgcagc agcagggcta tcgctttgtc 6420

tacacgctgc cgctctccgc cgacacgctg ctgatcgagg atacgcgcta cgccaatgtc 6480

ccgcagcgtg atgataatgc cctacgccag acggttaccg actatgctca cagcaaaggg 6540

tggcagctgg cccagcttga acgcgaggag accggctgtc tgccgattac cctggcgggt 6600

gacatccagg ctctgtgggc cgatgcgccg ggcgtgccgc gctcgggaat gcgggctggg 6660

ctatttcacc ctaccactgg ctattcgctg ccgctggcgg tggcccttgc cgacgcgatt 6720

gccgacagcc cgcggctggg cagcgttccg ctctatcagc tcacccggca gtttgccgaa 6780

cgccactggc gcaggcaggg attcttccgc ctgctgaacc ggatgctttt cctggccggg 6840

cgcgaggaga accgctggcg ggtgatgcag cgcttttatg ggctgccgga gcccaccgta 6900

gagcgctttt acgccggtcg gctctctctc tttgataagg cccgcatttt gacgggcaag 6960

ccaccggttc cgctgggcga agcctggcgg gcggcgctga accattttcc tgacagacga 7020

gataaaggat gatttaaaat gctagtaaat agtttaatcg tcatcttgag cgttattgcg 7080

atggagggca tcgccgcgtt tacccaccgc tacattatgc acggctgggg atggcgctgg 7140

catgagtcac accatacccc gcgcaagggc gtatttgagc taaacgatct ctttgcggtg 7200

gtgtttgccg gggtggctat cgcgctgatt gccgtgggca cggcgggcgt ttggcccctg 7260

cagtggattg gctgcggcat gacggtctat ggcctgctct acttcctggt gcacgacggt 7320

ctggtgcatc agcgctggcc cttccactgg atcccgcgcc ggggctacct gaagcggctc 7380

tacgtcgccc accgcctgca ccacgcggtg cgcggccggg agggctgcgt ctccttcggt 7440

tttatttacg cccgcaagcc tgccgaccta caggcgatcc tgcgtgaacg tcatggccgc 7500

Claims (5)

1. a zeaxanthin synthetic gene recombinant expression plasmid pET-Zea, is characterized in that described recombinant plasmid carries crtE gene, crtB gene, crtI gene, crtY gene, crtZ gene, and the structure of this recombinant plasmid is as follows:

2. zeaxanthin synthetic gene recombinant expression plasmid pET-Zea according to claim 1, is characterized in that the base sequence of described recombinant expression plasmid is as shown in SEQ ID NO:1.

3. a preparation method of zeaxanthin synthetic gene recombinant expression plasmid pET-Zea, its feature comprises the steps:

I) gene amplification:

Extract the genomic dna of Pantoea agglomerans ACCC10495, adopt pcr amplification crtE, crtB, crtI, crtY, crtZ gene coding region, corresponding primer is respectively P1/P2, P3/P4, P5/P6, P7/P8, P9/P10:

The base sequence of described P1 is 5 '-CAGCATATGATGGTGAGTGGCAGT-3 '

The base sequence of described P2 is 5 '-TTAATTGTTAACTCAGGCGATTTTCAT-3 '

The base sequence of described P3 is 5 '-TTAATTGTTAACATGAGCCAACCGCCG-3 '

The base sequence of described P4 is 5 '-GGGCCCCAATTGCTAAACGGGACGCTG-3 '

The base sequence of described P5 is 5 '-GGGCCCCAATTGATGAAAAAAACCGTT-3 '

The base sequence of described P6 is 5 '-GGAATTCGCTAGCGAATTTCAGGCTGGCGGTGG-3 '

The base sequence of described P7 is 5 '-GGAATTCGCTAGCGTGAGGGATCTGATT-3 '

The base sequence of described P8 is 5 '-CCCTTTAAAGGGTCATCCTTTATCTCG-3 '

The base sequence of described P9 is 5 '-ATTGCGGCCGCTTATTCGGGCGAAGA-3 '

The base sequence of described P10 is 5 '-CCCTTTAAAGGGATGCTAGTAAATAGT-3 ';

II) structure cloned plasmids:

The amplified production of crtE, crtB, crtI, crtY, crtZ is carried out respectively to 1.5% agarose gel electrophoresis, then reclaim respectively the band at 924bp, 930bp, 1459bp, 1161bp, 531bp place, be connected, transform bacillus coli DH 5 alpha with cloning vector pMD18-T respectively, filter out positive colony, after fermentation, extract and obtain pMD18-crtE, pMD18-crtB, pMD18-crtI, pMD18-crtY and pMD18-crtZ cloned plasmids;

III) structure recombinant expression plasmid pET-Zea:

Prepared cloned plasmids is carried out respectively to double digestion, the direction that checking gene order is connected with carrier: pMD18-crtE is with Hpa I and EcoR I double digestion; PMD18-crtB is with Hpa I and EcoR I double digestion; PMD18-crtI is with Mfe I and EcoR I double digestion; PMD18-crtY is with Nhe I and EcoR I double digestion; PMD18-crtZ, with Aha III and EcoR I double digestion, chooses the cloned plasmids of gene order and pMD18-T Opposite direction connection;

The plasmid pMD18-crtE that screening is obtained and pMD18-crtB through Hpa I with EcoR I double digestion, be connected and obtain pMD18-crtEB; By pMD18-crtEB and pMD18-crtI through Mfe I with EcoR I double digestion, be connected and obtain pMD18-crtEBI; By pMD18-crtEBI and pMD18-crtY through Nhe I with EcoR I double digestion, be connected and obtain pMD18-crtEBIY; By pMD18-crtEBIY and pMD18-crtZ through Aha III with EcoR I double digestion, be connected and obtain plasmid pMD18-crtEBIYZ;

Expression vector pET-28a is cut through Sna I and Bgl II enzyme, otch is filled, connects and to obtain carrier pET-28a-SB;

By plasmid pMD18-crtEBIYZ and carrier pET-28a-SB, respectively through Nde I and Not I double digestion, enzyme is cut product and is connected and obtain recombinant expression plasmid pET-Zea.

4. zeaxanthin synthetic gene recombinant expression plasmid pET-Zea is in the application detecting in target recipient zeaxanthin synthetic gene activity.

5. a method that detects target recipient zeaxanthin synthetic gene activity, is characterized in that:

By zeaxanthin synthetic gene amplification to be detected in target recipient; Amplified production and recombinant expression plasmid pET-Zea compare according to following manner;

(a) amplified production and recombinant expression plasmid pET-Zea are respectively through Nde I and Hpa I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtE gene in target recipient, it has crtE activity;

(b) amplified production and recombinant expression plasmid pET-Zea are respectively through Hpa I and Mfe I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtB gene in target recipient, it has phytoene synthetase activity;

(c) amplified production and recombinant expression plasmid pET-Zea are respectively through Mfe I and Nhe I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtB gene in target recipient, it has phytoene dehydrogenase activity;

(d) amplified production and recombinant expression plasmid pET-Zea are respectively through through Nhe I and Aha III double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, as the charateristic avsorption band that zeaxanthin detected show to carry crtY gene in target recipient, it has lycopene cyclase activity

(e) amplified production and recombinant expression plasmid pET-Zea are respectively through Aha III and Not I double digestion, enzyme is cut 16 ℃ of connections of product and is spent the night, connect product and transform e. coli bl21, after the fermentation of e. coli bl21 transformant, carry out HPLC analysis, the absorption peak that zeaxanthin detected shows to carry crtZ gene in target recipient, has Cartoene hydroxylase activity.

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