CN103805623B - A kind of chemical activators gene recombination plasmid and its production and use - Google Patents

Abstract

The invention discloses an astaxanthin synthetic gene recombinant expression plasmid pET-Ast, the base sequence of which is as SEQ? ID? NO:1 shown. At the same time, the preparation method of the recombinant expression plasmid pET-Ast and its application and application method in detecting the activity of target receptor astaxanthin synthetase are also disclosed. The astaxanthin synthetic gene recombinant expression plasmid pET-Ast provided by the present invention successfully realizes the accumulation of astaxanthin in E. coli cells after being introduced into the E. coli BL21 strain, thereby accurately and rapidly constructing astaxanthin Production strains. The method for preparing the astaxanthin synthetic gene recombinant expression plasmid pET-Ast provided by the invention is simple, easy to operate and low in cost. The method of the present invention can replace the relevant gene sequence in the recombinant expression plasmid pET-Ast with the gene sequence to be detected in the target receptor by enzyme cutting and ligation, and then detect whether astaxanthin is accumulated in the host cell body to achieve simple and effective Detection and verification of the function of the gene to be tested in the target receptor.

Description

A kind of astaxanthin synthetic gene recombinant plasmid and its preparation method and application

technical field

The invention relates to a recombinant plasmid and its preparation method and application, in particular to an astaxanthin synthetic gene recombinant plasmid and its preparation method and application.

Background technique

Astaxanthin is a ketocarotenoid that is not a source of vitamin A. Pharmacological and physiological studies in recent years have found that astaxanthin has strong biological antioxidant properties. In addition, it also has the functions of promoting antibody production, enhancing immunity and resisting ultraviolet radiation. Therefore, it is widely used in medicine, food, aquaculture, cosmetics, etc. It has broad application prospects. At present, the production methods of astaxanthin mainly include chemical synthesis, shrimp shell extraction and biotechnology. Due to the complex chemical synthesis process and the low content of astaxanthin in shrimp shell waste, the production costs of chemical synthesis and shrimp shell extraction are relatively high. Biotechnology usually uses Phaffia rhodozyme and Haematococcus pluvialis to produce astaxanthin. Existing research focuses on the breeding of high-yielding strains, the utilization of cheap medium, and the optimization of culture conditions. However, although traditional strain selection methods (such as: protoplast fusion, physical mutagenesis, chemical mutagenesis and other methods are easy to operate and can increase the content of astaxanthin in cells to a certain extent, they are random and poor in direction. In recent years, through the use of specific inhibitors, the biosynthetic pathway of astaxanthin in Haematococcus has been basically clarified, and its synthetic pathway is usually composed of pyruvate (pyruvate) and 3-glyceraldehyde (3-P) De novo synthesis, the five-carbon isopentenyl pyrophosphate (Isopentenypyrophosphate, IPP) is first formed, and then converted into isomers of dimethylallyl pyrophosphate (DMAPP). Three molecules of DMAPP and one molecule of IPP (isopentenypyrophosphate) undergoes a three-step condensation reaction to form 20-carbon GGPP (geranylgeranylpyrophosphate). Under the action of phytoene synthase (crtB>, 2 molecules of GGPP are condensed to form 40-carbon phytoene) Phytoene is catalyzed by phytoene desaturase (crtI) and '-carotene desaturase (ZDS) to form lycopene (lycopene) through 4-step dehydrogenation desaturation reaction, tomato Under the action of lycopene B cyclase (crtY), red pigment is further cyclized to form B-carotene. B-carotene c3' and C4' undergo hydroxylation and ketonylation respectively to finally form astaxanthin. B-carotene can be hydroxylated at the C-3,3' position to form the intermediate product B-cryptoxanthin (B-Cryptoxanthin) and zeaxanthin, and then oxidized at the C-4 position to form the intermediate product 3-dihydroxy- 2-ketone-B-carotene (Adonixanthin), and further oxidize Adonixanthin to finally form astaxanthin. 9-carotene can also be oxidized at c-4, 4' first to form intermediate products echinenone (Echinenone) and canthaxanthin, and then Hydroxylation at C-3 position forms intermediate product 4-diketone-3-hydroxyl-B-carotene (Adonimbuin), and Adonimbuin is further formed astaxanthin at C-3' position hydroxylation. Wherein B-carotene is ketonized Enzymes (Crt or CrtO) and B-carotene hydroxylase (CrtZ) control the ketoylation and hydroxylation processes of B-carotene, respectively. More than 150 enzymes currently encode 27 different enzymes in the astaxanthin synthesis route This gene has been cloned from the cells of bacteria, plants, algae and fungi, so the synthesis of astaxanthin by constructing astaxanthin bio-metabolism engineering has gradually been favored by researchers. The source astaxanthin synthesis gene is introduced into the receptor that does not synthesize astaxanthin itself, so as to form the high expression of astaxanthin synthase, or through the Haematococcus pluvialis or red algae that can synthesize astaxanthin itself Phaffia and others are the receptors of metabolic engineering, by changing the quantity, activity and regulation mechanism of astaxanthin synthase, the high-efficiency expression of astaxanthin can be realized. Since the synthesis steps of astaxanthin in microorganisms are many and complicated, the development of vectors capable of efficiently expressing various astaxanthin synthetases has become a research focus and difficulty. In addition, researchers still have a lot of confusion about the detection of the function of the main enzyme system in the synthetic and metabolic pathway of the target receptor astaxanthin during the research and development process, such as through in vitro biochemical reactions or the use of gene mutants for functional complementation. Its operation steps are more loaded down with trivial details, and efficiency is not high also. Therefore, how to obtain a method for efficiently detecting the activity of target receptor astaxanthin synthase is also a problem that researchers in this field are concerned about.

Contents of the invention

One of the purposes of the present invention is to provide a recombinant plasmid that efficiently expresses various astaxanthin synthetases, in order to accurately and rapidly construct astaxanthin production strains; the second purpose of the present invention is to provide a kind of recombinant plasmid Preparation method; the third purpose of the present invention is to provide a use of the recombinant plasmid, that is, the application of the recombinant plasmid in detecting the function of the main gene related to astaxanthin synthesis, more specifically, the recombination of the astaxanthin synthesis gene Application of expression plasmid pET-Ast in detection of target receptor astaxanthin synthase activity.

The purpose of the present invention is achieved by the following technical solutions:

The astaxanthin synthetic gene recombinant expression plasmid pET-Ast provided by the present invention carries crtE gene, crtB gene, crtI gene, crtY gene, crtS gene and crtR gene, and the structure of the recombinant expression plasmid is shown in Figure 1;

The recombinant expression plasmid is sequentially connected with the crtE gene, crtB gene, crtI gene, crtY gene, crtS gene and crtR gene on the expression vector pET-28a, wherein, there is NdeI between the expression vector pET-28a and the crtE gene Restriction site, HpaI restriction site between crtE gene and crtB gene, MfeI restriction site between crtB gene and crtI gene, NheI restriction site between crtI gene and crtY gene, crtY gene and crtY gene There is an AhaⅢ restriction site between the crtS gene, an ApaLI restriction site between the crtS gene and the crtR gene, and a NotⅠ restriction site between the crtR gene and the expression vector pET-28a.

The base sequence of the astaxanthin synthetic gene recombinant expression plasmid pET-Ast is shown in SEQ ID NO:1.

The crtE, crtY, crtI, and crtB genes are the GGPP synthetase gene, lycopene cyclase gene, phytoene dehydrogenase gene, and phytoene synthase gene of Pantoea agglomerans, respectively; crtS , crtR genes are the astaxanthin oxygenase gene and cytochrome P450 reductase gene of Phaffia rhodozyma Phaffiarhodozyma respectively.

The astaxanthin synthetic gene recombinant expression plasmid pET-Ast provided by the present invention successfully realizes the accumulation of astaxanthin in E. coli cells after being introduced into the E. coli BL21 strain, so that astaxanthin can be constructed accurately and rapidly Production strains.

The preparation method of the astaxanthin synthetic gene recombinant expression plasmid pET-Ast provided by the present invention comprises the following steps:

Ⅰ) Gene amplification:

The genomic DNA of PantoeaagglomeransACCC10495 was extracted, and the coding frames of crtE, crtB, crtI, and crtY genes were amplified by PCR, and the corresponding primers were P1/P2, P3/P4, P5/P6, and P7/P8:

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

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

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

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

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

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

The base sequence of P7 is 5'-GGAATTCGCTAGCGAATTGTGAGGGATCTGATT-3'

The base sequence of the P8 is 5'-CCCTTTAAAGGGTCATCCTTTATCTCG-3';

Use the RNA extraction kit to extract the total RNA of PhaffiarhodozymaCGMCC2.1557, use the HiFi-MMLV cDNA first strand synthesis kit to reverse transcribe the first strand of cDNA, and use RT-PCR to amplify the crtS and crtR gene coding regions, and the corresponding primers are P9/P10 , P11/P12:

The base sequence of P9 is 5'-CCCTTTAAAGGGATGTTCATCTTGGTC-3'

The base sequence of the P10 is 5'-CTAGTGCACTAGTCATTCGACCGGCTT-3'

The base sequence of P11 is 5'-CTAGTGCACTAGATGGCCACACTCTCCGAT-3'

The base sequence of P12 is 5'-ATTGCGGCCGCCTACGACCAGACGTCCATC-3';

Ⅱ) Construction of cloning plasmid:

The amplification products of crtE, crtB, crtI, crtY, crtS, and crtR were subjected to 1.5% gel electrophoresis, and then the bands at 924bp, 930bp, 1459bp, 1161bp, 1674bp, and 2825bp were recovered, and then respectively combined with the cloning vector pMD18- T-connected and transformed into Escherichia coli DH5α, screened out positive clones, extracted after fermentation to obtain cloning plasmids of pMD18-crtE, pMD18-crtB, pMD18-crtI, pMD18-crtY, pMD18-crtS and pMD18-crtR;

Ⅲ) Construction of recombinant expression plasmid pET-Ast:

The positive cloned plasmids were double-digested to verify the connection direction of the gene sequence and the vector: pMD18-crtE was digested with NdeI and EcoRI; pMD18-crtB was digested with HpaI and EcoRI; pMD18-crtI was digested with MfeI and EcoRI ; pMD18-crtY was digested with NheⅠ and EcoRI; pMD18-crtS was digested with AhaⅢ and EcoRI; pMD18-crtR was digested with ApaLI and EcoRI, and the cloning plasmid whose gene sequence was reversely connected with pMD18-T was selected;

The screened pMD18-crtE and pMD18-crtB were digested by HpaI and EcoRI and ligated to obtain pMD18-crtEB; the pMD18-crtEB and pMD18-crtI obtained by enzyme digestion were digested by MfeI and EcoRI and ligated to obtain pMD18- crtEBI; the pMD18-crtEBI and pMD18-crtY obtained by enzyme digestion were ligated by NheI and EcoRI double enzymes to obtain pMD18-crtEBIY; the pMD18-crtEBIY and pMD18-crtS obtained by enzyme digestion were ligated by AhaIII and EcoRI double enzymes pMD18-crtEBIYS; pMD18-crtEBIYS and pMD18-crtR obtained by digestion and ligation were digested with ApaLI and EcoRI to obtain pMD18-crtEBIYSR;

The expression vector pET-28a was digested with SnaI and BglII, and the incision was filled and ligated to obtain the vector pET-28a-SB;

The plasmid pMD18-crtEBIYSR and the vector pET-28a-SB were digested with NdeI and NotI, and the digested products were ligated and transformed into Escherichia coli DH5α to obtain the recombinant expression plasmid pET-Ast carrying the astaxanthin synthase gene.

The method for preparing the astaxanthin synthetic gene recombinant expression plasmid pET-Ast provided by the invention is simple, easy to operate and low in cost.

The recombinant plasmid provided by the invention can be used to detect the functions of the main genes related to astaxanthin synthesis. More specifically, it can be used to detect the activity of target receptor astaxanthin synthase.

The method for detecting the activity of the target receptor astaxanthin synthase provided by the present invention is:

Amplify the astaxanthin synthesis gene to be detected in the target receptor, and compare the amplified product with the recombinant expression plasmid pET-Ast in the following manner;

(a) The amplified product and the recombinant expression plasmid pET-Ast were digested with NdeI and HpaI respectively, and the digested products were ligated overnight at 16°C. The ligated product was transformed into E. coli BL21, and the E. coli BL21 transformant was fermented for HPLC analysis, such as detection The characteristic absorption peak of astaxanthin indicates that the target receptor carries the crtE gene, which has GGPP synthetase activity;

(b) The amplified product and the recombinant expression plasmid pET-Ast were digested by HpaI and MfeI respectively, and the digested products were ligated overnight at 16°C. The ligated product was transformed into E. coli BL21, and the E. coli BL21 transformant was fermented for HPLC analysis, such as detection The characteristic absorption peak of astaxanthin indicates that the target receptor carries the crtB gene, which has phytoene synthase activity;

(c) The amplified product and the recombinant expression plasmid pET-Ast were digested by MfeI and NheI respectively, and the digested products were ligated overnight at 16°C. The ligated product was transformed into E. coli BL21, and the E. coli BL21 transformant was fermented for HPLC analysis, such as detection The characteristic absorption peak of astaxanthin indicates that the target receptor carries the crtI gene, which has phytoene dehydrogenase activity;

(d) The amplified product and the recombinant expression plasmid pET-Ast were digested by NheⅠ and AhaⅢ respectively, and the digested products were ligated overnight at 16°C. The ligated products were transformed into E. coli BL21, and the E. coli BL21 transformants were fermented for HPLC analysis, as shown in The detection of the characteristic absorption peak of astaxanthin indicates that the target receptor carries the crtY gene, which has lycopene cyclase activity;

(e) The amplified product and the recombinant expression plasmid pET-Ast were digested with AhaⅢ and ApaLI respectively, and the digested product was ligated overnight at 16°C. The ligated product was transformed into E. coli BL21, and the E. coli BL21 transformant was fermented for HPLC analysis, such as detection The characteristic absorption peak of astaxanthin indicates that the target receptor carries the crtS gene, which has astaxanthin oxygenase activity;

(f) The amplified product and the recombinant expression plasmid pET-Ast were digested with ApaLI and NotⅠ respectively, and the digested product was ligated overnight at 16°C. The ligated product was transformed into E. coli BL21, and the E. coli BL21 transformant was fermented and analyzed by HPLC. The characteristic absorption peak of astaxanthin indicates that the target receptor carries the crtR gene, which has cytochrome P450 reductase activity.

The method of the present invention can replace the relevant gene sequence in the recombinant expression plasmid pET-Ast with the gene sequence to be detected in the target receptor by enzyme cutting and ligation, and then detect whether astaxanthin is accumulated in the host cell body, which can be simply and effectively Detection and verification of the function of the gene to be tested in the target receptor.

Description of drawings

Figure 1 is a map of the recombinant expression plasmid pET-Ast.

Fig. 2 is an agarose gel electrophoresis pattern of amplified products of crtS, crtR genes and crtE, crtI, crtY, crtB genes.

Among them: M1 represents DM5000marker; M2 represents DM2000marker; 1 represents crtE; 2 represents crtB; 3 represents crtY; 4 represents crtI; 5 represents the cDNA of crtS; 6 represents the cDNA of crtR.

Fig. 3 is an agarose gel electrophoresis profile of the recombinant expression plasmid pET-Ast double enzyme digestion detection.

Among them: M1 indicates DM5000marker; M2 indicates DM2000marker; 1 indicates NdeI and HpaI double enzyme digestion; 2 indicates HpaI and MfeI double enzyme digestion; 3 indicates MfeI and NheI double enzyme digestion; 4 indicates NheI and AhaIII double enzyme digestion; 5 indicates ApaLI and ApaLI AhaⅢ double enzyme digestion; 6 indicates NotⅠ and ApaLI double enzyme digestion.

Fig. 4 is a liquid chromatogram of astaxanthin in pET-Ast/BL21 recombinant Escherichia coli fermentation broth.

Figure 5 is a liquid chromatogram of astaxanthin standard solution.

Fig. 6 is a liquid chromatogram of astaxanthin in the fermented liquid after recombining the crtE gene of Pantoeaananatis MCCC1F01131 with the plasmid pET-Ast and transferring it into an Escherichia coli strain and fermentation.

Fig. 7 is a liquid chromatogram of astaxanthin in the fermentation broth after recombining the crtB gene of PantoeaananatisMCCC1F01131 with the plasmid pET-Ast and transferring it into an Escherichia coli strain and fermenting it.

Fig. 8 is a liquid chromatogram of astaxanthin in the fermentation broth after recombining the crtI gene of PantoeaananatisMCCC1F01131 with the plasmid pET-Ast and transferring it into an Escherichia coli strain and fermenting it.

Fig. 9 is a liquid chromatogram of astaxanthin in the fermentation broth after recombining the crtY gene of Pantoeaananatis MCCC1F01131 with the plasmid pET-Ast and transferring it into an Escherichia coli strain and fermenting it.

Fig. 10 is a liquid chromatogram of astaxanthin in the fermentation broth after the crtS gene of Phaffiarhodozyma AS2.1557 was recombined with the plasmid pET-Ast and transformed into an Escherichia coli strain and fermented.

Fig. 11 is a liquid chromatogram of astaxanthin in the fermentation broth after the crtR gene of Phaffiarhodozyma AS2.1557 was recombined with the plasmid pET-Ast and transformed into an Escherichia coli strain and fermented.

detailed description

Example 1: Construction of recombinant expression plasmid pET-Ast

(1) Gene amplification:

The total RNA of Phaffiarhodozyma CGMCC2.1557 (purchased from China Agricultural Microorganism Culture Collection Management Center) was extracted with the ultrapure RNA extraction kit (purchased from Kangwei Century Biotechnology Co., Ltd.), and the HiFi-MMLV cDNA first-strand synthesis kit (purchased from Kangwei Century Biotechnology Co., Ltd.) was reverse-transcribed to obtain the first strand of cDNA, and RT-PCR was used to amplify the coding regions of crtS and crtR genes, ExTaq DNA polymerase (purchased from Takara), and corresponding primers (synthesized by Beijing Sanbo Polygala) are shown in Table 1 .

Genomic DNA of Pantoea agglomerans ACCC10495 (purchased from China Agricultural Microorganism Culture Collection and Management Center) was extracted by phenol-form extraction method, and used as a template for PCR amplification. Using ExTaq DNA polymerase, the corresponding primers are shown in Table 1, and the coding frames of crtE, crtI, crtY, and crtB genes were respectively amplified according to conventional methods.

The amplified products were detected by 1.5% agarose gel electrophoresis, the results are shown in Figure 2, and then the bands at 924bp, 930bp, 1459bp, 1161bp, 1674bp, and 2825bp were recovered respectively for crtE, crtB, crtI, crtY, crtS, and crtR The expected amplified product was obtained. The expected amplified products were recovered and purified with the gel recovery kit (purchased from Beijing Sanbo Polygala), respectively ligated with the cloning vector pMD18-T (purchased from Takara) and transformed into Escherichia coli DH5α (purchased from Takara). Pick a single colony, use M13PrimerRV and M13PrimerM4 as primer pairs (purchased from Takara) to detect positive clones, screen out positive clone plasmids, and sequence and analyze the gene sequence by Beijing Sanbo Polygala Company. The amino acid sequence of each gene is shown in SEQ ID NO: 2～ SEQ ID NO:7.

Table 1: Primer sequence information required for amplifying crtS, crtR genes, crtE, crtI, crtY, crtB genes

Note: In Table 1, the double underlined part is the restriction endonuclease recognition sequence, and the single underlined part is the endonuclease protective base. The sequences of the above primers P1-P12 were designed according to GenBank accession number M87280, DQ202402 and EU884133.

(2) Construction of recombinant expression plasmids:

The positive cloned plasmids were double-digested to verify the connection direction of the gene sequence and the vector: pMD18-crtE was digested with NdeI and EcoRI; pMD18-crtB was digested with HpaI and EcoRI; pMD18-crtI was digested with MfeI and EcoRI ; pMD18-crtY was digested with NheⅠ and EcoRI; pMD18-crtS was digested with AhaⅢ and EcoRI; pMD18-crtR was digested with ApaLI and EcoRI, and the clone plasmid whose gene sequence was reversely connected with pMD18-T was selected.

The screened pMD18-crtE and pMD18-crtB were digested by HpaI and EcoRI to obtain pMD18-crtEB; the pMD18-crtEB and pMD18-crtI obtained by enzyme digestion were digested by MfeI and EcoRI to obtain pMD18-crtEBI; The pMD18-crtEBI and pMD18-crtY obtained by enzyme digestion were ligated by NheI and EcoRI double enzymes to obtain pMD18-crtEBIY; crtEBIYS; pMD18-crtEBIYS and pMD18-crtR obtained by digestion and ligation were digested with ApaLI and EcoRI to obtain pMD18-crtEBIYSR.

The expression vector pET-28a was digested with SnaI and BglII, and the incision was filled and ligated to obtain pET-28a-SB.

The plasmid pMD18-crtEBIYSR and the vector pET-28a-SB were digested with NdeI and NotI, and the digested products were ligated and transformed into Escherichia coli DH5α to obtain pET-Ast. See the sequence table and SEQ ID NO: 1 for the sequencing results. The plasmid map is shown in Figure 1 .

Example 2: Enzyme digestion verification of recombinant expression plasmid pET-Ast

The recombinant plasmid pET-Ast constructed in Example 1 was digested with NdeI and HpaI to verify the connection of crtE (Figure 3, lane 1); after double digestion with HpaI and MfeI, the connection of crtB was verified (Figure 3, lane 2) ; The connection of crtI was verified by double digestion with MfeⅠ and NheⅠ (Figure 3, lane 3); the connection of crtY was verified by double digestion with NheⅠ and AhaIII (Figure 3, lane 4); the connection of crtY was verified by double digestion with AhaⅢ and ApaLI The connection of crtS (Figure 3, lane 5); the connection of crtR was verified by NotI and ApaLI double digestion (Figure 3, lane 6).

Embodiment 3: the fermentation detection of recombinant bacterial strain

The recombinant plasmid pET-Ast was introduced into Escherichia coli BL21 (Escherichia coli strain BL21 was purchased from Takara Company) for expression, and the main components of the fermentation product were determined by high performance liquid chromatography (HPLC) to detect whether the constructed recombinant expression plasmid accumulated astaxanthin .

The specific conditions for high performance liquid chromatography in this example are: chromatographic column: HypersilODS25um, 250×4.6mm; mobile phase: methanol: acetone: water; UV detection wavelength: 480nm; flow rate: 1.0mL/min; 20 μL.

(1) Precisely weigh 10mg of astaxanthin, first dissolve it with a small amount of dichloromethane and then dilute it with acetonitrile to make a solution with a concentration of 0.1mg/mL, and use it as a standard solution for later use;

Introduce the recombinant plasmid pET-Ast into Escherichia coli BL21 (purchased from Takara Company) strain according to conventional methods to obtain recombinant Escherichia coli, after liquid fermentation at 37°C for 72 hours, freeze-dry the fermentation broth, and accurately weigh the recombinant Escherichia coli after freeze-drying Add a small amount of dichloromethane to dissolve, and dilute to a 100mL volumetric flask with acetonitrile, and use it as the test sample solution for later use.

(2) Identification

Take the prepared standard solution 1.5mL and place it in the sampling bottle, inject the solution into the liquid chromatograph for measurement, and the results are shown in Figure 5;

Take 100 μL of the test sample solution and inject it into the liquid chromatograph for determination, and the results are shown in Figure 4.

Result analysis: By comparing Figure 5 and Figure 4, it can be seen that there is accumulation of astaxanthin in the constructed recombinant E. coli cells.

Example 4: Detection of the function of the gene to be tested

With the crtE, crtB, crtI, crtY gene of known gene sequence and function in PantoeaananatisMCCC1F01131, and the crtS, crtR gene of known gene sequence and function of PhaffiarhodozymaAS2.1557 as the gene of function to be tested, with detection system of the present invention (i.e. implement The recombinant expression plasmid pET-Ast constructed in Example 1) was used for detection of gene function.

(1) Amplification of the gene to be tested

The genomic DNA of Pantoeaananatis MCCC1F01131 was extracted by phenolform extraction as a template for PCR amplification, and the coding regions of crtE, crtI, crtY, crtZ, and crtB genes were amplified with ExTaq DNA polymerase and corresponding primers (Table 2). The expected amplified products were respectively connected with the cloning vector pMD18-T to transform DH5α. The positive colonies were picked, and the gene sequences were sequenced and analyzed by Beijing Sanbo Polygala Company.

The total RNA of Phaffiarhodozyma AS2.1557 was extracted with the Ultrapure RNA Extraction Kit, and the first strand of cDNA was obtained by reverse transcription with the HiFi-MMLV cDNA First Strand Synthesis Kit, and the coding regions of crtS and crtR genes were respectively amplified by RT-PCR. The corresponding primers are shown in the table 2.

Table 2: Primer sequence information required for amplifying crtS, crtR genes and crtE, crtI, crtY, crtB genes

Note: In Table 2, the double underlined part is the restriction endonuclease recognition sequence, and the single underlined part is the endonuclease protective base. The primer sequences of P1'～P12' were designed according to GenBankaccessionnumber D90087.2, DQ202402 and EU884133.

(2) Functional detection of the gene to be tested:

(2.1) Functional detection of GGPP synthetase activity

The detection system and the crtE amplified product of PantoeaananatisMCCC1F01131 were digested by NdeI and HpaI respectively, and the digested product was ligated overnight at 16°C. The ligated product was transformed into E. coli BL21, and the E. coli BL21 strain containing the recombinant plasmid was fermented and analyzed by HPLC. The characteristic absorption peak of astaxanthin is shown in Figure 6. It shows that the Pantoeaananatis MCCC1F01131crtE gene to be tested has GGPP synthetase activity.

(2.2) Functional detection of phytoene synthase activity

The detection system and the crtB amplified product of PantoeaananatisMCCC1F01131 were digested by HpaI and MfeI respectively, and the digested products were ligated overnight at 16°C. The ligated product was transformed into E. coli BL21, and the E. coli BL21 strain containing the recombinant plasmid was fermented and analyzed by HPLC. The characteristic absorption peak of astaxanthin is shown in Figure 7. It shows that the Pantoeaananatis MCCC1F01131crtB gene to be tested has phytoene synthase activity.

(2.3) Functional detection of phytoene dehydrogenase activity

The amplified product of the plasmid pET-Ast of the detection system and the crtI of PantoeaananatisMCCC1F01131 were digested by MfeI and NheI respectively, and the digested products were ligated overnight at 16°C. HPLC analysis detected the characteristic absorption peak of astaxanthin as shown in Figure 8. It shows that the Pantoeaananatis MCCC1F01131crtI gene to be tested has phytoene dehydrogenase activity.

(2.4) Functional detection of lycopene cyclase activity

The plasmid pET-Ast of the detection system and the crtY amplification product of PantoeaananatisMCCC1F01131 were digested by NheⅠ and AhaⅢ respectively, and the digested products were ligated overnight at 16°C. The ligated products were transformed into Escherichia coli BL21, and the Escherichia coli BL21 strain containing the recombinant plasmid was fermented. HPLC analysis detected the characteristic absorption peak of astaxanthin as shown in Figure 9. It shows that the Pantoeaananatis MCCC1F01131crtY gene to be tested has lycopene cyclase activity.

(2.5) Functional detection of astaxanthin oxygenase activity

The plasmid pET-Ast of the detection system and the crtS amplification product of PhaffiarhodozymaAS2.1557 were digested by AhaⅢ and ApaLI respectively, and the digested products were ligated overnight at 16°C. The ligated products were transformed into Escherichia coli BL21, and the Escherichia coli BL21 strain containing the recombinant plasmid was fermented After HPLC analysis, the characteristic absorption peak of astaxanthin was detected as shown in Figure 10. It shows that the PhaffiarhodozymaAS2.1557crtS gene to be tested has astaxanthin oxygenase activity.

(2.6) Functional detection of cytochrome P450 reductase activity

The amplified products of the plasmid pET-Ast and PhaffiarhodozymaAS2.1557crtR of the detection system were digested by ApaLI and NotⅠ respectively, and the digested products were ligated overnight at 16°C. The ligated products were transformed into Escherichia coli BL21, and the Escherichia coli BL21 strain containing the recombinant plasmid was fermented. HPLC analysis detected the characteristic absorption peak of astaxanthin as shown in Figure 11. It shows that the Phaffiarhodozyma AS2.1557crtR gene to be tested has cytochrome P450 reductase activity.

The specific conditions of the HPLC analysis in the above steps (2.1) to (2.6) are consistent with the HPLC analysis conditions of Example 3.

Claims (4)

1. a chemical activators DNA recombinant expression plasmid pET-Ast, is characterized in that, described recombinant expression plasmid carries crtE gene, crtB gene, crtI gene, crtY gene, crtS gene and crtR gene, and the structure of this recombinant expression plasmid is as follows:

Recombinant expression plasmid described in it is on expression vector pET-28a, be connected with described crtE gene in turn, crtB gene, crtI gene, crtY gene, crtS gene and crtR gene, wherein, there is between expression vector pET-28a and crtE gene Nde I restriction enzyme site, there is between crtE gene and crtB gene Hpa I restriction enzyme site, there is between crtB gene and crtI gene Mfe I restriction enzyme site, there is between crtI gene and crtY gene Nhe I restriction enzyme site, there is between crtY gene and crtS gene Aha III restriction enzyme site, there is between crtS gene and crtR gene ApaL I restriction enzyme site, there is between crtR gene and expression vector pET-28a Not I restriction enzyme site,

Described chemical activators DNA recombinant expression plasmid pET-Ast, its base sequence is as shown in SEQIDNO:1.

2. the preparation method of chemical activators DNA recombinant expression plasmid pET-Ast described in claim 1, is characterized in that, comprise the steps:

I) gene amplification:

Extract the genomic dna of PantoeaagglomeransACCC10495, adopt pcr amplification crtE, crtB, crtI, crtY genes encoding frame, corresponding primer is respectively P1/P2, P3/P4, P5/P6, P7/P8:

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 '-GGAATTCGCTAGCGAATTGTGAGGGATCTGATT-3 '

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

RNA is selected to extract the total serum IgE of test kit extraction PhaffiarhodozymaCGMCC2.1557, cDNA first chain is obtained with HiFi-MMLVcDNA first chain synthetic agent box reverse transcription, adopt RT-PCR amplification crtS, crtR gene coding region, corresponding primer is P9/P10, P11/P12:

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

The base sequence of described P10 is 5 '-CTAGTGCACTAGTCATTCGACCGGCTT-3 '

The base sequence of described P11 is 5 '-CTAGTGCACTAGATGGCCACACTCTCCGAT-3 '

The base sequence of described P12 is 5 '-ATTGCGGCCGCCTACGACCAGACGTCCATC-3 ';

II) cloned plasmids is built:

The amplified production of crtE, crtB, crtI, crtY, crtS, crtR is carried out 1.5% gel electrophoresis respectively, then the band at 924bp, 930bp, 1459bp, 1161bp, 1674bp, 2825bp place is reclaimed respectively, then be connected with cloning vector pMD18-T respectively, transformation of E. coli DH5 α, filter out positive colony, extract after fermentation and obtain pMD18-crtE, pMD18-crtB, pMD18-crtI, pMD18-crtY, pMD18-crtS and pMD18-crtR cloned plasmids;

III) recombinant expression plasmid pET-Ast is built:

Positive colony plasmid carries out double digestion respectively, the direction that checking gene order is connected with carrier: pMD18-crtE is with Nde 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-crtS is with Aha III and EcoR I double digestion; PMD18-crtR, with ApaL I and EcoR I double digestion, chooses the cloned plasmids of gene order and pMD18-T Opposite direction connection;

PMD18-crtEB is connected to obtain through Hpa I after EcoR I double digestion by screening pMD18-crtE with pMD18-crtB obtained; Enzyme is cut and connects pMD18-crtEB with pMD18-crtI that obtain and be connected to obtain pMD18-crtEBI through Mfe I after EcoR I double digestion; Enzyme is cut and connects pMD18-crtEBI with pMD18-crtY that obtain and be connected to obtain pMD18-crtEBIY through Nhe I with EcoR I double digestion; Enzyme is cut and connects pMD18-crtEBIY with pMD18-crtS that obtain and be connected to obtain pMD18-crtEBIYS through Aha III with EcoR I double digestion; Enzyme is cut and connects pMD18-crtEBIYS with pMD18-crtR that obtain and be connected to obtain pMD18-crtEBIYSR through ApaL I with EcoR I double digestion;

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

By plasmid pMD18-crtEBIYSR and carrier pET-28a-SB through Nde I and Not I double digestion, digestion products connects, transformation of E. coli DH5 α, obtains recombinant expression plasmid pET-Ast.

3. chemical activators DNA recombinant expression plasmid pET-Ast described in claim 1 is detecting the application in target recipient chemical activators enzymic activity.

4. detect a method for target recipient chemical activators enzymic activity, it is characterized in that:

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

A () amplified production and recombinant expression plasmid pET-Ast are respectively through Nde I and Hpa I double digestion, digestion products 16 DEG C of connections are spent the night, connect product conversion e. coli bl21, HPLC analysis is carried out after the fermentation of e. coli bl21 transformant, as detected, namely the charateristic avsorption band of astaxanthin shows to carry crtE gene in target recipient, and it has crtE activity;

B () amplified production and recombinant expression plasmid pET-Ast are respectively through Hpa I and Mfe I double digestion, digestion products 16 DEG C of connections are spent the night, connect product conversion e. coli bl21, HPLC analysis is carried out after the fermentation of e. coli bl21 transformant, as detected, namely the charateristic avsorption band of astaxanthin shows to carry crtB gene in target recipient, and it has phytoene synthetase activity;

C () amplified production and recombinant expression plasmid pET-Ast are respectively through Mfe I and Nhe I double digestion, digestion products 16 DEG C of connections are spent the night, connect product conversion e. coli bl21, HPLC analysis is carried out after the fermentation of e. coli bl21 transformant, as detected, namely the charateristic avsorption band of astaxanthin shows to carry crtB gene in target recipient, and it has phytoene dehydrogenase activity;

D () amplified production and recombinant expression plasmid pET-Ast are respectively through through Nhe I and Aha III double digestion, digestion products 16 DEG C of connections are spent the night, connect product conversion e. coli bl21, HPLC analysis is carried out after the fermentation of e. coli bl21 transformant, as detected, namely the charateristic avsorption band of astaxanthin shows to carry crtY gene in target recipient, and it has lycopene cyclase activity;

E () amplified production and recombinant expression plasmid pET-Ast are respectively through Aha III and ApaL I double digestion, digestion products 16 DEG C of connections are spent the night, connect product conversion e. coli bl21, HPLC analysis is carried out after the fermentation of e. coli bl21 transformant, as detected, namely the charateristic avsorption band of astaxanthin shows to carry crtS gene in target recipient, and it has astaxanthin cyclooxygenase activity;

F () amplified production and recombinant expression plasmid pET-Ast are respectively through ApaL I and Not I double digestion, digestion products 16 DEG C of connections are spent the night, connect product conversion e. coli bl21, HPLC analysis is carried out after the fermentation of e. coli bl21 transformant, detect that namely the charateristic avsorption band of astaxanthin shows to carry crtR gene in target recipient, it has cytochrome P450 reductase activity;

Described recombinant expression plasmid pET-Ast is the chemical activators DNA recombinant expression plasmid pET-Ast described in claim 1.