CN110484550B - Carotenoid metabolism gene function identification method - Google Patents
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a carotenoid metabolic gene function identification method, which comprises the following steps: s1: cloning of the selectable marker elements: cloning of the plasmid from pGBK-T7 by AmprPromoter-driven kanamycin resistance gene KanrWith this element (pAmp)r::Kanr) As a subsequent screening marker; s2: cloning of the promoter: the promoter sequence of a RuBisCO large subunit gene (rbcL) in cyanobacteria Synechocystis sp.PCC6803 is cloned, and the promoter is used for driving an enzyme gene to be detected; s3: cloning of the enzyme genes: the carotenoid metabolism-related enzyme gene X to be identified from different organisms is cloned downstream of the rbcL promoter (rbcL:: X), driven by this promoter; s4 construction of the expression cassette: an entire gene expression cassette comprises a pAmp arranged in sequencer::KanrA selection marker element and an rbcL:: X element for expressing an enzyme protein. Compared with the traditional identification method, the method has the advantages of simplicity, convenience and directness, and can definitely and pertinently identify the gene function.
Description
Technical Field
The invention relates to the technical field of gene function identification, in particular to a carotenoid metabolic gene function identification method.
Background
Carotenoids are widely distributed in nature. They generally act as light traps and as photoprotective pigments, and in addition provide the plant pollen with a vivid color to aid seed transmission. For human beings, these compounds are beneficial to human health and are essential phytonutrients in the daily diet. Almost all photosynthetic organisms are able to synthesize the common carotenoids (lycopene and beta-carotene), while in higher plants more types of carotenoids occur, including lutein and its oxygenated derivatives. However, some specific carotenoids are only present in specific species, such as astaxanthin in haematococcus pluvialis and lactucin in lettuce. If one could identify enzymes that catalyze the synthesis of these unique carotenoids, it would be helpful to synthesize them in large quantities by genetic engineering and synthetic biological strategies. The enzyme activity identification work in the conventional biochemical research usually uses an aqueous solution system, and uses recombinase protein to carry out in-vitro enzyme activity reaction to determine the product. However, the same strategy is difficult to implement in the research of carotenoid synthesis pathway, because most of the enzymes involved in the synthesis pathway utilize carotenoid substrates and corresponding products which are fat-soluble, and the function of carotenoid metabolic enzyme is inconvenient to identify in an aqueous solution system. Therefore, we have proposed a method for in vivo identification of carotenoid metabolism gene function using cyanobacteria capable of synthesizing simple carotenoid components, which has been proposed to solve the above-mentioned problems.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a carotenoid metabolic gene function identification method.
The invention provides a carotenoid metabolic gene function identification method, which comprises the following steps:
s1: cloning of the selectable marker elements: cloning of the plasmid from pGBK-T7 by AmprPromoter-driven kanamycin resistance gene KanrWith this element (pAmp)r::Kanr) As a subsequent screening marker;
s2: cloning of the promoter: the promoter sequence of a RuBisCO large subunit gene (rbcL) in cyanobacteria Synechocystis sp.PCC6803 is cloned, and the promoter is used for driving an enzyme gene to be detected;
s3: cloning of the enzyme genes: the carotenoid metabolism-related enzyme gene X to be identified from different organisms is cloned downstream of the rbcL promoter (rbcL:: X), driven by this promoter;
s4 construction of the expression cassette: an entire gene expression cassette comprises a pAmp arranged in sequencer::KanrA selection marker element and an rbcL for expressing an enzyme protein;
s5: plasmid construction: at two ends of the constructed expression cassette, homologous arm sequences at two sides of the insertion site of the introduced synechocystis genome are amplified and introduced by a PCR technology, and the amplified fragments are cloned into a vector;
s6: culturing the cyanobacteria strain: selecting a proper cyanobacteria strain, and culturing the cyanobacteria strain;
s7 transformation of cyanobacteria: the plasmid cloned in S5 is transformed into cyanobacteria by a natural transformation method; the transformed cyanobacteria contained 50. mu.g/ml-1Screening on BG-11 medium plates of kanamycin under the same other culture screening conditions as S6;
s8: and (3) pigment identification: when cyanobacteria were grown to OD in S7 liquid medium700When the concentration is 2.0, 5ml of the bacterial solution is centrifuged to collect cells, the accumulated carotenoids are extracted with methanol, and the pigments are separated and identified by high performance liquid chromatography.
Preferably, in the S4, when two enzyme genes need to be expressed simultaneously, a ribosome binding sequence in a prokaryotic polycistron is inserted between the open reading frames of the two genes, so that the rbcL promoter can drive the expression of the two enzyme genes at the downstream of the rbcL promoter simultaneously. Preferably, in the S6, the culture condition is BG-11 culture medium, 28-30 ℃, 30-35 mu mol photon m-2·s-1。
Preferably, in S5, the synechocystis genome insertion site is selected to be the middle of the gene encoding carotene ketolase CrtO, and disruption of normal expression of the gene helps to further simplify carotene metabolism and promote accumulation of β -carotene in synechocystis.
Preferably, in the S6, the culture condition is BG-11 culture medium, 28-30 ℃, 30-35 mu mol photon m-2·s-1。
Preferably, in the S7, the transformed cyanobacteria contain 50 μ g/ml-1Kanamycin was screened on BG-11 medium plates. Selecting the selected transgenic strain containing 50. mu.g/ml-1Culturing and growing in BG-11 liquid culture medium of kanamycin; the growth condition is BG-11 culture medium, 28-30 deg.C, 30-35 μmol photon. m-2·s-1。
The invention has the beneficial effects that: generally, enzyme function identification is carried out by in vitro enzyme activity measurement using recombinant proteins. However, since many carotenoids are fat-soluble organic compounds, similar biochemical reactions cannot be carried out in an aqueous environment using recombinant proteins. The method takes living cyanobacteria as a reaction environment, fully utilizes the advantage that the cyanobacteria can synthesize simpler carotenoid components as a subsequent detection working substrate, and can conveniently and quickly identify the functions of carotenoid metabolic genes. In addition, the method utilizes simple operation of gene expression in cyanobacteria, and avoids the difficulty of expression, classification and purification of recombinant protein. Therefore, compared with the traditional identification method, the method has the advantages of simplicity, convenience and directness, and can definitely and pertinently identify the gene function.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example one
The embodiment provides a carotenoid metabolic gene function identification method, which comprises the following steps:
s1: cloning of the selectable marker elements: cloning of the plasmid from pGBK-T7 by AmprPromoter-driven kanamycin resistance gene KanrWith this element (pAmp)r::Kanr) As a subsequent screening marker;
s2: cloning of the promoter: the promoter sequence of a RuBisCO large subunit gene (rbcL) in cyanobacteria Synechocystis sp.PCC6803 is cloned, and the promoter is used for driving an enzyme gene to be detected;
s3: cloning of the enzyme genes: zeaxanthin epoxidase (AtZEP), the gene responsible for the identification of the carotenoid metabolism-related enzyme Arabidopsis thaliana, was cloned downstream of the rbcL promoter (rbcL:: AtZEP), driven by the promoter;
s4 construction of the expression cassette: an entire gene expression cassette comprises a pAmp arranged in sequencer::KanrScreening marker element and protein for expressing enzymeThe white rbcL comprises an AtZEP element, wherein a BamHI site is reserved in the process of constructing a gene expression cassette, so that more genes can be conveniently introduced subsequently;
s5: plasmid construction: introducing homologous arm sequences at two sides of an insertion site of a synechocystis genome through PCR (polymerase chain reaction) amplification at two ends of a constructed expression cassette, cloning an amplified fragment into a vector, selecting a gene coding carotene ketolase CrtO in the synechocystis genome as the insertion site, wherein the insertion site of the gene expression cassette is in the middle section of the gene, and the designed homologous arm sequences destroy the normal expression of the gene and can help to accumulate beta-carotene;
s6: culturing the cyanobacteria strain: selecting a proper cyanobacteria strain, and culturing the cyanobacteria strain;
s7 transformation of cyanobacteria: the plasmid cloned in S5 is transformed into cyanobacteria by a natural transformation method; the transformed cyanobacteria contained 50. mu.g/ml-1Screening on BG-11 medium plates of kanamycin under the same other culture screening conditions as S6;
s8: and (3) pigment identification: when cyanobacteria were grown to OD in S7 liquid medium700When the concentration is 2.0, 5ml of the bacterial solution is centrifuged to collect cells, the accumulated carotenoids are extracted with methanol, and the pigments are separated and identified by high performance liquid chromatography.
In this example, in S6, BG-11 medium was used as the culture condition, and 30-35. mu. mol. photon. m was used at 28-30 ℃-2·s-1S7, the transformed cyanobacteria contained 50. mu.g.ml-1BG-11 medium plate screening of kanamycin, S7, selection of the transgenic strain obtained by screening, and screening of the strain containing 50. mu.g/ml-1Culturing and growing in BG-11 liquid culture medium of kanamycin; the growth condition is BG-11 culture medium, 28-30 deg.C, 30-35 μmol photon. m-2·s-1In the embodiment, the zeaxanthin epoxidase (AtZEP) algae strain of arabidopsis thaliana is cloned, and the occurrence of violaxanthin in the product is identified by high performance liquid chromatography separation compared with a non-transgenic control algae strain, so that the system is proved to be sensitive and reliable.
Example two
The embodiment provides a carotenoid metabolic gene function identification method, which comprises the following steps:
s1: cloning of the selectable marker elements: cloning of the plasmid from pGBK-T7 by AmprPromoter-driven kanamycin resistance gene Kanr, and the element (pAmp)r::Kanr) As a subsequent screening marker;
s2: cloning of the promoter: the promoter sequence of a RuBisCO large subunit gene (rbcL) in cyanobacteria Synechocystis sp.PCC6803 is cloned, and the promoter is used for driving an enzyme gene to be detected;
s3: cloning of the enzyme genes: the gene related to carotenoid metabolism to be identified, lycopene epsilon-cyclase (lycopen epsilon-cyclase, AtLUT2) of Arabidopsis thaliana was cloned downstream of the rbcL promoter (rbcL:: AtLUT2), driven by the promoter;
s4 construction of the expression cassette: an entire gene expression cassette comprises a pAmp arranged in sequencer::KanrThe kit comprises a screening marker element and an rbcL for expressing an enzyme protein, wherein AtLUT2 is used for reserving a BamHi site in the process of constructing a gene expression cassette so as to facilitate the subsequent introduction of more genes;
s5: plasmid construction: introducing homologous arm sequences at two sides of an insertion site of a synechocystis genome through PCR (polymerase chain reaction) amplification at two ends of a constructed expression cassette, cloning an amplified fragment into a vector, selecting a gene coding carotene ketolase (CrtO) in the synechocystis genome, wherein the insertion site of the gene expression cassette is in the middle section of the gene, and the designed homologous arm sequences destroy the normal expression of the gene and can help to accumulate carotene;
s6: culturing the cyanobacteria strain: selecting a proper cyanobacteria strain, and culturing the cyanobacteria strain;
s7 transformation of cyanobacteria: the plasmid cloned in S5 is transformed into cyanobacteria by a natural transformation method; the transformed cyanobacteria contained 50. mu.g/ml-1Screening on BG-11 medium plates of kanamycin under the same other culture screening conditions as S6;
s8: and (3) pigment identification: when cyanobacteria were grown to OD in S7 liquid medium700When the concentration is 2.0, 5ml of the bacterial solution is collected by centrifugation, and the cells are extracted with methanolExtracting the accumulated carotenoid, and separating and identifying the pigment by using a high performance liquid chromatography.
In this example, in S6, BG-11 medium was used as the culture condition, and 30-35. mu. mol. photon. m was used at 28-30 ℃-2·s-1S7, the transformed cyanobacteria contained 50. mu.g.ml-1BG-11 medium plate screening of kanamycin, S7, selection of the transgenic strain obtained by screening, and screening of the strain containing 50. mu.g/ml-1Culturing and growing in BG-11 liquid culture medium of kanamycin; the growth condition is BG-11 culture medium, 28-30 deg.C, 30-35 μmol photon. m-2·s-1The algae strain with cloned lycopene epsilon-cyclase (AtLUT2) of arabidopsis thaliana is obtained in the embodiment, compared with the algae strain without transgenosis, alpha-carotene is identified by high performance liquid chromatography separation, and the synthesis of the alpha-carotene after the addition of the lycopene epsilon-cyclase is reasonable because the cyanobacteria can catalyze the beta-cyclization of lycopene, thereby proving that the system is sensitive and reliable.
EXAMPLE III
The embodiment provides a carotenoid metabolic gene function identification method, which comprises the following steps:
s1: cloning of the selectable marker elements: cloning of the plasmid from pGBK-T7 by AmprPromoter-driven kanamycin resistance gene KanrWith this element (pAmp)r::Kanr) As a subsequent screening marker;
s2: cloning of the promoter: the promoter sequence of a RuBisCO large subunit gene (rbcL) in cyanobacteria Synechocystis sp.PCC6803 is cloned, and the promoter is used for driving an enzyme gene to be detected;
s3: cloning of the enzyme genes: the gene related to carotenoid metabolism to be identified, carotene epsilon-hydroxylase (AtLUT1), of Arabidopsis thaliana was cloned downstream of AtLUT2 in example 3 by ribosome binding site rbs sequence in tandem (rbcL:: AtLUT2:: rbs:: AtLUT1), and two inserted enzyme genes were expressed by rbcL promoter binding rbs sequence;
s4 construction of the expression cassette: an entire gene expression cassette comprisesOne pAmpr::KanrScreening a marker element and an rbcL (expressed protein: AtLUT2: rbs: AtLUT1 element) for expressing an enzyme protein, wherein a BamHI site is reserved in the process of constructing a gene expression cassette, so that more genes can be conveniently introduced subsequently;
s5: plasmid construction: introducing homologous arm sequences at two sides of an insertion site of a synechocystis genome through PCR (polymerase chain reaction) amplification at two ends of a constructed expression cassette, cloning an amplified fragment into a vector, selecting a gene coding carotene ketolase (CrtO) in the synechocystis genome, wherein the insertion site of the gene expression cassette is in the middle section of the gene, and the designed homologous arm sequences destroy the normal expression of the gene and can help to accumulate carotene;
s6: culturing the cyanobacteria strain: selecting a proper cyanobacteria strain, and culturing the cyanobacteria strain;
s7 transformation of cyanobacteria: the plasmid cloned in S5 is transformed into cyanobacteria by a natural transformation method; the transformed cyanobacteria contained 50. mu.g/ml-1Screening on BG-11 medium plates of kanamycin under the same other culture screening conditions as S6;
s8: and (3) pigment identification: when cyanobacteria were grown to OD in S7 liquid medium700When the concentration is 2.0, 5ml of the bacterial solution is centrifuged to collect cells, the accumulated carotenoids are extracted with methanol, and the pigments are separated and identified by high performance liquid chromatography.
In this example, in S4, when two enzyme genes need to be expressed simultaneously, a ribosome binding sequence in a prokaryotic polycistron is inserted between the open reading frames of the two genes, so that the rbcL promoter can simultaneously drive the expression of the two enzyme genes at the downstream, and in S6, the culture conditions are BG-11 medium, 28-30 ℃, 30-35 mu mol photon m-2·s-1S7, the transformed cyanobacteria contained 50. mu.g.ml-1BG-11 medium plate screening of kanamycin, S7, selection of the transgenic strain obtained by screening, and screening of the strain containing 50. mu.g/ml-1Culturing and growing in BG-11 liquid culture medium of kanamycin; the growth condition is BG-11 culture medium, 28-30 deg.C, 30-35 μmol photon. m-2·s-1This example is based on example 2The carotene epsilon-hydroxylase (AtLUT1) of arabidopsis thaliana is further cloned, a ribosome binding sequence in a polycistron of a prokaryote is utilized to express the carotene epsilon-hydroxylase and AtLUT2 together, and synthesis of lutein is detected in the screened cyanobacteria through high performance liquid chromatography.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (1)
1. A method for identifying the function of a carotenoid metabolism gene is characterized by comprising the following steps:
s1: cloning of the selectable marker elements: cloning of the plasmid from pGBK-T7 by AmprPromoter-driven kanamycin resistance gene KanrWith this element (pAmp)r::Kanr) As a subsequent screening marker;
s2: cloning of the promoter: the promoter sequence of a RuBisCO large subunit gene (rbcL) in cyanobacteria Synechocystis sp.PCC6803 is cloned, and the promoter is used for driving an enzyme gene to be detected;
s3: cloning of the enzyme genes: the carotenoid metabolism-related enzyme gene X to be identified from different organisms is cloned downstream of the rbcL promoter (rbcL:: X), driven by this promoter;
s4 construction of the expression cassette: an entire gene expression cassette comprises a pAmp arranged in sequencer::KanrA selection marker element and an rbcL for expressing an enzyme protein;
s5: plasmid construction: at two ends of the constructed expression cassette, homologous arm sequences at two sides of the insertion site of the introduced synechocystis genome are amplified and introduced by a PCR technology, and the amplified fragments are cloned into a vector;
s6: culturing the cyanobacteria strain: selecting a proper cyanobacteria strain, and culturing the cyanobacteria strain;
s7 transformation of cyanobacteria: the plasmid cloned in S5 is transformed into cyanobacteria by a natural transformation method; the transformed cyanobacteria contained 50. mu.g/ml-1Screening on BG-11 medium plates of kanamycin under the same other culture screening conditions as S6;
s8: and (3) pigment identification: when cyanobacteria were grown to OD in S7 liquid medium700When the concentration is 2.0, 5ml of bacterial liquid is taken for centrifugal collection of cells, the carotenoid accumulated is extracted by methanol, and the pigment is separated and identified by high performance liquid chromatography;
in the S4, when two enzyme genes need to be expressed simultaneously, a ribosome binding site (rbs) in a polycistron of a prokaryote is inserted between open reading frames of the two genes, so that an rbcL promoter can drive the expression of the two enzyme genes at the downstream of the rbcL promoter simultaneously;
in the S5, the selected synechocystis genome insertion site is the middle end of a gene coding carotene ketolase CrtO, and the disruption of the normal expression of the gene is helpful for further simplifying the carotene metabolism in synechocystis and promoting the accumulation of beta-carotene;
in the S6, the culture condition is BG-11 culture medium at 28-30 deg.C and 30-35 μmol photon m-2·s-1；
In the S7, the transformed cyanobacteria contained 50. mu.g/ml-1Screening on BG-11 medium plate containing kanamycin, selecting the transgenic strain obtained by screening, and screening on BG-11 medium plate containing 50 mug/ml-1Culturing and growing in BG-11 liquid culture medium of kanamycin; the growth condition is BG-11 culture medium, 28-30 deg.C, 30-35 μmol photon. m-2·s-1。
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