CN114686501A - Prokaryotic expression and application of gardenia phytoene synthase gene (GjPSY) - Google Patents
Prokaryotic expression and application of gardenia phytoene synthase gene (GjPSY) Download PDFInfo
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- CN114686501A CN114686501A CN202210478959.0A CN202210478959A CN114686501A CN 114686501 A CN114686501 A CN 114686501A CN 202210478959 A CN202210478959 A CN 202210478959A CN 114686501 A CN114686501 A CN 114686501A
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Abstract
The invention discloses a construction method of a prokaryotic expression vector of a gardenia phytoene synthase gene (GjPSY), and also provides an optimal induced expression condition for prokaryotic expression of the gardenia phytoene synthase gene. The invention lays a foundation for the subsequent research on the function of the gene of the gardenia phytoene synthase and the biosynthesis regulation of the gardenia yellow pigment compounds.
Description
Technical Field
The invention belongs to the technical field of molecular breeding, and provides construction of a prokaryotic expression vector of a gardenia phytoene synthase GjPSY gene, and also provides an optimal prokaryotic induced expression condition of the gardenia phytoene synthase gene.
Background
Gardenia is taken as a traditional Chinese medicine, is the first medical and edible dual-purpose resource issued by Ministry of health, and has the effects of clearing heat, promoting urination, cooling blood, detoxifying and the like. Modern researches find that gardenia contains iridoid (jasminoidin), flavonoid (gardenia), triterpenes (gardenia acids), organic acid esters (chlorogenic acid and crocetin), volatile oil and other components. The gardenia fruit has high nutritional and medicinal values, and not only contains rich gardenia yellow pigment, geniposide, gardenia oil, organic acid and flavonoid compounds, but also is rich in microelements such as iron, silicon, manganese, boron, copper and the like. As a common traditional Chinese medicine, gardenia plants have wide research prospects in terms of chemical components and pharmacological action. Modern pharmacological research shows that cape jasmine has the functions of diminishing inflammation, clearing away heat, relieving pain, protecting liver, benefiting gallbladder, lowering blood fat, resisting thrombus, protecting nerve, tranquilizing, hypnotizing, etc. The application of gardenia nowadays is mainly divided into two aspects: in medicine, gardenia fruits are used for Chinese patent medicine production, preparation of traditional Chinese medicines, deep processing and extraction of effective components (such as geniposide, ursolic acid and the like) in the gardenia fruits to serve as medicine intermediates, experimental reagents and the like; in food, the method is mainly used for extracting gardenia yellow pigment or extracting effective components to synthesize gardenia red pigment, gardenia blue pigment and the like. With the research on gardenia, more and more new pharmacological effects can be discovered. Therefore, gardenia still has development value and is worthy of further intensive research.
The gardenia yellow pigment is a natural edible yellow pigment which has high safety, strong tinting strength, lower cost, internationally allowable production and use, has no toxic or side effect, is easy to dissolve in polar solvents such as water, ethanol and the like, and is difficult to dissolve in non-polar solvents such as benzene, gasoline and the like. The gardenia yellow pigment has strong thermal stability and good stability below 80 ℃. The gardenia yellow pigment is a mixture comprising crocin, crocetin, chlorogenic acid, flavone, geniposide and the like. Research shows that the gardenia yellow pigment has the effects of promoting bile secretion, enhancing the detoxifying function of the liver and the like, and can reduce the content of bilirubin and cholesterol in blood. Gardenia jasminoides ellis, as a traditional Chinese medicinal material, has currently occupied an important position in the production of natural edible pigments (gardenia yellow pigment and gardenia blue pigment). In recent years, the sales increase rate of natural edible pigments in the international market is kept above 10% every year, and the natural edible pigments gradually occupy the market share of synthetic pigments, so that the market prospect is very good.
Biosynthesis of gardenia yellow pigment requires a high coordination of various pathways, including the upstream methylerythritol phosphate (MEP) pathway, the midstream carotenoid biosynthesis pathway, and the downstream crocin biosynthesis pathway. Phytoene Synthase (PSY) is the first rate-limiting enzyme in the synthesis process of carotenoids, catalyzes 2 molecules of geranylgeranyl pyrophosphate (GGPP) to condense to form phytoene, and the phytoene is catalyzed by Phytoene Dehydrogenase (PDS) to form zeta-carotene and then continuously catalyzed by zeta-carotene dehydrogenase (ZDS) to form lycopene. Although there are many phytoene synthase genes isolated from various plants, animals and microorganisms at present, no one at home has disclosed or published the construction method of the prokaryotic expression vector (pQE30) of gardenia phytoene synthase GjPSY and its induction expression conditions before the application of the present invention.
Disclosure of Invention
1. The invention provides a construction method of a prokaryotic expression vector of a gardenia phytoene synthase gene (GjPSY). The method comprises the following steps: screening GjPSY gene according to data of gardenia transcriptome, and designing a specific primer; secondly, taking gardenia fruits as a material to extract RNA and carrying out reverse transcription to obtain cDNA; cloning core cDNA sequence of GjPSY gene, transferring into Escherichia coli DH5 alpha after connecting pQE30 vector, selecting positive clone and sequencing;
2. the invention provides an optimal prokaryotic expression condition of a gardenia phytoene synthase gene GjPSY. The method comprises the following steps: firstly, transferring GjPSY-pQE30 into M15; designing an IPTG inducible expression system and carrying out inducible expression; thirdly, carrying out electrophoresis; and fourthly, determining the optimal prokaryotic induction expression condition of the GjPSY according to the electrophoresis result.
Drawings
FIG. 1 is an electrophoresis diagram of PCR amplification of a core cDNA fragment of a gardenia phytoene synthase (GjPSY) gene, wherein M is DL2000 Marker, and Lane 1 is the PCR product of the core cDNA fragment of the GjPSY gene.
FIG. 2 is an electrophoresis diagram of the prokaryotic expression protein of the GjPSY gene.
Table 1 shows the optimal expression conditions for prokaryotic expression of the GjPSY gene.
Detailed description of the preferred embodiments
1. Experimental materials: the research respectively adopts roots, stems, leaves and fruits of a gardenia jasminoides cultivar Linhai I in different growth periods, namely a young fruit period (20 days after blooming), a young fruit period (54 days after blooming), a yellow fruit period (87 days after blooming), a red fruit period (129 days after blooming) and a mature period (155 days after blooming), and the roots, the stems, the leaves and the fruits are quickly frozen by liquid nitrogen and stored at the temperature of minus 80 ℃ for later use.
2. Total RNAs of roots, stems, leaves and fruits of gardenia jasminoides in 3 growth stages were extracted using RNAprep Pure plantarplus Kit of TIANGEN, respectively, and first strands of cDNAs were synthesized using PrimeScript II 1st Strand cDNA Synthesis Kit of TAKARA, and the concentrations thereof were measured and stored at-20 ℃ for gene cloning and RT-qPCR.
3. A candidate gene sequence of gardenia phytoene synthase is obtained according to the analysis of the data of the transcription group of gardenia 'Linhai No. I', and primers (P1: 5'-AGTTATCTTCGTTCCGCC-3'; P2: 5'-GCTCTGCTATGCCCTTCT-3') are designed according to the sequence.
4. And performing PCR amplification by using cDNA reverse transcription of total RNA of gardenia fruits as a template. The PCR amplification reaction system is as follows:
the reaction program of the core fragment PCR amplification reaction of the gardenia phytoene synthase gene GjPSY is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 57 ℃ for 30s, and 72 ℃ for 2 min; 5min at 72 ℃.
5. The PCR product was an approximately 1400bp fragment, which was ligated with pM18-T vector and sequenced by Botanhuan Biotech (Shanghai) Ltd.
6. The primers (P3: CGGGGTACCATGTCTTGCAGAAATATC; P4: AAGCTTGTCTTACTCTTCACCAACATCCCT) for adding enzyme cutting sites for connecting pQE30 are designed, and pCR amplification is carried out by taking GjPSY-pM18-T as a template.
7. Recovering PCR product (GjPSY (pQE30)) from gel, connecting with pM18-T vector, and transforming into Escherichia coli DH5 alpha; and (5) selecting a single clone to extract plasmids.
8. GjPSY (pQE30) -pM18-T and pQE30 were digested with KpnI and HindIII, and the digested products were recovered as a gel.
9. The recovered gel products GjPSY (pQE30) and pQE30 were ligated and transformed into E.coli M15.
10. Selecting monoclonal shake bacteria, upgrading the quality particles and sequencing to obtain M15 strain transformed into GjPSY-pQE 30.
11. The GjPSY-pQE30 induction expression conditions were designed (Table 1).
TABLE 1 IPTG inducible expression System
12. Collecting the bacteria cultured under different inducing conditions, and performing protein gel electrophoresis.
13. And determining the optimal induction expression condition of GjPSY-pQE30 according to the protein electrophoresis result. The optimal prokaryotic induction expression conditions of GjPSY are as follows: IPTG concentration of 0.8mmol/L, temperature of 28 ℃ and bacteria shaking time of 4 h.
Claims (2)
1. A method for constructing a prokaryotic expression vector of a gardenia phytoene synthase gene (GjPSY) is characterized in that a core cDNA sequence of the GjPSY gene is screened and cloned according to gardenia transcriptome data, and the fragment is connected with a pQE30 vector to construct a prokaryotic expression vector GjPSY-pQE 30.
2. An optimal prokaryotic expression condition of a gardenia phytoene synthase gene GjPSY is characterized in that: the GjPSY-pQE30 vector is expressed in Escherichia coli M15; the optimal induction expression conditions are as follows: IPTG concentration of 0.8mmol/L, temperature of 28 ℃ and bacteria shaking time of 4 h.
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Non-Patent Citations (4)
| Title |
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| 李宁;王柏柯;杨生保;唐亚萍;王强;杨涛;帕提古丽;余庆辉;: "21种植物八氢番茄红素合成酶的生物信息学分析", 新疆农业科学, no. 12, pages 2157 - 2165 * |
| 陈建荣;毛凯权;陈果;刘芷翾;邓文月;刘芳;: "栀子(Gardenia jasminoides)GGPPS基因小亚基的克隆及表达分析", 分子植物育种, no. 10, pages 3199 - 3206 * |
| 高蓝等: "栀子GjPSY基因的克隆及原核表达", 《生物技术》, vol. 23, no. 3, pages 1 - 1 * |
| 高蓝等: "栀子八氢番茄红素合成酶基因的分离及表达分析", 《热带亚热带植物学报》, vol. 21, no. 5, pages 439 * |
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