A grape polyketide synthase and its application in achieving plant luminescence

Abstract

The present invention relates to a glucosamine synthase and its application in achieving plant luminescence. The glucosamine synthase can synthesize milkvine, and then synthesize luciferin through other proteins to make the plant luminescent. The results obtained by the present invention significantly improve the luminescence intensity of the plant, and through a mobile phone or camera lens, visible photos of luminous plants can be obtained without long-term exposure; the luminous phenotype of the plant can be easily observed with the naked eye.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology and relates to a grape polyketide synthase and an application thereof in realizing plant luminescence. Background Art

[0002] Plant luminescence technology originated from the observation and exploration of bioluminescence phenomena in nature. In the early days, it was done by exogenously adding fluorescent pigments and fluorescent proteins. This method requires a continuous supply of external chemicals, so its application was limited.

[0003] The main defects of the existing technology are: the luminous intensity of fruiting plants is low, and long exposure is required under the camera lens to obtain visible photos of luminous plants; under naked eye conditions, the human eye must adapt to dark conditions before observing the weak plant luminous phenotype.

[0004] In recent years, fungal luminescence systems have been shown to be more suitable for working in plant cell environments. Through the Agrobacterium-mediated method, the fungal luminescence gene cluster is introduced into the plant nuclear genome, and caffeic acid in the plant metabolic pathway is used to convert it into luciferin, allowing the plant to emit light autonomously. However, the key gene in the fungal luminescence system, the Hisps gene, has a large gene sequence, which limits its application. The applicant provides a polyketide synthase in grapes, which is only 1 / 3 the size of Hisps. After replacing Hisps in the fungal luminescence gene cluster gene with grape polyketide synthase, the luminescence effect is significantly enhanced.

[0005] The fungal luminescent gene cluster consists of: hispidin synthase, hispidin-3-hydroxylase, luciferase and caffeoylpyruvate hydrolase.

[0006] The process of catalyzing the formation of fluorescein using caffeic acid as substrate is as follows:

[0007] Step 1: Caffeic acid reacts with propionyl-CoA to form milk lysine

[0008] Catalytic enzyme: Hispidin Synthase (Hisps)

[0009] Reaction description: Caffeic acid combines with propionyl-CoA to generate hispidin under the catalysis of hispidin synthase.

[0010] Caffeic acid + 2-propionyl-CoA + 2ATP → milk echinopsine + 2CO 2 +H 2 O+2AMP

[0011] Step 2: Hydroxylation of milk tadine to form 3-hydroxy milk tadine

[0012] Catalytic enzyme: Hispidin Hydroxylase (H3H)

[0013] Reaction description: Milk tree alkaloid undergoes hydroxylation reaction under the action of milk tree alkaloid hydroxylase to generate 3-hydroxy milk tree alkaloid milk tree alkaloid + O 2 +NADPH+H + →3-Hydroxymilk echinopsin + H 2 O+NADP +

[0014] Step 3: Oxidation of 3-hydroxylactine to form caffeoylpyruvate

[0015] Catalytic enzyme: Luciferase (LUZ)

[0016] Reaction description: 3-Hydroxylactocin is oxidized to generate luciferin under the catalysis of luciferase, accompanied by luminescence.

[0017] 3-Hydroxymilk echinopsine +O 2 → Caffeoylpyruvic acid + CO 2 +Light

[0018] Through these three steps, caffeic acid and pyruvate are converted into caffeoylpyruvate through enzymatic reactions to achieve bioluminescence.

[0019] Step 4: Oxidation of 3-hydroxylactine to form caffeoylpyruvate

[0020] Catalytic enzyme: caffeoyl pyruvate hydrolase (CPH)

[0021] Reaction description: Caffeoylpyruvate generates caffeic acid under the catalysis of caffeoylpyruvate hydrolase, thereby realizing the regeneration of caffeic acid and achieving the purpose of cyclic luminescence.

[0022] Caffeoylpyruvate + H 2 O→pyruvate + caffeic acid. Summary of the invention

[0023] In view of this, the object of the present invention is to provide a glucone synthase and its application in achieving plant luminescence.

[0024] In order to achieve the above object, the present invention provides the following technical solutions:

[0025] 1. A grape polyketide synthase (VvPKS), the amino acid sequence of which is shown in SEQ ID NO.1.

[0026] As one of the preferred technical solutions, the 3D domain of the glucone synthase is as follows Figure 1 shown.

[0027] 2. A gene encoding the aforementioned gluconeol synthase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0028] 3. Use of the aforementioned grape polyketide synthase or gene in achieving plant luminescence.

[0029] As one of the preferred technical solutions, plant luminescence is achieved by synthesizing milk tree pine and then synthesizing luciferin.

[0030] The beneficial effects of the present invention are:

[0031] The grape polyketide synthase used in the present invention is derived from grape (Vitis vinifera L.) of the genus Vitis in the family Vitaceae.

[0032] Compared with Hisps in the prior art, the glucosidase synthase of the present invention can synthesize milkvine, and the activity of VvPKS in catalyzing the production of Hispidin is about 1.5 times that of Hisps, but the molecular weight is significantly lower than that of Hisps. Other genes of the fungal luminescent gene cluster are used to synthesize luciferin to make plants glow.

[0033] The results obtained by the present invention are that the luminous intensity of plants is significantly improved, and visible photos of luminous plants can be obtained through a mobile phone or camera lens without long-term exposure; the luminous phenotype of plants can be easily observed with the naked eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0035] Figure 1 The three-dimensional structure of gluconeol synthase.

[0036] Figure 2 These are photos of luminous plants, where A is the bright field and B is the dark field.

[0037] Figure 3 A comparison chart of Hispidin content and luminescence intensity, where A is the Hispidin content and B is the luminescence intensity.

[0038] Figure 4 is the gene structure, where A is VvPKS and B is Hisps.

[0039] Figure 5 This is the DNA map of the pCAMBIA2300 vector.

[0040] Figure 6 To construct a vector T-DNA map, A is a 2300-VvPKS-luminescent gene cluster vector, and B is a 2300-Hisps-luminescent gene cluster vector. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0042] Example

[0043] Construction of vector pCAMBIA2300 and experimental methods of plant transformation

[0044] 1. Gene cloning and vector construction

[0045] Gene synthesis and acquisition: The sequences of Luz gene (SEQ ID NO.3), H3H gene (SEQ ID NO.4), CPH gene (SEQ ID NO.5), and VvPKS encoding gene were obtained by gene synthesis (Beijing Qingke Biotechnology Co., Ltd.), and primers were designed to connect the genes to the pCAMBIA2300 vector (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., DNA map see Figure 5 , 2× high-fidelity PCR mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd. (Cat. No.: P520-01). PCR amplification: PCR amplification of genes was performed using high-fidelity polymerase (Table 1).

[0046] Table 1. Gene cloning reaction system

[0047]

[0048] After the PCR reaction, the DNA was excised and recovered according to the instructions of the Novagen gel recovery kit. The recovered product was sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing to determine the gene sequence. The primer sequences are shown in Table 2.

[0049] Table 2. Primer list

[0050]

[0051] Double digestion: pCAMBIA2300 vector was double digested with restriction endonucleases EcoRI and HindIII (the endonucleases were purchased from New England Biolabs). (Table 3)

[0052] Table 3. pCAMBIA2300 vector double restriction enzyme digestion

[0053]

[0054]

[0055] Ligation reaction: A seamless cloning kit (purchased from Beijing Biomed Gene Technology Co., Ltd.) was used to connect the amplified gene with the restriction-digested vector. Each gene carries a 35S promoter and a CaMV poly(A) terminator.

[0056] Vector transformation of Escherichia coli: The ligation product was transformed into competent Escherichia coli DH5α (purchased from Beijing Bomade Gene Technology Co., Ltd.) by heat shock method, and positive clones were screened on LB plates containing 50 mg / L Kan.

[0057] The synthesized VvPKS-luminescent gene cluster, the nucleotide sequence is shown in SEQ ID NO.7, and the gene structure is shown in Figure 4 As shown in A, each gene contains an enhanced CaMV 35S promoter and a CaMV poly A terminator. The promoter + gene + terminator is an expression frame of a gene. The applicant connects the expression frames of all genes required for luminescence in series, which is the sequence synthesized by the applicant. The constructed 2300-VvPKS-luminescent gene cluster vector T-DNA sequence is shown in SEQ ID NO.9, and the map is shown in Figure 6 As shown in A.

[0058] 2. Conversion and screening

[0059] E. coli culture: Select positive clones and culture them in liquid form using 10 ml of LB containing 50 mg / L kan.

[0060] Plasmid extraction: The recombinant plasmid was extracted using a plasmid extraction kit (the kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd.).

[0061] Plasmid storage: Freeze the plasmid in a refrigerator at -20℃ for future use.

[0062] 3. Bacterial testing and Agrobacterium transformation

[0063] Transformation of Agrobacterium: The plasmid was transformed into Agrobacterium GV3101 competent cells (purchased from Beijing Bomade Gene Technology Co., Ltd.) according to the instruction manual.

[0064] Screening of transformed Agrobacterium: The transformed Agrobacterium was screened on YEP plates containing antibiotics (50 mg / L Kan + 50 mg / L Gen + 25 mg / L Rif) (Kan, kanamycin, Gen, gentamicin, Rif, rifampicin, all purchased from Beijing Coolbo Technology Co., Ltd.).

[0065] Agrobacterium culture and suspension preparation: Select positive Agrobacterium clones for liquid culture, and adjust the concentration of the suspension to OD 0. 600 =0.6-0.8.

[0066] 4. Plant transformation

[0067] Preparation of plant materials: Select tobacco leaves suitable for transformation and cut them into 0.5 cm*0.5 cm size.

[0068] Leaf disc transformation: Soak plant leaf discs in Agrobacterium suspension at 28°C for 30 min and then transfer to co-cultivation medium (MS + 1 mg / L 6-BA + 30 g / L sucrose + 6 g / L agar powder + 100 μM acetosyringone, pH 5.8).

[0069] Co-cultivation and screening: Culture in the dark at 28°C on co-cultivation medium for 2 days, then transfer to selection medium (MS + 1 mg / L 6-BA + 30 g / L sucrose + 6 g / L agar powder + 100 mg / L Kan, pH 5.8) containing selective antibiotics.

[0070] Regeneration and rooting: After about 2-3 weeks, resistant shoots were selected and induced to root in rooting medium (MS + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L agar powder + 100 mg / L Kan, pH 5.8).

[0071] Transplantation and growth of tobacco: The rooted tobacco was transplanted into soil (peat soil: vermiculite: perlite = 5:3:1) and managed in a greenhouse with a light intensity of 6000Lx and 16h / day.

[0072] Transgenic identification: The transplanted tobacco was sampled and DNA was extracted using the CTAB method and PCR positive identification was performed. (Table 4)

[0073] Table 4. Transgenic identification

[0074]

[0075] Comparative Example

[0076] Gene synthesis and acquisition: The sequences of Luz gene (SEQ ID NO.3), H3H gene (SEQ ID NO.4), CPH gene (SEQ ID NO.5), and HIsps gene (SEQ ID NO.6) were obtained by gene synthesis, and primers were designed to connect the genes to the pCAMBIA2300 vector (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.). 2× high-fidelity PCR mix was purchased from Nanjing Novogene Biotechnology Co., Ltd. (Cat. No.: P520-01).

[0077] The synthesized Hisps-luminescent gene cluster has a nucleotide sequence as shown in SEQ ID NO.8 and a gene structure as shown in Figure 4 The constructed 2300-Hisps-luminescent gene cluster vector T-DNA sequence is shown in SEQ ID NO.10, and the map is shown in Figure 6 As shown in B.

[0078] The rest is the same as the embodiment.

[0079] Experimental Results

[0080] 1. Detection of milk tree alkaloid content (HPLC method)

[0081] Leaves of wild-type tobacco and transgenic plants of the embodiment and comparative example were collected and immediately frozen in liquid nitrogen.

[0082] Grind and freeze-dry approximately 1 g of frozen tissue into a 50 ml centrifuge tube, and store the freeze-dried material at -20° C. Each sample was prepared and analyzed three times.

[0083] About 50 mg of lyophilized powder was weighed, treated with 7 ml of 70% methanol aqueous solution in an ultrasonic bath for 30 minutes, and then centrifuged at 4,000 rpm for 10 minutes. The supernatant was collected, filtered with a Phenex GF / PVDF syringe filter (30 mm in diameter, 0.45 μm in pore size), and then analyzed on an LCMS instrument. The analysis was performed by a Shimadzu 8030 system consisting of HPLC coupled to a PDA and a triple quadrupole mass spectrometer (HPLC-DAD-ESI-TQ MS). Chromatographic separation was performed on a Discovery C18 column 4.6×150 mm, 5 μm in gradient mode with mobile phase components A (0.3% acetic acid aqueous solution) and B (acetonitrile). The gradient run was as follows: 0-4 minutes 10-40% B, 4-5 minutes 40-80%, 5-10.5 minutes, isocratic elution with 100% B, and then returned to the initial conditions. The column temperature was 40°C, the flow rate was 1 ml / min, and the injection volume was 20 μl.

[0084] Test results see Figure 3 Figure B: As can be seen from Figure A, the content of hispidin produced by VvPKS is much higher than that of Hisps, and the catalytic ability of VvPKS is about 1.5 times that of Hisps.

[0085] 2. Luminous intensity detection

[0086] Through the mobile phone or camera lens, you can obtain visible photos of luminous plants, and the luminous phenotype of plants can be easily observed with the naked eye. Figure 2 , where A is bright field and B is dark field (Iso 6400, aperture F1.2mm, exposure time 2s).

[0087] Use LightScout full-spectrum photon meter (Shanghai Zequan Technology) to align the detection port with the tobacco leaves to be tested (close to the tobacco leaves) in a dark environment, and detect the accumulated amount of photons at different times. The test results are shown in Figure 3 Middle B, Figure 2 B in.

[0088] As can be seen from the figure, the luminescence brightness of the previous luminescence system FBP using the HIsps gene is not as strong as that of the luminescence system using the VvPKS. The luminescence intensity after replacement is about 4 times that of the FBP system.

[0089] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. An application of a glucone synthase in achieving tobacco luminescence, characterized in that: The amino acid sequence of the gluconeol synthase is shown in SEQ ID NO.

1.

2. Application of a gene encoding glucosyl ketone synthase in achieving tobacco luminescence, characterized in that: The nucleotide sequence of the gene encoding glucone synthase is shown in SEQ ID NO.2.

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