A Wucai BcLUT5 gene and its vector, recombinant bacteria, cells and applications

Abstract

The present invention provides a Wucai BcLUT5 gene, the nucleotide sequence of which is shown in SEQ ID NO.1. The Wucai BcLUT5 gene promotes the yellowing of Wucai leaves by positively regulating carotenoid accumulation. The present invention also provides an expression vector, a recombinant bacterium, and a cell containing the BcLUT5 gene. At the same time, the present invention also provides an application of the BcLUT5 gene in cultivating a new Wucai yellowing variety and a specific method for promoting the yellowing of Wucai leaves. The present invention screened out a BcLUT5 gene that is positively correlated with carotenoid accumulation in Wucai, and further verified the role of BcLUT5 in carotenoid accumulation and promoting the yellowing of plants through transient expression in tobacco, laying a foundation for exploring the molecular mechanism of Wucai yellowing, while providing new gene resources for the creation of breeding materials, and also providing important scientific basis and germplasm materials for the breeding of Wucai varieties with different leaf colors.

Description

Technical Field

[0001] The present invention relates to the field of genetic breeding technology, and in particular to a Wucai BcLUT5 gene and its vector, recombinant bacteria, cells and applications. Background Art

[0002] During the aging process of most plant leaves, the leaf color changes from green to yellow. Studies have shown that the fading of green is due to the degradation of chlorophyll in the leaves, while the accumulation of carotenoids is the main cause of leaf yellowing.

[0003] Carotenoids are a general term for an important natural pigment. According to their chemical structures, carotenoids can be divided into two categories: carotene (containing only carbon and hydrogen) and xanthophylls (containing oxygen-containing functional groups such as hydroxyl, keto, carboxyl, and methoxy). There are two pathways for their synthesis: 2-C-methyl-D-erythritol 4-phosphate (2-C-Methyl-D-erythritol 4-phosphate, MEP) pathway and mevalonate pathway (MVA) pathway. Among them, the MEP pathway usually occurs in plant cell organelles and fungi. The MVA pathway is a metabolic pathway that uses acetyl-CoA as a raw material to produce mevalonate, and finally increases isoprenyl pyrophosphate and dimethylallyl pyrophosphate, which usually occurs in the cytosol of plant cells and bacteria. Carotenoids are the main source of vitamin A in the body, and also have antioxidant, immunomodulatory, anti-cancer, and anti-aging effects. The human body cannot synthesize carotenoids by itself and must be ingested from the outside.

[0004] Wucai (Brassica campesttris L.ssp.chinesis L.) is a subspecies of Chinese cabbage (B.campestris L.), which is native to China and mainly distributed in the Yangtze and Huaihe River basins. Wucai is a low-temperature resistant vegetable that can grow normally under growth conditions of around 4°C. It is rich in multiple vitamins and elements beneficial to the human body, and can enhance the body's immune and disease resistance. Among the existing Wucai, there are some Wucai varieties, such as the W7-2 variety, whose leaves are green at normal temperature, but the leaves will turn yellow after being exposed to low temperatures. After turning yellow, Wucai can still maintain normal growth and taste better.

[0005] Therefore, exploring the molecular mechanism of the yellowing of Wucai and providing a gene related to the accumulation of carotenoids in Wucai are the current research focuses. This will provide new genetic resources for the creation of breeding materials, and also provide important scientific basis and germplasm materials for the breeding of Wucai varieties with different leaf colors. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a BcLUT5 gene related to carotenoid accumulation in Wucai and its vector, recombinant bacteria, cells and application.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A Wucai BcLUT5 gene, the nucleotide sequence of the Wucai BcLUT5 gene is shown as SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the Wucai BcLUT5 gene promotes the yellowing of Wucai leaves by positively regulating carotenoid accumulation.

[0010] An overexpression vector contains the above-mentioned Wucai BcLUT5 gene.

[0011] As one of the preferred embodiments of the present invention, the overexpression vector is pCAMBIA2300-BcLUT5.

[0012] A recombinant bacterium contains the above-mentioned Wucai BcLUT5 gene.

[0013] As one of the preferred embodiments of the present invention, the overexpression vector pCAMBIA2300-BcLUT5 constructed by the Wucai BcLUT5 gene is introduced into the bacterial cell to obtain the product.

[0014] A genetically engineered host cell, wherein the host cell contains the overexpression vector pCAMBIA2300-BcLUT5 constructed by the above-mentioned Wucai BcLUT5 gene.

[0015] An application of the above-mentioned Wucai BcLUT5 gene in cultivating a new Wucai yellowing variety.

[0016] A method for promoting yellowing of Wucai leaves, comprising overexpressing a BcLUT5 gene in Wucai by a genetic engineering method; the nucleotide sequence of the Wucai BcLUT5 gene is shown in SEQ ID NO.1, and promoting yellowing of Wucai leaves by positively regulating the accumulation of carotenoids in Wucai.

[0017] The advantages of the present invention over the prior art are as follows: the present invention screened out the BcLUT5 gene which is positively correlated with carotenoid accumulation in wucai, and further verified the role of BcLUT5 in carotenoid accumulation and promoting yellowing of plants through transient expression in tobacco, which laid a foundation for exploring the molecular mechanism of yellowing of wucai, provided new gene resources for the creation of breeding materials, and provided important scientific basis and germplasm materials for the breeding of wucai varieties with different leaf colors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1It is the transient expression analysis and subcellular localization map of BcLUT5 in Example 5 (in the figure, Figure A is the color change map of the part of the leaf overexpressing BcLUT5, and Figure B is the subcellular localization map);

[0019] Figure 2 It is a color difference value diagram and a zeaxanthin content diagram after transient expression of BcLUT5 in Example 5 (in the figure, Figure A is a color difference value diagram of L*, a*, and b* after transient expression of BcLUT5, and Figure B is a zeaxanthin content diagram). DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described in detail below. The embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagents, kits and other materials used in the following embodiments are all commercially available products conventional in the art unless otherwise specified. The experimental methods used in the following embodiments are conventional methods in the art unless otherwise specified, and will not be repeated.

[0021] Example 1

[0022] Wucai BcLUT5 gene:

[0023] The present invention screened out the BcLUT5 gene positively correlated with carotenoid accumulation in Wucai, and the nucleotide sequence was shown as SEQ ID NO.1.

[0024] Example 2

[0025] Cloning and purification of BcLUT5 gene from Wucai:

[0026] 1. Wucai W7-2 was selected as the material. W7-2 gradually experienced low temperatures in autumn and winter during its growth period, and its leaves turned yellow. W7-2 seedlings were planted in a greenhouse at 26±2℃ (daytime) and 20±2℃ (nighttime) with a light intensity of 300μmol m -2 s -1 , relative humidity 70-80%. Seedlings with 7-8 leaves were transplanted to 6±2℃ (daytime) and -1±2℃ (nighttime), 300μmol / m -2 s -1 The cells were cultured in a low-temperature growth room with a light intensity of 300 μmol m -2 s -1 , relative humidity 70-80% for 12 days. After transfer to the growth chamber, collect the third fully expanded young leaf from the center every 3 days.

[0027] 2. Total RNA extraction and cDNA synthesis were performed on the plant leaves at the three stages before yellowing (G), after yellowing (Y) and after regreening (RG). The specific method is as follows:

[0028] The RNA of samples from three periods was extracted using the TakaRa RNA kit. After quantification, the RNA was reverse transcribed using the TRAN reverse transcription kit. The system is shown in Table 1.

[0029] Table 1 Reverse transcription system

[0030]

[0031] 3. The DNA obtained by reverse transcription of plant leaves in the above steps before yellowing (G), after yellowing (Y) and after regreening (RG) is used as the Wucai genome, and the nucleotide sequence of the BcLUT5 gene is obtained in the Wucai genome, as shown in SEQ ID NO.1.

[0032] Homologous arm primers (primer F: SEQ ID NO.2; primer R: SEQ ID NO.3) were designed according to the gene sequence and pCAMBIA2300 vector sequence using CE-Design software and sent to Universal Company for synthesis of primer sequences.

[0033] Samples were added according to the system in Table 2, and the PCR program was 95°C for 3 min, 95°C for 15 s, 60°C for 15 s, 72°C for 45 s, and 72°C for 10 min. After the reaction, the PCR product was identified by 1% agarose gel electrophoresis. The target DNA fragment was recovered using the TIANGEN agarose gel DNA recovery kit.

[0034] Table 2 PCR amplification system

[0035]

[0036] Example 3

[0037] Construction of overexpression vector pCAMBIA2300-BcLUT5 and transformation of E. coli:

[0038] Add samples according to the system in Table 3, gently pipette to mix, connect at 37℃ for 30min, and then quickly put on ice. Add 10μL of the connection product pCAMBIA2300-BcLUT5 to 100μL of E. coli, gently pipette to mix, let stand on ice for 5min, heat shock at 42℃ for 45-60s, quickly transfer to ice, and let stand for 2min. Then add 700μL of LB without antibiotics to the centrifuge tube, mix well, and resuscitate at 37℃ 200rpm for 20min, take 100μL of the resuscitation solution and evenly spread it on the culture medium containing 100mg / mL Kan antibiotics, and invert and culture overnight at 37℃ incubator. The next day, pick 8 single colonies in a centrifuge tube with 500μL LB containing 100mg / mL Kan, and shake at 37℃ for 5h until the bacterial solution is turbid.

[0039] Table 3 Vector connection system

[0040] Components volume pCAMBIA2300 4μL Target gene 1μL 5×CE Buffer 2μL Exnase II 1μL <![CDATA[dd H2O]]> Up to 10μL

[0041] Take the bacterial solution obtained in the previous step, use the gene upstream primer F: SEQ ID NO.2 and the vector downstream primer R: SEQ ID NO.4 to add samples according to the system in Table 4, and the PCR program is 95℃5min, 95℃15s, 60℃15s, 72℃45s, 72℃5min. After the reaction, the PCR product is identified by 1% agarose gel electrophoresis and sent to Universal Company for sequencing. If the alignment is successful, the gene is successfully connected to the vector. Return the bacterial solution and plasmid, mix the bacterial solution with glycerol in a ratio of 1:1, freeze it with liquid nitrogen, and store it at -80℃.

[0042] Table 4 Bacterial liquid PCR system

[0043] Components volume Taq enzyme 5μL Bacterial liquid 1μL Upstream primer F 0.2μL Downstream primer R 0.2μL <![CDATA[dd H2O]]> Up to 10μL

[0044] Example 4

[0045] Agrobacterium transformation:

[0046] Take 100μL Agrobacterium GV3101 competent cells and add 5μL pCAMBIA2300-BcLUT5 plasmid, pipette and mix, place on ice, liquid nitrogen, 37℃ water bath, and ice for 5min each, add 700μL LB without antibiotics, and shake at 28℃ for 3h. After centrifugation at 6000rpm for 1min, collect the bacteria, take 100μL supernatant, gently pipette and resuspend the bacteria, and spread it on LB plates containing 100mg / mL Kan and 25mg / mL rif, then invert and culture in a 28℃ incubator for 2 days. Pick 8 single colonies in a 1.5mL centrifuge tube containing "100mg / mL Kan, 2mg / mL rif, 500μL LB", and shake at 28℃ for 24h until the bacterial solution is turbid. Samples were added according to the system in Table 4, and the PCR program was 95°C for 3 min, 95°C for 15 s, 60°C for 15 s, 72°C for 45 s, and 72°C for 10 min. After the reaction, the PCR product was identified as qualified by 1% agarose gel electrophoresis, and then mixed with bacterial solution and glycerol in a ratio of 1:1, frozen with liquid nitrogen, and stored at -80°C.

[0047] Example 5

[0048] Tobacco transient expression analysis and subcellular localization of BcLUT5 gene:

[0049] 1. Experimental Methods

[0050] pCAMBIA2300-BcLUT5 and pCAMBIA2300 Agrobacterium (pCAMBIA2300-Empty as a control) were shaken overnight at 28°C until the OD value of the bacterial solution reached 0.8-1.0. Centrifuge at 4000rpm for 8min, remove the supernatant, suspend with 3mL of ddH2O, centrifuge at 4000rpm for 8min, suspend with Activation buffer (MES and MgCl2 are mixed in a ratio of 5:1), and centrifuge at 4000rpm for 8min. Using Activation buffer as a control, measure the OD value of the bacterial solution, and adjust the OD value of the bacterial solution to 0.8-1.0 with Activation buffer. Add AS (acetosyringone) to the bacterial solution at a ratio of bacterial solution: AS = 1000:1. After static culture at 28°C in the dark for 3h, inject Nicotiana benthamiana. After injection, treat in the dark for 1 day, culture under light for about 2 days, and observe the localization results with laser confocal microscopy. In addition, the leaf phenotypes after injection of tobacco were observed, and the leaf color difference values ​​and total chlorophyll content were measured.

[0051] 1. Color difference measurement

[0052] After planting, the leaf chromaticity value was measured on the upper surface of the leaf every 3 days using a colorimeter (DS-700D). Six plants were measured and the measurement was repeated 3 times at the same leaf position. The CIELAB color coordinate system was used for color data analysis. Among them, the color difference value has three representative color values: L*, a*, and b*; L* represents brightness (black and white), a* represents red and green, and b* represents yellow and blue.

[0053] 2. Determination of zeaxanthin (carotenoid) content

[0054] The results were determined by enzyme-linked immunosorbent assay (ELISA kit).

[0055] (1) Preparation: Grind fresh leaves into powder for sample preparation.

[0056] (2) Sample addition: Set up standard wells, blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), and wells for the samples to be tested. Add 50 μL to the standard wells on the enzyme-labeled plate, add 40 μL of sample diluent to the wells for the samples to be tested, and then add 10 μL of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well on the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0057] (3) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0058] (4) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 min.

[0059] (5) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water and set aside.

[0060] (6) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard. Repeat this process 5 times and pat dry.

[0061] (7) Color development: Add 50uL of color developer A to each well, then add 50uL of color developer B, gently shake to mix, and incubate at 37℃ in the dark for 15 min.

[0062] (8) Termination: Add 50uL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0063] (9) Measurement: Use the blank well as the zero setting and measure the absorbance (OD) value of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0064] (10) Calculation: Draw a standard curve on coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration of the sample from the standard curve based on the OD value of the sample; then multiply it by the dilution factor; or use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to obtain the actual concentration of the sample.

[0065] 2. Experimental Results

[0066] The results are as follows Figure 1 , 2 , as shown in Table 5.

[0067] Table 5 Color difference value and total chlorophyll content results

[0068] L*Value a*Value b*Value Control 45.31 -11.56 31.74 pCAMBIA2300-BcLUT5 47.31 -11.65 35.25

[0069] By transiently expressing BcLUT5 in Nicotiana benthamiana, it was found that compared with the control, Nicotiana benthamiana overexpressing BcLUT5 showed yellowing phenomenon ( Figure 1 A); color difference values ​​and total chlorophyll content are shown in Table 5 and Figure 2 As shown, compared with the control, the L*Value value increased by 4.41%, the b*Value value increased by 9.95%, and the a*Value value did not change significantly; the subcellular localization results showed that BcLUT5 was localized in chloroplasts ( Figure 1 B).

[0070] In summary, the present invention screened out the BcLUT5 gene, which is positively correlated with carotenoid accumulation, in Wucai, and further verified the role of BcLUT5 in carotenoid accumulation and promoting yellowing of plants through transient expression in tobacco, laying a foundation for exploring the molecular mechanism of yellowing of Wucai, providing new gene resources for the creation of breeding materials, and providing important scientific basis and germplasm materials for the breeding of Wucai varieties with different leaf colors.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A kind of black vegetable BcLUT5 The application of the gene in breeding new yellowing varieties of Wucai is characterized by: Wucai BcLUT5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. By overexpressing the gene, the accumulation of zeaxanthin in Wucai is positively regulated, and the yellowing of Wucai leaves is promoted.

2. A method for promoting yellowing of Wucai leaves, characterized in that: Through genetic engineering, BcLUT5 Gene overexpression; BcLUT5 The nucleotide sequence of the gene is shown in SEQ ID NO.1, which positively regulates the accumulation of zeaxanthin in Wucai and promotes the yellowing of Wucai leaves.

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