Phoebe bournei PbbHLH30 gene, its encoded protein and application

Abstract

The present invention belongs to the field of plant molecular biology, and specifically relates to the PbbHLH30 gene of Phoebe fujianensis, the protein encoded by it and its application. The full length of the cDNA sequence of the gene is shown in SEQ ID No.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.2. Tobacco is transformed by Agrobacterium tumefaciens-mediated method to obtain overexpression tobacco plants. Molecular detection is performed on the tobacco with overexpression of the gene, anthocyanin extraction and expression analysis of the PbbHLH30 gene are performed on the tobacco with overexpression of the gene, and the results show that the anthocyanin content in the PbbHLH30 overexpression tobacco is significantly higher than the anthocyanin content in the leaves of the control tobacco.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology and relates to a Phoebe bournei PbbHLH30 gene, a protein encoded by the gene and an application thereof. Background Art

[0002] Phoebe bournei belongs to the genus Phoebe in the Lauraceae family. It is a rare native broad-leaved tree species in China and one of the original species of "golden silk nanmu". It has high ornamental value due to its diverse leaf colors and upright trunk. Phoebe bournei also has extremely high commercial value. Its wood is used to make noble buildings, furniture and sculptures due to its famous corrosion resistance and beautiful texture. Its extracts contain a variety of active substances, such as anthocyanins and lignans.

[0003] Studies have shown that anthocyanins not only provide plants with rich and diverse colors, but also reduce damage to plants from insects, pathogens, ultraviolet (UV) radiation and abiotic stress. There is also evidence that anthocyanins extracted from plants are beneficial to human health. As anticancer agents and antioxidants, they have strong antioxidant and anti-cancer capabilities, protect nerves, lower blood lipids and blood sugar, prevent a variety of chronic diseases and are used to treat related diseases.

[0004] The anti-cancer effect of anthocyanins has been widely studied by scientific researchers. Anthocyanins play an anti-cancer role by regulating the carbohydrate, lipid and protein metabolism of cells and inflammation, oxidative stress and apoptosis signaling pathways. For example, anthocyanins in grapes prevent tumor necrosis factor ɑ from inducing NF-κB activation by inhibiting IκBɑ phosphorylation, thereby resisting the invasion of human colon cancer cells in a dose-dependent manner. Anthocyanins can scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as superoxide anions (O 2 - ), peroxide radical (RCOO), hydrogen peroxide, hydroxyl radical (OH), and peroxynitrite anion (ONOO -) and so on. For example, anthocyanidins isolated from bilberry have a strong ability to scavenge superoxide anions and peroxynitrite anions. People with metabolic syndrome who take 320 mg of anthocyanidins per day can significantly inhibit the expression of pro-inflammatory factor genes related to the NF-κB pathway in the human body and enhance the expression of PPAR-γ genes to reduce the risk of inflammation. Cyanidin-3-O-glucoside extracted from raspberries can inhibit the production of pro-inflammatory factors IL-6, TNF-α, IL-1B, MCP-1 and Inos in mouse bone marrow macrophages induced by lipopolysaccharide. Other studies have shown that the intake of anthocyanidins can protect the central nervous system. Anthocyanidins can cross the blood-brain barrier to reach multiple functional areas of the brain, reduce the risk of brain dysfunction, and prevent cerebral ischemia and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Anthocyanidin extracts can also reduce the sugar content in serum and urine, and can prevent the generation of oxygen free radicals and reduce lipid oxidation.

[0005] Anthocyanins are common flavonoid compounds, and one of the core transcription factors regulating anthocyanin biosynthesis is bHLH protein, which can activate the expression of enzyme genes in the anthocyanin biosynthesis pathway. For example, a bHLH gene is highly expressed in black wolfberry, which may lead to anthocyanin accumulation in black wolfberry by upregulating the flavonoid-anthocyanin biosynthesis pathway. In addition, WD40 interacts with MYB and bHLH to enhance anthocyanin accumulation in bayberry. In Arabidopsis, the anthocyanin content of bHLH113 overexpressing plants was significantly higher than that of the wild type, especially in stems and branches during flowering. We previously found rich anthocyanins in red leaves of Phoebe fujianensis, which may be related to the highly expressed PbbHLH30 gene. We overexpressed the PbbHLH30 gene in tobacco for the first time to verify its biological function in anthocyanin accumulation and provide genetic resources for artificial regulation of anthocyanin content. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the Phoebe bournei PbbHLH30 gene, the protein encoded by the gene and the application thereof.

[0007] First, the present invention provides Phoebe bournei PbbHLH30 protein, which is:

[0008] 1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

[0009] 2) A protein derived from 1) with equivalent activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2.

[0010] The present invention also provides a gene encoding the Phoebe bournei PbbHLH30 protein.

[0011] Preferably, the sequence of the gene is shown as SEQ ID No.1.

[0012] The invention also provides an overexpression vector, a host cell and an engineering bacterium containing the gene.

[0013] The invention also provides the use of the gene in increasing the anthocyanin content accumulated in plants.

[0014] In a specific embodiment of the present invention, the gene is transferred into the plant genome and overexpressed in transgenic plants to increase the accumulation of anthocyanins in the plants.

[0015] The present invention also provides a method for increasing the accumulation of anthocyanins in plants, wherein a vector containing the gene is transformed into a plant genome by an Agrobacterium-mediated method to obtain a transgenic plant. The transgenic plant overexpresses the gene. The transgenic tobacco leaves accumulate a higher content of anthocyanins than the control leaves.

[0016] The present invention cloned the PbbHLH30 gene of Phoebe fujianensis, and transformed tobacco by Agrobacterium tumefaciens-mediated method to obtain tobacco with overexpressed gene, and performed molecular detection on it. The expression analysis of PbbHLH30 was performed on the overexpressed tobacco, and the results showed that PbbHLH30 was significantly highly expressed in transgenic tobacco. Anthocyanins of control tobacco and overexpressed tobacco were further extracted, and the absolute content of anthocyanins was detected by ultraviolet-visible spectrophotometer. The results showed that the anthocyanin content detected in the overexpressed (OE) tobacco was significantly higher than that in the wild-type (WT) and empty-load (EV) tobacco, and the contents were 0.156ng / g, 0.031ng / g and 0.029ng / g, respectively; in short, we overexpressed the PbbHLH30 gene of Phoebe fujianensis in tobacco for the first time and obtained a higher concentration of anthocyanins, that is, it is feasible to artificially regulate the synthesis of anthocyanins in plants, which has great application prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the total RNA of the leaves of Phoebe bournei of the present invention.

[0018] Figure 2 To PCR amplify the PbbHLH30 gene of Phoebe bournei.

[0019] Figure 3 PCR detection in Phoebe bournei PbbHLH30 gene overexpressing plants.

[0020] Figure 4 To identify the expression level of PbbHLH30 in positive strains (fluorescence quantitative PCR).

[0021] Figure 5 Analysis of anthocyanin content in leaves of tobacco overexpressing PbbHLH30. DETAILED DESCRIPTION

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] 1 Materials

[0026] 1.1 Experimental Materials

[0027] The Phoebe fujianensis materials were taken from the Experimental Forest Farm in Qingyuan County, Lishui City, Zhejiang Province.

[0028] The wild type of tobacco, Nicotiana benthamiana, was cultured in the growth room of the intelligent laboratory building of Zhejiang Agriculture and Forestry University under the growth conditions of 25°C and 16 h / d of light.

[0029] 1.2 Experimental reagents and instruments

[0030] High-fidelity DNA polymerase, various restriction endonucleases, markers, and DNA gel recovery kits were purchased from Takara Bioengineering (Dalian) Co., Ltd.; plasmid extraction kits and competent Escherichia coli were purchased from Beijing TransGen Biotech Co., Ltd.; the PCR instrument was a US PE9700 PCR instrument; and the clean bench was purchased from Suzhou Chengjing Purification Technology Co., Ltd.

[0031] 1.3 Primer synthesis and sequencing

[0032] Primer synthesis and sequencing were completed by Zhejiang Youkang Biotechnology Co., Ltd.

[0033] 2 Methods

[0034] 2.1 Extraction of total RNA from Phoebe bournei

[0035] M5 Plant RNeasy Complex Mini Kit was used to extract total RNA from Phoebe bournei. The steps are as follows:

[0036] (1) Take 1 ml of lysis buffer CLB into a centrifuge tube (if CLB has precipitation or precipitate, place it in a 65°C water bath to re-dissolve it), add 5% β-mercaptoethanol to the lysis buffer CLB (1 ml CLB plus 50 μl β-mercaptoethanol). Invert to mix and preheat in a 65°C water bath;

[0037] (2) Take about 0.1 g of the sample, put it into a mortar pre-cooled with liquid nitrogen, and grind it into a fine powder with liquid nitrogen;

[0038] (3) Transfer 100-200 mg of fine powder to a preheated lysis buffer CLB (with β-mercaptoethanol) centrifuge tube. Immediately vortex vigorously for 30-60 seconds or pipette and mix until a satisfactory homogenate is obtained;

[0039] (4) Return to the 65°C water bath for a short period of time (5-10 min), occasionally inverting 1-2 times to aid lysis;

[0040] (5) Centrifuge the lysate at 13,000 rpm for 10 min to precipitate the insoluble fragments;

[0041] (6) Take the supernatant of the lysate (you can take more supernatant as long as it does not exceed the capacity of the genomic DNA removal column, which can increase the yield) and transfer it to a new centrifuge tube. Add half the volume of the supernatant (0.5 volume) of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Mix it immediately by pipetting and do not centrifuge.

[0042] (7) Immediately proceed to step 3 of the procedure;

[0043] (8) Add the mixture (less than 720 μl each time, which can be added twice at most) to a genome cleanup column, centrifuge at 13,000 rpm for 2 minutes, and discard the waste liquid;

[0044] (9) Place the genomic DNA cleanup column in a clean 2 ml centrifuge tube, add 500 μl of lysis buffer RLT Plus to the genomic DNA cleanup column, centrifuge at 13,000 rpm for 30 seconds, collect the filtrate, use a micropipette to more accurately estimate the volume of the filtrate (usually about 450-500 μl, the volume lost during filtration should be subtracted), add 0.5 times the volume of anhydrous ethanol, and mix immediately by pipetting. Do not centrifuge;

[0045] (10) Immediately add the mixture (less than 720 μl each time, which can be added in two times) to an adsorption column RA (the adsorption column is placed in a collection tube) and centrifuge at 13,000 rpm for 2 minutes, and discard the waste liquid;

[0046] (11) Add 700 μl of deproteinized solution RW1, let stand at room temperature for 1 min, centrifuge at 13,000 rpm for 30 s, and discard the waste liquid;

[0047] (12) Add 500 μl of Rinse Buffer RW, centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid. Add 500 μl of Rinse Buffer RW, and repeat;

[0048] (13) Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 min to remove as much rinse solution as possible;

[0049] (14) Take out the adsorption column RA and place it in an RNase-free centrifuge tube. Add 30-50 μl RNase-free H2O to the middle part of the adsorption membrane according to the expected RNA yield (preheating in a 70°C water bath can increase the yield). Leave it at room temperature for 1 min and centrifuge at 12,000 rpm for 1 min.

[0050] (15) If the expected RNA yield is >30 μg, add 30-50 μl RNase-Free H2O and repeat step 9. Combine the two washes or use the first eluate and add it back to the column and repeat the step.

[0051] 2.2 Synthesis of the first strand of reverse transcribed cDNA

[0052] The first-strand cDNA was synthesized by reverse transcription of the total RNA according to the instructions of PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa).

[0053] (1) Prepare the following mixed solution for genome removal reaction:

[0054]

[0055] The reaction procedure was: 42°C, 2 min; 4°C, hold.

[0056] (2) Prepare the following mixture for reverse transcription reaction:

[0057]

[0058] The reaction procedure was: 37°C, 15 min; 85°C, 5 sec; 4°C, hold.

[0059] 2.3 Target gene cloning

[0060] 2.3.1 Gene cloning

[0061] Specific primers for PbbHLH30 were designed (Table 1), and the amplification system and procedure are shown in Table 2.

[0062] Table 1 Cloning primers of PbbHLH30

[0063]

[0064] Table 2 Cloning system

[0065]

[0066] Reaction program: 98°C, 30 sec, 52°C, 5 sec, 72°C, 1 min, 35 cycles; 72°C, 1 min; 16°C, hold.

[0067] 2.3.2 Target fragment recovery

[0068] 1% agarose gel was prepared and the PCR product was detected by agarose gel electrophoresis. If the bands were correct, the target fragments were excised and recovered according to the instructions of MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian).

[0069] (1) Prepare a sterilized 2 mL centrifuge tube and weigh the empty tube.

[0070] (2) Use a clean scalpel to cut out the gel block containing the target fragment under ultraviolet light, chop the gel block into pieces and place it in a 2 mL centrifuge tube, weigh and calculate the volume of the gel block (with 1 mg = 1 μL as the standard).

[0071] (3) Add 3 times the volume of the gel to the gel block and dissolve the gel block at room temperature, shaking intermittently to mix.

[0072] (3) When the gel is completely dissolved, add isopropanol to a final concentration of 20%.

[0073] (4) Add the solution from the previous step to the Spin Column adsorption column, place it on the Collection Tube, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0074] (5) Add 700 μL of Buffer WB to the adsorption column, incubate at room temperature at 12,000 rpm for 1 min, and discard the waste liquid;

[0075] (6) Repeat step (5).

[0076] (6) Idle: 12000 rpm, 1 min.

[0077] (7) Place the adsorption column in a sterilized 1.5 mL centrifuge tube, add 30 μL of sterile water (preheated to 65°C) to the adsorption membrane, let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, and store the collected solution at -20°C.

[0078] 2.3.3 Target fragment ligation and transformation

[0079] (1) Ligation: Use pEASY Blunt Zero vector (Transgene, -Blunt ZeroCloning Kit) and the target gene, mix the following solutions, mix gently, and centrifuge briefly. PCR: 25℃, 30min.

[0080]

[0081] (2) Transformation: Take Trans T1 (Transgene, Beijing) competent E. coli, melt it in an ice bath, add 2 μL of the above ligation product, place it in an ice bath for 30 min, heat shock it in a 42°C water bath for 30 s, then quickly and steadily transfer the centrifuge tube to an ice bath for 2 min, add 500 μL of LB medium without antibiotics, and culture it at 37°C at 200 rpm for 1 h. Incubate it at 4000 rpm for 2 min, remove part of the supernatant, keep 100 μL of the bacterial solution, mix the bacteria again by pipetting, spread it on a solid LB medium (containing 50 mg / L Kana), and culture it upside down at 37°C for 12 h.

[0082] (3) Bacterial test: Pick a single white colony on the plate, add 500 μL of liquid LB medium (containing 50 mg / L Kana), and culture at 37°C with a shaker at 200 rpm for 3-5 hours. Take 1 μL of bacterial solution as a template for PCR detection. The primers are gene cloning primers. The system and procedure are as follows.

[0083]

[0084]

[0085] The reaction program was: 94°C, 5 min; 94°C, 30 sec, 52°C, 30 sec, 72°C, 1 min, 35 cycles; 72°C, 5 min; 16°C, hold.

[0086] (4) Detect the PCR products of the bacterial solution by agarose gel electrophoresis, and select the positive clones and send them to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing.

[0087] (5) After the SnapGene sequencing results are correct, the plasmid is extracted using the Transgene EasyPure PlasmidMiniPrep Kit, and the obtained positive plasmid is used to construct an overexpression vector of the PbbHLH30 gene.

[0088] 2.4 Construction of overexpression vector using homologous recombination

[0089] according to II One Step Cloning Kit was used to construct the overexpression vector by homologous recombination.

[0090] (1) First, add the pK2W7-eYGFPuv-3xFLAG linker sequence to the target gene-specific primers, and use the positive plasmid obtained in the previous step as a template to prepare the following PCR system (20 μL):

[0091]

[0092] Reaction program: 98°C, 30 sec, 54°C, 5 sec, 72°C, 1 min, 35 cycles; 72°C, 5 min; 16°C, hold.

[0093] (2) After ensuring that the product is a single band, the target fragment was excised and recovered according to the instructions of MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian).

[0094] (3) The overexpression vector pK2W7-eYGFPuv-3xFLAG was linearized by double restriction digestion, and the following PCR system was configured using the pK2W7-eYGFPuv-3xFLAG plasmid as a template:

[0095]

[0096] Reaction procedure: 37°C, 2h; 16°C, hold.

[0097] (4) Refer to the instructions of MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) to excise the target fragment.

[0098] (5) Genetic transformation: The overexpression vector used was pK2W7-eYGFPuv-3xFLAG, and the overexpression vector of PbbHLH30 gene was constructed. The following mixed solution was prepared and incubated at 25°C overnight.

[0099]

[0100] The optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol) The optimal amount of insert fragment used = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0101] Reaction procedure: 37°C, 30 min; cool to 4°C or immediately cool on ice.

[0102] (2) Take 2 μL to transform Trans T1 (Transgene, Beijing) competent E. coli, and then pick a single clone for bacterial liquid PCR detection. The primers are gene cloning primers, and the PCR system is as follows:

[0103]

[0104]

[0105] The reaction program was: 94°C, 5 min; 94°C, 30 sec, 52°C, 30 sec, 72°C, 1 min, 35 cycles; 72°C, 5 min; 16°C, hold.

[0106] (3) PCR products were detected by agarose gel electrophoresis, and positive clones were selected after transformation into Trans T1 (Transgene, Beijing) competent Escherichia coli and sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. Plasmids of strains with correct sequencing results were extracted and set aside.

[0107] 2.5 Liquid nitrogen transformation of Agrobacterium

[0108] (1) Take out Agrobacterium GV1301 (Wei Di Biotechnology) stored at -80°C and thaw it on ice;

[0109] (2) Take 0.5 μg of expression vector plasmid and add it to the competent Agrobacterium. After flicking to mix, place it on ice, in liquid nitrogen, in a 37°C water bath, and in an ice-water mixture for 5 min each.

[0110] (3) Add 700 μL of fresh LB liquid medium without any resistance to the centrifuge tube, and place the bacterial liquid in a shaker (28°C, 200 rpm) for 4-6 h;

[0111] (4) Centrifuge (6000 rpm, 2 min), discard part of the supernatant, and make the remaining liquid volume in the tube about 70-100 μL, and pipette evenly;

[0112] (5) The bacterial solution was evenly spread on LB solid medium containing antibiotics (50 mg / L Kana and 50 mg / L Rif) and incubated in an inverted incubator at 28°C for 48-72 h;

[0113] (6) Pick a single clone on the plate for bacterial liquid PCR detection. The primers are gene cloning primers. The PCR system is as follows:

[0114]

[0115] The reaction program was: 94°C, 5 min; 94°C, 30 sec, 46°C, 30 sec, 72°C, 20 sec, 30 cycles; 72°C, 5 min; 16°C, hold.

[0116] (7) Add the same volume of 50% glycerol to the monoclonal bacterial suspension with positive test results and store it in a -80°C refrigerator.

[0117] 2.6 Agrobacterium-mediated transformation of tobacco

[0118] 2.6.1 Leaf disc method for tobacco transformation

[0119] Take a healthy tobacco leaf in a culture bottle, remove the main veins and uneven parts of the leaf edge, cut half a leaf into three or four small pieces, and place it on a pre-culture medium with the wound facing down for dark culture for two days. Prepare the Agrobacterium GV3101 bacterial solution containing the PbbHLH30 overexpression vector prepared in Example 1, and infect tobacco leaves using the leaf disc method, shaking at 28°C at 150rpm for 20min; discard the liquid in the clean bench, place the leaf on filter paper to absorb the surface moisture, and place it on a co-culture medium with the wound facing down for dark culture for three days. Place the leaf on the co-culture medium with the wound facing up on the screening culture medium, cut it off after the callus grows, and place it on a new screening culture medium for continued culture. After the seedlings grow out, cut them from the base and insert them into the rooting culture medium, and they will take root in about a week; clean the root culture medium, and then transplant it to soil culture, and pay attention to moisturizing for two weeks.

[0120] 2.6.2 Fluorescence quantitative PCR identification

[0121] Total RNA was extracted from transgenic tobacco leaves using the M5 Plant RNeasy Complex Mini Kit, and then reverse transcribed into cDNA using the PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa, Dalian, China).

[0122] ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for fluorescence quantitative PCR. The instrument used was Bio-Rad CFX-96. A 20 μL system mixture was prepared: 2×ChamQ SYBR qPCR Master Mix 10 μL, Primer-F 0.4 μL, Primer-R 0.4 μL, 50×ROX Reference Dye 1 0.4 μL, cDNA 1 μL and ddH 2 O7.8μL; the reaction program is: 95℃30s; 95℃10s, 60℃30s, 40 cycles; 65℃5s, 95℃5min (melting curve is set according to the instrument type). The primers used for fluorescence quantitative PCR are shown in Table 3, and U6 is selected as the internal reference gene.

[0123] Table 3 Fluorescence quantitative PCR primers

[0124]

[0125]

[0126] 2.7 Analysis of anthocyanin content in transgenic plants

[0127] Grind 0.5 g of leaves into 10 ml of methanol (containing 1% HCL) extract, shake well, cover with a lid to avoid light, and ultrasonicate for 1 hour. After centrifugation (5000 rpm, 10 minutes), the supernatant is used for anthocyanin content determination. Then, 2 ml of the supernatant is aspirated and mixed with 8 ml of potassium chlorate-hydrochloric acid buffer (pH 1.0) and sodium acetate-glacial acetic acid buffer (pH 4.5), respectively, and the mixture is incubated in a dark room at 40 degrees for 30 minutes. Use a UV-visible spectrophotometer (Thermo Scientific Co., Ltd, USA) to measure the absorbance of the supernatant of the mixture at 520 nm to measure the anthocyanin content, and measure the correction at 700 nm. All absorbance measurements were performed at room temperature, with distilled water as a blank control.

[0128] Obtain absorbance and calculate anthocyanin content according to the formula:

[0129] ΔA=(A 520 -A 700 )Ph4.5-(A 520 -A 700 )Ph1

[0130] Anthocyanin content (mg·100g -1 ) = ΔA·M·DF·V·100 / m·ε·LM: Molecular weight of cyanidin-3-glucoside, 449.2 g·moL -1 ;

[0131] DF: dilution factor; take 1ml to 10ml volumetric flask, DF = 10V: total volume of extract, mL;

[0132] m: weight, g;

[0133] ε: extinction coefficient of cyanidin-3-glucoside, 26 900 L·mol -1 cm -1 ;

[0134] L: optical path, 1 cm.

[0135] 3. Experimental results

[0136] 3.1 Total RNA extraction and analysis of Phoebe bournei leaves

[0137] M5 Plant RNeasy Complex Mini Kit was used to extract total RNA from Phoebe bournei ( Figure 1), and the OD260 / 280 ratio of the extracted RNA was determined by UV spectrophotometer to be between 1.8 and 2.1, indicating that the total RNA purity was good; the agarose gel electrophoresis results showed that the 18s and 28s bands of the RNA sample were very clear, which could be inferred that the RNA was not degraded and met the requirements of the next experiment.

[0138] 3.2 Cloning and vector construction of Phoebe bournei PbbHLH30 sequence

[0139] PbbHLH30 was amplified from Phoebe cDNA by high-fidelity PCR to obtain a 2127 bp fragment ( Figure 2 ), the amplified fragment was connected to the overexpression vector pK2W7-eYGFPuv-3xFLAG and transformed into Escherichia coli. After sequencing by Zhejiang Youkang Biotechnology Co., Ltd., the sequence was completely correct. One of the clones was selected to extract the plasmid and transformed into Agrobacterium, and then transformed into tobacco.

[0140] 3.3 Screening of transgenic tobacco positive strains

[0141] The transgenic plants were obtained after the PbbHLH30 gene of Phoebe bournei was transformed into tobacco and identified by PCR. Figure 3 ), and the expression level of PbbHLH30 in transgenic plants was detected by fluorescence quantitative PCR ( Figure 4 ), indicating that the expression level was 4.32-fold.

[0142] 3.4 Determination of anthocyanin content in transgenic tobacco

[0143] The anthocyanin content in the leaves of PbbHLH30 overexpressing plants was significantly higher than that in the wild type and empty vector ( Figure 5 ), suggesting that the structural genes of the plant anthocyanin biosynthesis pathway are regulated by PbbHLH30, which promotes the accumulation of tobacco anthocyanins. In summary, overexpression of the PbbHLH30 gene in Phoebe bournei can significantly increase the anthocyanin content in tobacco.

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of the gene encoding PbbHLH30 protein of Phoebe bournei in improving the accumulation of anthocyanins in plants, in, The amino acid sequence of the protein is shown in SEQ ID No.

2.

2. The use according to claim 1, It is characterized in that The gene is transferred into the plant genome and overexpressed in transgenic plants to increase the accumulation of anthocyanins in the plants.

3. The use according to claim 1 or 2, It is characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. A method for increasing anthocyanin accumulation in plants, comprising: transferring a vector containing a gene encoding PbbHLH30 protein of Phoebe bournei into the plant genome by Agrobacterium-mediated method, and screening to obtain transgenic plants. in, The amino acid sequence of the protein is shown in SEQ ID No.

2.

5. The method according to claim 4, It is characterized in that The transgenic plants overexpress the gene.

6. The method according to claim 4 or 5, It is characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.1.

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