CN116199759A

CN116199759A - Phoebe bournei PbMYB201 gene, coded protein and application thereof

Info

Publication number: CN116199759A
Application number: CN202310200866.6A
Authority: CN
Inventors: 张俊红; 王立; 付宁宁; 张毓婷; 童再康
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2023-02-22
Filing date: 2023-02-22
Publication date: 2023-06-02

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to a phoebe bournei PbMYB201 gene, a coded protein and application thereof. The full length of cDNA sequence of the gene is shown as SEQ ID No.1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2. And transforming the arabidopsis through an agrobacterium tumefaciens mediated method to obtain an overexpression arabidopsis plant. The result shows that the stem and leaf of the PbMYB201 over-expressed Arabidopsis thaliana has obviously red color compared with the stem and leaf of the Arabidopsis thaliana of the control group. And molecular detection is carried out on the arabidopsis thaliana, anthocyanin extraction and PbMYB201 gene expression analysis are carried out on the arabidopsis thaliana with over-expressed genes, and the result shows that the anthocyanin content in the leaf of the control arabidopsis thaliana is obviously lower than that in the arabidopsis thaliana with over-expressed PbMYB 201.

Description

Phoebe bournei PbMYB201 gene, coded protein and application thereof

Technical Field

The invention belongs to the technical field of plant biology, and relates to a phoebe bournei PbMYB201 gene, a coded protein and application thereof.

Background

Phoebe bournei belongs to Lauraceae genus plant, is a natural rare broad-leaved tree species with Chinese character, is one of original plants of golden silk Phoebe, and has high ornamental value due to diversity of leaf colors and upright trunks. Minnan also has extremely high commercial value, its wood being used for the production of expensive buildings, furniture and sculptures due to its remarkable durability and beautiful texture, and its extract containing various active substances such as anthocyanins and lignans, etc.

It has been shown that anthocyanins not only provide plants with a rich variety of colors, but also reduce the damage of plants from insects, pathogens, ultraviolet (UV) radiation and abiotic stress. There is also evidence that anthocyanin extracted from plants is good for human health, has strong antioxidant and anticancer ability, protects nerves, reduces blood lipid and blood sugar, prevents various chronic diseases and is used for treating related diseases as an anticancer agent and an antioxidant.

The anticancer effect of anthocyanin has been widely studied by researchers, and anthocyanin plays an anticancer role by regulating carbohydrate, lipid and protein metabolism, inflammation, oxidative stress, apoptosis signaling pathway and the like of cells. For example, anthocyanin in grape prevents tumor necrosis factor alpha from inducing NF- κB to be activated by inhibiting IκBα phosphorylation, and resists invasion of human colon cancer cells in a metering dependent manner. The anthocyanin can scavenge Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), such as superoxide anion (O) ₂ ^- ) Peroxide Radical (RCOO), hydrogen peroxide, hydroxyl radical (OH) and peroxynitrite anion (ONOO) ^- ) Etc. For example, anthocyanin isolated from cowberry fruit has strong scavenging effect on superoxide anionThe ability of ions and peroxynitrite anions. The ingestion of 320mg anthocyanin per day by a human suffering from metabolic syndrome can significantly inhibit the expression of NF- κB pathway-related pro-inflammatory factor genes and enhance PPAR- γ gene expression in the human to reduce the risk of inflammation. procyanidin-3-O-glucoside extracted from raspberry can inhibit lipopolysaccharide from inducing the production of proinflammatory factors IL-6, TNF-alpha, IL-1B, MCP-1 and Inos of mouse bone marrow macrophages. Also, it has been shown that ingestion of anthocyanin can protect the central nervous system, and anthocyanin can cross the blood brain barrier to reach multiple functional areas of the brain, reduce risk of brain dysfunction, and prevent cerebral ischemia and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Anthocyanin extract also can reduce sugar content in serum and urine, and can prevent oxygen free radical generation and reduce lipid oxidation.

Anthocyanin is a common flavonoid compound, and the core of anthocyanin biosynthesis regulation is a transcription factor of R2R3-MYB protein, and the transcription factor can activate the expression of enzyme genes in an anthocyanin biosynthesis pathway. For example, from stage S1 to S2, expression levels of MYB are significantly down-regulated, potentially limiting the flavonoid-anthocyanin biosynthetic pathway, resulting in reduced anthocyanin accumulation in the leaves of crape myrtle (Lagerstroemia indica). In addition, mrWD40-1 interacts with MYB and bHLH to enhance anthocyanin accumulation in Myrica rubra. MYB transcription factors control the anthocyanin content in the lamina of Nicotiana benthamiana (N.benthamiana). The MYB transcription factors for regulating anthocyanin biosynthesis are the MYB transcription factors, but MYB transcription factors for regulating anthocyanin accumulation in phoebe bournei are not reported yet. The early stage of the method discovers abundant anthocyanin in the photinia fraseri leaves, which is possibly related to the PbMYB201 gene with high expression, the PbMYB201 gene is overexpressed in the arabidopsis for the first time, the biological function of the PbMYB201 gene in anthocyanin accumulation is verified, and gene resources are provided for regulating and controlling the anthocyanin content for people.

Disclosure of Invention

In order to solve the problems, the invention provides a phoebe bournei PbMYB201 gene, a coded protein and application thereof.

Firstly, the invention provides a phoebe bournei PbMYB201 protein, which is:

1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)

2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

The invention also provides a gene for encoding the phoebe bournei PbMYB201 protein.

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

The invention also provides an over-expression vector containing the gene, a host cell and engineering bacteria.

The invention also provides application of the gene in improving plant anthocyanin accumulation.

In one embodiment of the invention, the gene is transferred into the genome of a plant and overexpressed in the transgenic plant to increase plant anthocyanin accumulation.

The invention also provides a method for improving anthocyanin accumulation of plants, which adopts an agrobacterium-mediated method to transform a vector containing the genes into a plant genome, and screens to obtain transgenic plants. Wherein the gene is overexpressed in the transgenic plant and a higher amount of anthocyanin is accumulated compared to control plants.

The invention clones the PbMYB201 gene of photinia Min, stably converts Arabidopsis thaliana by an agrobacterium tumefaciens mediated method to obtain the Arabidopsis thaliana with over-expressed gene, carries out molecular detection on the Arabidopsis thaliana, and carries out expression analysis on the PbMYB201 of the over-expressed Arabidopsis thaliana, and the result shows that the PbMYB201 is obviously and highly expressed in the transgenic Arabidopsis thaliana. Further extracting anthocyanin from control Arabidopsis thaliana and over-expressed Arabidopsis thaliana, and detecting the absolute content of anthocyanin by using an ultraviolet-visible spectrophotometer. The result shows that the anthocyanin content detected in the over-expression arabidopsis thaliana is obviously higher than that detected in the control arabidopsis thaliana, and the anthocyanin content is respectively 0.1286ng/g and 0.0239ng/g; in a word, the PbMYB201 gene of photinia Min is over-expressed in arabidopsis for the first time, so that anthocyanin with higher concentration is obtained, namely, artificial regulation and control of anthocyanin synthesis in plants is feasible, and the method has a wide application prospect and economic benefit.

Drawings

FIG. 1 shows total RNA of the root, stem and leaf of Phoebe bournei.

FIG. 2 shows PCR amplification of the Phoebe bournei PbMYB201 gene.

FIG. 3 shows PCR detection of the overexpression vector bacterial liquid of the Phoebe bournei PbMYB201 gene.

FIG. 4 is a comparison of transgenic Arabidopsis phenotypes with wild type Arabidopsis. A: transgenic arabidopsis thaliana; b: wild type Arabidopsis thaliana; and C, anthocyanin extracting solution of transgenic arabidopsis thaliana and wild arabidopsis thaliana.

FIG. 5 is a PCR identification of positive lines and analysis of PbMYB201 expression levels. A: identifying positive strains by PCR; b: the expression level of PbMYB201 was analyzed by fluorescent quantitative PCR.

Fig. 6 is an anthocyanin content analysis of PbMYB201 over-expressed strains.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

1. Material

1.1 Experimental materials

Phoebe bournei material was taken from Qingyuan laboratory forest farm in Lishui, zhejiang province.

Arabidopsis thaliana was grown in an intelligent laboratory building Arabidopsis thaliana growth chamber from the university of Zhejiang agriculture and forestry using Columbia wild type (Col-0) under conditions of 25℃for 16h/d.

1.2 Experimental reagents and instruments

The high-fidelity DNA polymerase, various restriction enzymes, markers and DNA gel recovery kits are all purchased from Takara Bao bioengineering (Dalian) Co., ltd; plasmid extraction kit and E.coli competence were purchased from Beijing full gold biotechnology (TransGen Biotech) Co., ltd; the PCR instrument is an American PE9700 PCR instrument; the ultra clean bench was purchased from su-zhou honest clean purification technologies.

1.3 primer Synthesis and sequencing

Primer synthesis and sequencing were all done by Zhejiang have well-being biotechnology limited.

2 method

2.1 extraction of Phoebe bournei Total RNA

The M5 Plant RNeasy Complex Mini Kit is adopted to extract the total RNA of phoebe bournei, and the steps are as follows:

(1) 1ml of the lysate CLB was taken into a centrifuge tube (if the CLB had precipitated or precipitated, it was first redissolved in a water bath at 65 ℃) and 5% beta-mercaptoethanol (1 ml of CLB plus 50. Mu.l of beta-mercaptoethanol) was added to the lysate CLB. After being mixed reversely, the mixture is preheated in a water bath at 65 ℃;

(2) Taking about 0.1g of sample, putting the sample into a mortar precooled by liquid nitrogen, and grinding the liquid nitrogen into fine powder;

(3) Transfer 100-200mg of the fine powder into a pre-heated lysate CLB (with β -mercaptoethanol added) centrifuge tube. Immediately and vigorously vortex for 30-60 seconds or blow with a suction head to mix and crack evenly to obtain satisfactory homogenization results;

(4) Short-time putting back into 65 ℃ water bath (5-10 min), and occasionally reversing the middle for 1-2 times to help cracking;

(5) Centrifuging the lysate at 13,000rpm for 10 minutes, precipitating uncleaved fragments;

(6) Lysate supernatant (more supernatant can be taken without exceeding the capacity of the genomic DNA removal column, which can increase yield) is transferred to a new centrifuge tube. Absolute ethanol (0.5 vol) was added to half the volume of the supernatant, at which time precipitation was possible, but the extraction process was not affected, and immediately, the mixture was blown and homogenized without centrifugation.

(7) Step 3 of immediately connecting the operation steps;

(8) The mixture (less than 720. Mu.l each time, and possibly in two additions) was loaded into a genome clearance column and centrifuged at 13,000rpm for 2 minutes, and the waste liquid was discarded;

(9) Placing the genome DNA removal column in a clean 2ml centrifuge tube, adding 500 μl of lysate RLT Plus into the genome removal column, centrifuging at 13,000rpm for 30 seconds, collecting filtrate, accurately estimating the volume of the filtrate (usually about 450-500 μl, the lost volume should be subtracted during filtration) by using a micropipette, adding 0.5 times of absolute ethanol, immediately blowing and mixing, and not centrifuging;

(10) Immediately adding the mixture (less than 720. Mu.l each time, and possibly two times) to an adsorption column RA (the adsorption column is placed in a collecting tube), centrifuging at 13,000rpm for 2 minutes, and discarding the waste liquid;

(11) Adding 700 μl deproteinized solution RW1, standing at room temperature for 1min, centrifuging at 13,000rpm for 30 seconds, and discarding the waste liquid;

(12) Mu.l of rinse liquid RW (please check whether absolute ethanol has been added |) was added, centrifuged at 13,000rpm for 30 seconds and the waste liquid was discarded. 500 μl of rinse RW was added and repeated;

(13) Placing the adsorption column RA back into the empty collecting pipe, centrifuging at 13,000rpm for 2 minutes, and removing the rinsing liquid as much as possible;

(14) Taking out the adsorption column RA, placing into an RNase Free centrifuge tube, adding 30-50 μl of RNase-Free H2O (which can increase the yield by heating in water bath at 70deg.C in advance) at the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 1min, and centrifuging at 12,000rpm for 1min;

(15) If RNA yield is expected>30. Mu.g, 30-50. Mu.l RNase-Free H were added ₂ O repeat step 9, combining the two washes, or repeat step one time with the first eluent added back to the column.

2.2 Synthesis of first strand of reverse transcribed cDNA

The synthesis of the first strand of reverse transcribed cDNA was performed by Phoebe bournei RNA (Total RNA) with reference to the instructions of PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa).

(1) The following mixture was prepared for genome removal reaction:

the reaction procedure is: 42 ℃ for 2min;4 ℃, hold.

(2) The reverse transcription reaction was carried out by preparing the following mixture:

the reaction procedure is: 37 ℃ for 15min;85 ℃,5sec;4 ℃, hold.

2.3 cloning of the target Gene

2.3.1 Gene cloning

Specific primers for PbMYB201 were designed (Table 1), and the amplification system and procedure are shown in Table 2.

Table 1 cloning primers for PbMYB201

TABLE 2 cloning System

The reaction procedure: cycling for 35 times at 98 ℃,30sec,52 ℃,5sec,72 ℃ for 1min; 72 ℃ for 1min;16 ℃, hold.

2.3.2 recovery of the target fragment

1% agarose gel was prepared, the PCR products were detected by agarose gel electrophoresis, and if the band was correctly referred to MiniBEST Agarose Gel DNA Extraction Kit (Takara, dalian) the target fragment was excised and recovered.

(1) Prepare sterilized 2mL centrifuge tube and weigh empty tube mass.

(2) The slab was cut under an ultraviolet lamp with a clean scalpel, the slab was cut and placed into a 2mL centrifuge tube, and the slab volume was calculated by weighing (1 mg = 1 μl as standard).

(3) And adding Buffer GM with the volume of 3 times of gel into the gel block, dissolving the gel block at room temperature, and intermittently vibrating and mixing.

(3) After the gel was completely dissolved, isopropanol was added at a final concentration of 20%.

(4) Adding the solution obtained in the previous step into Spin Column, placing on Collection Tube, centrifuging at 12000rpm for 1min, and discarding the waste liquid.

(5) Adding 700 mu L of rinsing solution Buffer WB (absolute ethyl alcohol is added) into the adsorption column, and discarding waste liquid at the room temperature of 12000rpm and 1min;

(6) Repeating the step (5).

(6) Air separation 12000rpm,1min.

(7) The adsorption column was placed in a sterilized 1.5mL centrifuge tube, 30. Mu.L of sterilized water (preheated to 65 ℃) was added to the adsorption film, the mixture was allowed to stand at room temperature for 1min, centrifuged at 12000rpm for 1min, and the collected solution was kept at-20 ℃.

2.3.3 ligation and transformation of fragments of interest

(1) And (3) connection: the pEASY blue Zero vector (Transgene,

blunt Zero Cloning Kit) are ligated with the gene of interest, mixed with the following solutions, gently mixed, and centrifuged briefly. And (2) PCR:25℃for 30min.

(2) Conversion: 2. Mu.L of the above-mentioned ligation product was added after the competent E.coli of Trans T1 (Transgene, beijing) was melted in an ice bath, the ice bath was left for 30min, the tube was rapidly and smoothly transferred to the ice bath for 30s, 500. Mu.L of LB medium containing no antibiotics was added, and the tube was cultured at 200rpm in a shaker at 37℃for 1h. 2min at 4000rpm, removing part of the supernatant, reserving 100 mu L of bacterial liquid, blowing and mixing the bacterial liquid again, plating on a solid LB culture medium (containing 50mg/L Kana), and culturing for 12h at 37 ℃ in an inverted mode.

(3) And (3) bacterial inspection: a white single colony was picked up on the plate, and 500. Mu.L of liquid LB medium (containing 50mg/L Kana) was added thereto, followed by culturing at 200rpm in a shaker at 37℃for 3-5 hours. PCR detection was performed using 1. Mu.L of the bacterial liquid as a template, and the primers were gene cloning primers, and the system and procedure were as follows.

The reaction procedure is: 94 ℃ for 5min; cycling at 94 ℃,30sec,52 ℃,30sec,72 ℃ for 1min for 35 times; 72 ℃ for 5min;16 ℃, hold.

(4) And detecting bacterial liquid PCR products by agarose gel electrophoresis, and selecting positive clones to be sent to Zhejiang Jingkang biotechnology Co.

(5) And (3) after the snapge comparison and sequencing result is correct, extracting plasmids by using Transgene EasyPure Plasmid MiniPrep Kit, and constructing an overexpression vector of the PbMYB201 gene by using the obtained positive plasmids.

2.4 construction of the overexpression vector by homologous recombination

According to

II One Step Cloning Kit the over-expression vector is constructed by homologous recombination.

(1) Firstly, adding a kit containing a pK2W7-eYGFPuv-3xFLAG linker sequence on the basis of a target gene specific primer, and preparing the following PCR system (20 mu L) by using the positive plasmid obtained in the previous step as a template:

the reaction procedure: cycling for 35 times at 98 ℃,30sec,54 ℃,5sec,72 ℃ for 1min; 72 ℃ for 5min;16 ℃, hold.

(2) After ensuring that the product is a single band, the target fragment is subjected to gel cutting recovery by referring to MiniBEST Agarose Gel DNA Extraction Kit (Takara, dalian) instruction book.

(3) The overexpression vector pK2W 7-eYGFPv-3 xFLAG was subjected to double-enzyme tangentially, and the following PCR system was prepared using the plasmid pK2W 7-eYGFPv-3 xFLAG as a template:

the reaction procedure: 37 ℃ for 2h;16 ℃, hold.

(4) The desired fragment was subjected to gel cutting recovery by reference to MiniBEST Agarose Gel DNA Extraction Kit (Takara, dalian) instructions.

(5) The genetic transformation over-expression vector is selected from pK2W 7-eYGFPv-3 xFLAG, and constructs the over-expression vector of the PbMYB201 gene. The following mixed solutions were prepared at 25℃overnight.

Optimal cloning vector usage= [0.02×cloning vector base pair number ] ng (0.03 pmol) optimal insert usage= [0.04×insert base pair number ] ng (0.06 pmol)

The reaction procedure: 37 ℃ for 30min; cooling to 4 ℃ or immediately cooling on ice.

(2) 2. Mu.L of the strain is used for transforming Trans T1 (Transgene, beijing) escherichia coli competence, then a monoclonal strain is selected for bacterial liquid PCR detection, and the primers are gene cloning primers, wherein a PCR system is as follows:

(3) And detecting PCR products by agarose gel electrophoresis, transforming into Trans T1 (Transgene, beijing) escherichia coli competence, and selecting positive clones to be sent to Zhejiang Kakon biotechnology Co. Extracting the strain plasmid with correct sequencing result for later use.

2.5 conversion of Agrobacterium by liquid nitrogen method

(1) Taking out Agrobacterium GV1301 (a unique organism) stored at-80deg.C, and thawing on ice;

(2) Adding 0.5 mug of expression vector plasmid into the competence of agrobacterium, flicking and uniformly mixing, and respectively and sequentially placing the mixture on ice, in liquid nitrogen, in a water bath at 37 ℃ and in an ice-water mixture for 5min;

(3) Adding 700 mu L of fresh LB liquid medium without any resistance into a centrifuge tube, and placing the bacterial liquid into a shaking table (28 ℃ C., 200 rpm) for shake culture for 4-6h;

(4) Centrifuging (6000 rpm,2 min), pouring out part of the supernatant to make the volume of the residual liquid in the tube about 70-100 mu L, and blowing uniformly;

(5) Uniformly coating bacterial liquid on LB solid medium containing antibiotics (50 mg/L Kana and 50mg/L Rif), and inversely culturing in a biochemical incubator at 28 ℃ for 48-72 hours;

(6) And (3) selecting a monoclonal on the plate for bacterial liquid PCR detection, wherein primers are gene cloning primers, and a PCR system is as follows:

the reaction procedure is: 94 ℃ for 5min; cycling at 94 ℃,30sec,46 ℃,30sec,72 ℃,20sec, 30 times; 72 ℃ for 5min;16 ℃, hold.

(7) Adding 50% glycerol with the same volume into the monoclonal bacterial liquid with positive detection result, and storing in a refrigerator at-80 ℃.

2.6 Agrobacterium-mediated transformation of Arabidopsis thaliana

2.6.1 inflorescence dip-dyeing method for transforming Arabidopsis thaliana

(1) Wild type Arabidopsis seeds (COL-0) were sown on 1/2MS medium, vernalized for two days at 4℃and cultured in Arabidopsis growth chamber. And when four cotyledons grow, the cotyledons are moved into a soil seedling raising basin, and are continuously placed into a growth chamber for cultivation. About 3 weeks minus the main stem of arabidopsis thaliana, inhibiting apical dominance and promoting vegetative growth. After the arabidopsis is extracted out of a plurality of side branches and enters a full bloom stage, the existing fruit pods are cut off by scissors and the soil is thoroughly irrigated by water the day before the arabidopsis is infected, and then the inflorescence dip-dyeing method is adopted to infect the arabidopsis.

(2) Taking positive clone bacterial liquid according to the following formula 1:30 (Kana at 50mg/L and Rif at 50 mg/L) were added and shake-cultured overnight in a shaker (28 ℃ C., 200 rpm).

(3) 200 mu L of the bacterial liquid in the last step is added with 200mL of fresh LB resistant medium (50 mg/L Kana and 50mg/L Rif), and shake-cultured in a shaking table (28 ℃ C., 200 rpm) until the OD value reaches 0.8-1.0.

(4) Pouring bacterial liquid into a special container for dyeing, adding 5% of sucrose and 0.02% of surfactant Silwet-77, shaking and mixing uniformly;

(5) Soaking inflorescences of Col-0 into the dye liquor for 50s, and sucking redundant infection liquor on the surface of Arabidopsis thaliana by using a paper towel in a light press manner;

(6) Placing Arabidopsis thaliana into a seedling raising basin, and placing the Arabidopsis thaliana into an Arabidopsis thaliana growth chamber for dark culture for 2d;

(7) Placing Arabidopsis thaliana under light for normal growth, and collecting T0 generation seeds after the Arabidopsis thaliana is pod-grown Huang Liekai;

(8) Taking a proper amount of transgenic arabidopsis T0 seeds to be screened, adding the transgenic arabidopsis T0 seeds into a 1.5mL centrifuge tube, sequentially adding 75% ethanol, oscillating for 10min, and oscillating for 10min with absolute ethanol for sterilization.

(9) The seeds are poured into sterilized filter paper on an ultra-clean workbench to be dried, then the seeds are sown in a screening culture medium (1/2MS+50 mg/L Kana), vernalized for two days at 4 ℃ and placed in a growth chamber for culture.

(10) After about one week, T1-generation Arabidopsis thaliana with true leaves grown thereon was transferred to a medium, and after 2d of culture with a transparent cover for moisture preservation, the medium was cultured normally. And harvesting seeds after the arabidopsis is mature, and independently harvesting the arabidopsis by a single plant, and marking the arabidopsis as different numbers.

(11) And continuing to screen until a T3 generation homozygous strain is obtained according to a T0 generation seed screening method.

2.6.2 screening of transgenic Positive strains of Arabidopsis thaliana

DNA detection was performed on plants grown with true leaves in the screening medium using polymeric Mei super light speed mix MF848-01 (Mei 5bio, beijing) as follows:

(1) The yellow gun head is burned and melted by fire in advance to be self-made grinding pestle for standby.

(2) 1-2mm2 Arabidopsis thaliana leaves were placed in a 0.2 mM PCR tube, 20. Mu.L of lysate was added, and the wall was broken by rotary extrusion with a grinding pestle.

(3) The PCR tube is placed in a PCR instrument at 98 ℃ for 3-5min, and vortex vibration is carried out fully.

(4) Centrifuging at 12000rpm for 1-2min, taking 1 μl of supernatant as DNA template for PCR detection, wherein the system is as follows:

the reaction procedure is: 95 ℃ for 3min; cycling for 35 times at 94 ℃,25sec,52 ℃,25sec,72 ℃ for 1min; 72 ℃ for 5min;16 ℃, hold.

2.6.3 fluorescent quantitative PCR identification

Total RNA was extracted from the selected Arabidopsis positive plants using M5 Plant RNeasy Complex Mini Kit, and cDNA was reverse transcribed using PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa, dalian, china).

Fluorescent quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, nanjing, china) and 20. Mu.L of a system mixture was prepared using Bio-Rad CFX-96: 2X ChamQ SYBR qPCR Master Mix. Mu. L, primer-F0.4. Mu. L, primer-R0.4. Mu.L, 50X ROX Reference Dye 1.4. Mu. L, cDNA 1. Mu.L and ddH ₂ O7.8 μl; the reaction procedure is: 95 ℃ for 30s; cycling for 40 times at 95 ℃ for 10s and 60 ℃ for 30s; 65℃for 5s and 95℃for 5min (dissolution profile is set according to the type of instrument). The primers used for the fluorescent quantitative PCR are shown in Table 3, and U6 was selected as the reference gene.

TABLE 3 fluorescent quantitative PCR primers

2.7 analysis of the content of transgenic plant anthocyanin

0.5 g of leaves and stems of Arabidopsis thaliana was chopped and put into 10ml of methanol (containing 1% HCL) extract, shaken well, covered with a cover against light, and sonicated for 1 hour. After centrifugation (5000 rpm,10 minutes), the supernatant was used for anthocyanin content determination. Then, 2ml of the supernatant was 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 was incubated in a dark room at 40℃for 30 minutes. The absorbance of the mixture supernatant was measured at 520 nm, the anthocyanin content was measured and the correction was measured at 700 nm using an ultraviolet visible spectrophotometer (Thermo Scientific co., ltd, USA). All absorbance measurements were performed at room temperature with distilled water as a blank.

Obtaining absorbance and calculating anthocyanin content according to a formula:

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

anthocyanin content (mg. 100 g) ^-1 )＝ΔA·M·DF·V·100/m·ε·L

M: molecular weight of cyanidin-3-glucoside 449.2 g.mol ^-1 ；

DF: a dilution factor; take 1ml to 10ml volumetric flask, df=10

V: total volume of the extracting solution, mL;

m: weight, g;

epsilon: extinction coefficient of cyanidin-3-glucoside, 26 900L mol ^-1 ·cm ^-1 ；

L: optical path, 1cm.

3. Experimental results

3.1 analysis of total RNA extraction from leaves of Phoebe bournei

Extracting total RNA of Phoebe bournei by M5 Plant RNeasy Complex Mini Kit (figure 1), and measuring OD260/280 ratio of the extracted RNA by an ultraviolet spectrophotometer to be 1.8-2.1, which shows that the total RNA has better purity; the agarose gel electrophoresis result shows that the 18s and 28s bands of the RNA sample are very clear, and the RNA can be deduced to be not degraded, so that the requirement of the next experiment is met.

3.2 cloning and vector construction of Phoebe bournei PbMYB201 sequence

PbMYB201 is amplified from Phoebe bournei cDNA by a high-fidelity enzyme PCR amplification method to obtain a 917bp fragment (figure 2), the amplified fragment is connected to an over-expression vector pK2W 7-eYGFPv-3 xFLAG to transform escherichia coli, a single clone is selected to obtain positive clone (figure 3) through PCR verification, sequencing is carried out through Zhejiang Katsuzkya Co., ltd, the sequence is completely correct, one clone is selected to extract plasmid, agrobacterium is transformed, and arabidopsis thaliana is transformed.

3.3 screening of transgenic Positive strains of Arabidopsis thaliana

And (3) carrying out PCR identification on the T1 generation after the PbMYB201 of the photinia Min is converted into the Arabidopsis thaliana to obtain a positive strain (figure 4A), obtaining a T3 generation homozygous strain, detecting the PbMYB201 expression level (figure 4B) in each strain by using quantitative PCR, and selecting two strains OE2 and OE3 with higher expression to carry out subsequent analysis.

3.4 transgenic Arabidopsis thaliana flower phenotype

The phenotype of the T3 generation homozygous lines OE2 and OE3 (FIG. 5A) showed a pronounced reddening compared to Col (FIG. 5B). The anthocyanin extracts for OE2 and OE3 also differed significantly from Col (fig. 5C).

3.5 determination of the anthocyanin content of transgenic Arabidopsis thaliana

The anthocyanin content measurement shows that the anthocyanin content in the control arabidopsis leaves is 0.0239ng/g, and the anthocyanin content in the over-expression strain leaves is 0.1286ng/g, which is obviously higher than that of the wild type (figure 6), and shows that the plant anthocyanin biosynthesis pathway structural gene is regulated by PbMYB201, so that the accumulation of the arabidopsis anthocyanin is promoted. In conclusion, the PbMYB201 gene of the phoebe bournei is over-expressed, so that the anthocyanin content of the arabidopsis thaliana can be remarkably improved.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.

Claims

1. Phoebe bournei PbMYB201 protein, which is:

1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)

2. A gene encoding the phoebe bournei PbMYB201 protein of claim 1.

3. The gene of claim 2, wherein the sequence is set forth in SEQ ID No. 1.

4. A vector comprising the gene of claim 2 or 3.

5. A host cell comprising the vector of claim 4.

6. An engineered bacterium comprising the gene of claim 2 or 3.

7. Use of the gene of claim 2 or 3 for increasing plant accumulation of anthocyanin.

8. The use according to claim 7, wherein the gene is transferred into the genome of a plant and overexpressed in a transgenic plant to increase plant anthocyanin accumulation.

9. A method for improving anthocyanin accumulation in plants, which adopts an agrobacterium-mediated method to transfer a vector containing the gene of claim 2 or 3 into a plant genome, and screening to obtain transgenic plants.

10. The method of claim 9, wherein said gene is overexpressed in said transgenic plant and a higher level of anthocyanin is accumulated as compared to a control plant.