Application of gene BcWRKY1 in regulating yellow-green phenotype of Wucai leaves

Abstract

The present invention provides an application of a gene BcWRKY1 in regulating the yellow-green phenotype of leaves of Wucai, and relates to the technical field of gene engineering, wherein the nucleotide sequence of the gene BcWRKY1 is shown in SEQ ID NO.4, and the expression of the gene BcWRKY1 is inhibited and the expression of the gene BcCLH2 is promoted, so that the leaves of Wucai are changed from green to yellow; and the expression of the gene BcWRKY1 is promoted and the expression of the gene BcCLH2 is inhibited, so that the leaves of Wucai are changed from yellow to green; the nucleotide sequence of the gene BcCLH2 is shown in SEQ ID NO.1; the gene provided by the present application is helpful for the genetic breeding of Wucai.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of a gene BcWRKY1 in regulating the yellow-green phenotype of Wucai leaves. Background Art

[0002] Wucai (Brassica campesttris L.ssp.chinesis (L.) Makino var.rosularis Tsenet Lee) is a subspecies of Chinese cabbage (B.campestris L.), native to China, mainly distributed in the Yangtze and Huaihe River Basin. It is a nutritious vegetable rich in minerals and vitamins, and displays colorful leaves. Wucai yellow variety W7-2, the inner leaves remain green at normal temperature during the adult stage, and after experiencing low temperatures, the inner leaves gradually turn yellow. After turning yellow, Wucai is soft and juicy, and tastes more delicious.

[0003] Therefore, it is an urgent problem to study the molecular mechanism of the yellowing of wucai and provide new ideas or theoretical basis for cultivating yellow wucai varieties. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides an application of a gene BcWRKY1 in regulating the yellow-green phenotype of Wucai leaves, so as to solve the problem that the molecular mechanism of the yellowing of Wucai in the prior art is unclear.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] In a first aspect of the present invention, a gene BcWRKY1 is provided for use in regulating the yellow-green phenotype of Wucai leaves. The nucleotide sequence of the gene BcWRKY1 is shown in SEQ ID NO.4.

[0007] Furthermore, the application includes:

[0008] By inhibiting the expression of gene BcWRKY1 and promoting the expression of gene BcCLH2, the leaves of Wucai turn from green to yellow, or

[0009] By promoting the expression of the gene BcWRKY1 and inhibiting the expression of the gene BcCLH2, the leaves of the vegetable turned from yellow to green.

[0010] The nucleotide sequence of the gene BcCLH2 is shown in SEQ ID NO.1.

[0011] Furthermore, the inhibiting the expression of the gene BcWRKY1 includes knocking out the gene BcWRKY1.

[0012] Furthermore, the method of promoting the expression of the gene BcWRKY1 includes constructing an overexpression vector of the gene BcWRKY1.

[0013] Furthermore, the Wucai variety is W7-2 or WS-1.

[0014] A second aspect of the present invention provides a method for regulating chlorophyllase activity in leaves of Wucai, the method comprising:

[0015] By inhibiting the expression of the gene BcWRKY1, promoting the expression of the gene BcCLH2, the chlorophyllase activity in the leaves of Wucai is increased, or

[0016] The expression of the gene BcWRKY1 is promoted and the expression of the gene BcCLH2 is inhibited, thereby reducing the chlorophyllase activity in the leaves of Wucai.

[0017] The third aspect of the present invention also provides a use of a gene BcWRKY1, a protein encoded by the gene BcWRKY1 or a biological material containing the gene BcWRKY1 in preparing a product for promoting the transformation of Wucai leaves from green to yellow or from yellow to green; the nucleotide sequence of the gene BcWRKY1 is shown in SEQ ID NO.4.

[0018] Furthermore, the biological material is a plasmid, an expression vector or a transgenic cell.

[0019] The fourth aspect of the present invention also provides an application of the gene BcCLH2 in regulating the yellow-green phenotype of Wucai leaves.

[0020] Furthermore, the application includes:

[0021] Promote the expression of gene BcCLH2, causing the leaves of Wucai to change from green to yellow; or

[0022] Inhibit the expression of the gene BcCLH2, causing the leaves of the vegetable to change from yellow to green.

[0023] The application of a gene BcWRKY1 provided by the present invention in regulating the yellow-green phenotype of Wucai leaves has the following beneficial effects compared with the prior art:

[0024] The provided gene BcWRKY1 is related to the regulation of the yellow-green phenotype of Wucai leaves. By inhibiting or overexpressing the gene BcWRKY1, it can be used to control the yellow-green phenotype of Wucai leaves, thereby helping to cultivate new Wucai varieties with the target leaf phenotype and providing new ideas for genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 is the agarose gel electrophoresis diagram of the gene BcCLH2;

[0027] Figure 2 Comparison chart of RT-qPCR analysis and chlorophyllase activity determination of gene BcCLH2 at three stages;

[0028] Figure 3 The transient expression analysis and subcellular localization map of the gene BcCLH2 in tobacco;

[0029] Figure 4 This is a diagram of transient expression analysis of the gene BcCLH2 in Wucai;

[0030] Figure 5 This is the self-activation concentration screening diagram of pAbAi-BcCLH2;

[0031] Figure 6 This is the result of screening the yeast cDNA library of Wucai with pAbAi-BcCLH2;

[0032] Figure 7 Validation of the interaction map between BcWRKY1 and BcCLH2 in yeast one-hybrid experiments;

[0033] Figure 8 The interaction map between BcWRKY1 and BcCLH2 was verified for the dual luciferase reporter experiment. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] In order to solve the problem that the mechanism of yellowing of black cabbage is unclear in the prior art, the main idea of ​​this application is:

[0036] The invention provides a gene BcWRKY1 related to the yellow-green phenotype of wucai leaves. By upregulating or inhibiting the expression of the gene, the leaves of wucai can be effectively regulated to change from green to yellow or from yellow to green, thereby providing a new idea for the genetic breeding of wucai.

[0037] The present application uses the following examples to functionally verify the gene BcWRKY1 provided in the present application.

[0038] Example 1

[0039] In this embodiment, Wucai W7-2 variety was selected as the material (the leaves of W7-2 turned yellow after gradually experiencing low temperatures in autumn and winter during its growth period).

[0040] Perform the following operations on Wucai W7-2:

[0041] W7-2 seedlings were grown in a greenhouse at 26 ± 2 °C (day) and 20 ± 2 °C (night) with a light intensity of 300 μmol m -2 s -1 , relative humidity is 70-80%; during this process, the leaves of Wucai remain green (the corresponding state of the Wucai leaves obtained in this process is before turning yellow (G)).

[0042] Seedlings with 7 to 8 leaves were transplanted at 6 ± 2 °C (daytime) and -1 ± 2 °C (nighttime) under a 300 μmol m -2 s -1 The leaves of the black cabbage were cultured in a low-temperature growth room with a light intensity of 70-80% and a relative humidity of 12 days. During this process, the leaves of the black cabbage gradually turned from green to yellow (the corresponding state of the black cabbage leaves obtained in this process was after turning yellow (Y)).

[0043] Then, the temperature was set to 26 ± 2 °C (day) and 20 ± 2 °C (night), and the light intensity was 300 μmol m -2 s -1 , relative humidity was 70-80%, and continued for 12 days; during this process, the leaves of Wucai gradually turned from yellow to green (the corresponding state of the Wucai leaves obtained in this process is regreening (RG)).

[0044] During the above process, three fully expanded young leaves were collected from the center every 3 days.

[0045] 1. Total RNA extraction and cDNA synthesis were performed on plant leaves at three stages: before yellowing (G), after yellowing (Y), and after regreening (RG). The specific method is as follows:

[0046] The RNA of samples from three periods was extracted using the TakaRa RNA extraction kit. After quantification, the RNA was reverse transcribed using the TRAN reverse transcription kit. The system is shown in Table 1.

[0047] Table 1

[0048]

[0049] 2. Cloning, recovery and purification of the Wucai gene BcCLH2

[0050] The nucleotide sequence of gene BcCLH2 was obtained from the Wucai genome (as shown in SEQ ID NO.1), specifically:

[0051] Homologous arm primers (F: SEQ ID NO.5; R: SEQ ID NO.6) were designed using CE-Design software according to the gene sequence and pCAMBIA2300 vector sequence, and sent to General Company for synthesizing primer sequences. Samples were added according to the system in Table 2, and the PCR program was 95°C for 5 min, 95°C for 30 s, 60°C for 30 s, 72°C for 2 min, and 72°C for 5 min. After the reaction, the PCR product was identified by 1% agarose gel electrophoresis ( Figure 1 ). The target DNA fragment (nucleotide sequence as shown in SEQ ID NO.1) was recovered using the TIANGEN agarose gel DNA recovery kit.

[0052] Table 2

[0053]

[0054] 3. Connect the vector and transform E. coli

[0055] Add samples according to the system in Table 3, pipette and mix, connect at 37℃ for 30min, quickly put on ice, and obtain the connection product (including pCAMBIA2300-BcCLH2 and pCAMBIA2300). Add 10μL of the connection product to 100μL of E. coli, gently pipette and 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, then put the culture medium in the incubator and invert and culture at 37℃ overnight. The next day, pick 8 single colonies in a centrifuge tube of 500μL LB with 100mg / mL Kan, shake on a shaker at 37℃ for 5h until the bacterial solution is turbid, and obtain the bacterial solution.

[0056] Table 3

[0057]

[0058] Take the bacterial solution obtained in the previous step, use the gene upstream primer F: SEQ ID NO.5 and the vector downstream primer R: SEQ ID NO.7 to add samples according to the system in Table 4, and the PCR program is 95℃5min, 95℃30s, 60℃30s, 72℃2min, 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℃.

[0059] Table 4

[0060]

[0061] 4. Agrobacterium transformation

[0062] Take 100 μL of Agrobacterium competent cells, then add 5 μL of the ligation product (pCAMBIA2300-BcCLH2) obtained above, pipette and mix, place on ice for 5 min, liquid nitrogen for 5 min, 37°C water bath for 5 min, add 700 μL of LB without antibiotics, shake at 28°C for 3 h. Collect bacteria at 6000 rpm for one minute, keep 100 μL of supernatant and gently pipette and resuspend the bacteria, apply it to LB plates containing 100 mg / mL Kan (kanamycin) and 25 mg / mL rif (rifampicin), invert and culture in a 28-degree incubator for 2 days, pick 8 single colonies in a centrifuge tube containing 500 μL LB with 100 mg / mL Kan and 25 mg / mL rif, and shake at 28°C for 24 h until the bacterial solution becomes turbid. Samples were added according to the system in Table 4, and the PCR program was 95°C for 5 min, 95°C for 30 s, 60°C for 30 s, 72°C for 2 min, and 72°C for 5 min. After the reaction, the PCR product was identified as qualified by 1% agarose gel electrophoresis, and then the bacterial solution was mixed with glycerol in a ratio of 1:1, frozen with liquid nitrogen, and stored at -80°C to obtain Agrobacterium transformants.

[0063] Example 2

[0064] 1. Determination of the expression level of Wucai gene BcCLH2 at different stages

[0065] The expression levels of the gene BcCLH2 in the three periods of G, Y and RG were measured by the following method:

[0066] According to the nucleotide sequence of the gene BcCLH2 shown in SEQ ID NO.1, the fluorescent primers of the gene BcCLH2 (BcCLH2-F: SEQ ID NO.8; BcCLH2-R: SEQ ID NO.9) were designed using Oligo7 software to detect the expression of the gene BcCLH2, and Actin from Wucai was used as the internal reference gene. The primers were F: SEQ ID NO.10; R: SEQ ID NO.11, and the primer sequences were sent to General Biotechnology Company for synthesis. The qRT-PCR system was: Green qPCR SuperMix 10μL, BcCLH2-F 0.4μL, BcCLH2-R 0.4μL, cDNA 1μL, DEPC 8.2μL. qRT-PCR program: 95℃30s, 95℃5s, 60℃15s, 72℃10s, image acquisition, 40 cycles, melting curve 58℃to 95℃5s, image acquisition.

[0067] 2. Chlorophyllase activity determination

[0068] Weigh 0.1g sample, add 1mL PBS, and grind with a grinder. After grinding thoroughly, centrifuge at 2000rpm for 20 minutes, and aspirate the supernatant into a 1.5ml centrifuge tube. Set up standard wells and sample wells, and add 50μL of standard of different concentrations to each standard well; first add 40μL of sample diluent to the sample well to be tested on the ELISA plate, and then add 10μL of the sample to be tested, and gently shake to mix; add 100μL of ELISA reagent to each well, except for the blank plate; seal the plate with a sealing film and incubate at 37℃ for 60 minutes; dilute the 20-fold concentrate 20 times with distilled water and set aside. Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds and then discard it, repeat 5 times, and pat dry; first add 50μL of color developer A and then 50μl of color developer B to each well, gently shake and mix, and color develop at 37℃ in the dark for 15 minutes; add 50μL of stop solution to each well to stop the reaction; adjust the blank well to 0, and measure the absorbance of each well at a wavelength of 450nm. The measurement should be carried out 15 minutes after adding the stop solution.

[0069] The results of qRT-PCR analysis were as follows Figure 2 As shown in A, the expression level of gene BcCLH2 was significantly upregulated from G stage to Y stage, and was significantly downregulated from Y stage to RG stage. The results of chlorophyllase activity assay were as follows: Figure 2 B, it can be found that the activity of chlorophyllase increased significantly from the G period to the Y period, and decreased from the Y period to the RG period.

[0070] 3. Analysis of transient expression and subcellular localization of BcCLH2 gene in tobacco

[0071] Preparation of Activation buffer: MES (2-(N-morpholino)ethanesulfonic acid) and MgCl2 were mixed in a ratio of 5:1 and sterilized at high temperature. The Agrobacterium transformant obtained in Example 1 was shaken overnight in a 28°C shaker. The OD value of the bacterial solution reached 0.8-1.0. Centrifuged at 4000rpm for 8min, removed the supernatant, first suspended with 3ml of ddH2O, centrifuged at 4000rpm for 8min, then suspended with Activation buffer, and centrifuged at 4000rpm for 8min. Using Activation buffer as a control, the OD value of the bacterial solution was measured, and the OD value of the bacterial solution was adjusted to 0.8-1.0 with Activation buffer. AS (acetosyringone) was added to the bacterial solution at a ratio of bacterial solution: AS = 1000:1. After static culture at 28°C in the dark for 3h, it was injected into Nicotiana benthamiana. After injection, it was treated in the dark for 1 day, cultured under light for about 2 days, and its localization results were observed by laser confocal microscopy. In addition, the leaf phenotypes after injection were observed, and the leaf color difference and total chlorophyll content were measured; these included:

[0072] (1) Color difference measurement

[0073] Determination of color value: The color value of the leaves was measured using a colorimeter (DS-700D) on the upper surface of the leaves every 3 days after planting. Ten 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. In addition, there are two color differences: value H* (Hue angle) and value C* (Chroma).

[0074] (2) Determination of chlorophyll content

[0075] The content of photosynthetic pigments (Chl a, Chl b) was determined by the Arnon method: grind the fresh leaves into powder, then add 2.5 ml of 80% acetone and a small amount of calcium carbonate and quartz sand, and grind into a homogenate. Add 3 ml of acetone and let it stand in the dark for 3 to 5 seconds. Take a piece of filter paper and place it in a funnel, wet it with ethanol, pour the extract into the funnel along the glass rod, and filter it into a 25 ml volumetric flask. Use a dropper to absorb 95% ethanol to wash the mortar, and the cleaning solution should also be filtered into a volumetric flask, and use 95% ethanol to elute the pigment around the filter paper. After the filter paper and residue have all turned white, finally use 95% ethanol to make up to the scale. Use TU1950 UV-visible spectrophotometer (PER-SEE, Beijing, China) to measure the absorbance at wavelengths of 646 and 663 nm. Among them, the content of Chla (mg / g) and Chl b (mg / g) is calculated as follows:

[0076] CChl a mg / L=12.21×A663-2.81×A646;

[0077] CChl b mg / L=20.13×A646-5.03×A663;

[0078] Chl a mg / g=CChl a mg / L×V(L) / Mfresh;

[0079] Chl b mg / g=CChl b mg / L×V(L) / Mfresh.

[0080] The test results are as follows Figure 3 As shown in the figure, by transiently expressing the gene BcCLH2 in Nicotiana benthamiana, it was found that compared with the control, the Nicotiana benthamiana overexpressing the gene BcCLH2 showed chlorosis ( Figure 3 A), it was found that overexpression of the gene BcCLH2 could turn Nicotiana benthamiana from green to yellow; the color difference value and total chlorophyll content are shown in Table 5. Compared with the control, the L*Value value was increased by 8.07%, the b*Value value was increased by 18.07%, the a*Value value did not change significantly, and the total Chl content was decreased by 52.6%. The subcellular localization results showed that the gene BcCLH2 was located in the chloroplast ( Figure 3 B).

[0081] From the above, it can be concluded that overexpression of the gene BcCLH2 can cause Nicotiana benthamiana to turn from green to yellow.

[0082] Table 5

[0083]

[0084] 4. Analysis of transient expression of gene BcCLH2 in Wucai

[0085] The leaves of the evergreen Wucai variety 'WS-1' were injected according to the tobacco transient expression method, and the leaf phenotypes after injection were observed, and the leaf color difference values ​​and total chlorophyll content were measured.

[0086] By transiently expressing the gene BcCLH2 in Wucai, the results are as follows Figure 4 As shown in Table 6, compared with the control, the leaves of Wucai overexpressing the gene BcCLH2 turned yellow. The color difference value and total chlorophyll content are shown in Table 6. Compared with the control, the L*Value, a*Value and b*Value values ​​were significantly increased, and the total Chl content was significantly decreased. These results show that the gene BcCLH2 plays a key role in the chlorophyll degradation of Wucai inner leaves (i.e., the overexpression of the gene BcCLH2 causes the leaves of Wucai to turn from green to yellow).

[0087] Table 6

[0088]

[0089] Example 3

[0090] 1. Screening of BcCLH2 gene yeast library

[0091] The promoter sequence of gene BcCLH2 (nucleotide sequence is shown in SEQ ID NO.2) was extracted from the Wucai gene, and according to the cis-acting element analysis, the -1800 to -1000 fragment (nucleotide sequence is shown in SEQ ID NO.3) was selected to construct a bait vector for library screening, including:

[0092] The pAbAi-BcCLH2 recombinant plasmid was constructed according to the method in Example 1, wherein the primer sequences were F: SEQ ID NO.12; R: SEQ ID NO.13; after the construction was completed, 1 ug of the pAbAi-BcCLH2 recombinant plasmid was linearized using the BbsI restriction endonuclease, and the specific enzyme digestion system is shown in Table 7:

[0093] Table 7

[0094]

[0095] After one hour of enzyme digestion, the sample was recovered. Samples were added according to the system in Table 8, pipetted and mixed, and placed in a water bath at 30°C for 30 minutes (inverted 6-8 times for 15 minutes to mix), and in a water bath at 42°C for 15 minutes (inverted 6-8 times for 7.5 minutes to mix). Centrifuge at 5000rpm for 40 seconds, discard the supernatant, resuspend in 400μL of dd H2O, centrifuge for 30 seconds, discard the supernatant, 50μL of dd H2O, and spread on SD / -Ura plates. After 3-5 days of culture, 4-5 monoclonal clones on the SD / -Ura plates were selected for colony PCR identification; wherein, the following pretreatment was also performed before the carrier DNA was added: insert it into a 95°C metal bath for 5 minutes, and quickly insert it into ice after heating. After agarose gel electrophoresis, confirm whether the size of the inserted band is normal. Select the correctly identified monoclonal clone for AbA concentration screening. Identification primers: pAbAi-F: SEQ ID NO.14; pAbAi-R: SEQ ID NO.15.

[0096] Table 8

[0097]

[0098] The bait vector pAbAi-BcCLH2 was selected and cultured in 50ml YPDA (adenine sulfate yeast extract peptone glucose medium) at 30°C, 230-250rpm until OD600 reached 0.4-0.5. Then, Y1H Gold yeast cells were collected and resuspended with 3ml 1.1×PEG / LiAc. 10μg yeast library plasmid (the yeast library was constructed by Shanghai Ouyi Biomedical Technology Co., Ltd.) and 50μL Carrier DNA were added and mixed. Then 2.5ml 1×PEG / LiAc was added and cultured in a 30°C water bath for 45min; 160μLDMSO (dimethyl sulfoxide) was added and cultured in a 42°C water bath for 20min; 3ml YPD (yeast extract peptone glucose medium) was added and incubated at 30°C for 90min. Finally, the cells were suspended in 1ml 0.9% NaCl solution. Dilute the cultured bacteria 10 times, 100 times, 1000 times, and 10000 times, and spot them on SD / -leu plates and SD / -leu / 150μL AbA* plates, respectively, and incubate at 30℃ for 3-5 days. When the single clone grows to 1-2mm, clone identification is performed. The results are as follows:

[0099] The recombinant plasmid pAbAi-BcCLH2 can grow on SD / -Ura plates, indicating that the recombinant plasmid can successfully transform yeast cells, and 150ng / ml AbA can inhibit the growth of background bacteria ( Figure 5 Y1HGold (pAbAi-BcCLH2) was screened in the yeast library. A total of 33 clones were screened in the SD / -Leu / AbA (150 ng / ml) screening. Figure 6 ). The gene BcWRKY1 (transcription factor, corresponding nucleotide sequence as SEQ ID NO.4) was screened out by sequencing the positive clones.

[0100] 3. Yeast one-hybrid and dual-luciferase reporter assays validate the interaction between BcWRKY1 and BcCLH2

[0101] (1) Yeast one-hybrid assay

[0102] The prey vector pGADT7-BcWRKY1 was constructed according to the method in Example 1, and the primer sequences were F: SEQ ID NO.16; R: SEQ ID NO.17. The bait vector pAbAi-BcCLH2 was selected and cultured in 50 ml YPDA (adenine sulfate yeast extract peptone glucose medium) at 30°C, 230-250 rpm until OD600 reached 0.4-0.5. Then, pAbAi-BcCLH2 yeast cells were collected and resuspended with 3 ml 1.1×PEG / LiAc. 1 μg pGADT7-BcWRKY1 / pGADT7 recombinant plasmid and 5 μL Carrier DNA were added and mixed. Then 2.5 ml 1×PEG / LiAc was added and cultured in a 30°C water bath for 30 min; 20 μL DMSO was added and cultured in a 42°C water bath for 15 min; high-speed centrifugation was performed for 15 s, and the supernatant was discarded. Finally, the cells were suspended in 100 μL 0.9% NaCl solution. The cultured bacteria were diluted 10-fold, 100-fold, 1000-fold, and 10,000-fold, and spotted on SD / -leu plates and SD / -leu / 150μL AbA* plates, respectively, and incubated at 30°C for 3-5 days.

[0103] The results are as follows Figure 7 As shown, pAbAi-BcCLH2+pGADT7 and pAbAi-BcCLH2+pGADT7-BcWRKY1 were able to grow bacteria on SD / -leu plates, proving that pGADT7-BcWRKY1 and pGADT7 were successfully transformed into Y1H Gold yeast cells; pAbAi-BcCLH2+pGADT7-BcWRKY1 grew white colonies on SD / -leu / 150μLAbA* plates, while pAbAi-BcCLH2+pGADT7 did not, proving that gene BcWRKY1 can interact with gene BcCLH2.

[0104] (2) Dual luciferase reporter assay

[0105] According to the method in Example 1, LUC-BcCLH2 recombinant plasmid and pCAMBIA2300-BcWRKY1 were constructed, and the primer sequences were LUC-BcCLH2-F: SEQ ID NO.18, LUC-BcCLH2-R: SEQ ID NO.19 and pCAMBIA2300-BcWRKY1-F: SEQ ID NO.20, pCAMBIA2300-BcWRKY1-R: SEQ ID NO.21. LUC-BcCLH2, pCAMBIA2300-BcWRKY1 and pCAMBIA2300 Agrobacterium transformants were shaken in a shaker at 28°C overnight, and the OD value of the bacterial solution reached 0.8-1.0, centrifuged at 4000rpm for 8min, removed the supernatant, first suspended with 3ml of ddH2O, centrifuged at 4000rpm for 8min, then suspended with Activation buffer, centrifuged at 4000rpm for 8min, and obtained three bacterial solutions. Using Activation buffer as a control, the OD value of each bacterial solution was determined, and the OD value of each bacterial solution was adjusted to 0.8-1.0 with Activation buffer to obtain three target bacterial solutions. The target bacterial solution corresponding to LUC-BcCLH2 and the target bacterial solution corresponding to pCAMBIA2300-BcWRKY1 were mixed in a ratio of 1:1, and the target bacterial solution corresponding to the control group LUC-BcCLH2 and the target bacterial solution corresponding to the pCAMBIA2300 Agrobacterium transformant were mixed in a ratio of 1:1 to obtain two mixed bacterial solutions; finally, AS (acetosyringone) was added to each mixed bacterial solution at a ratio of mixed bacterial solution: AS = 1000:1. After static culture at 28°C in the dark for 3 hours, the samples were injected simultaneously at different positions on the same leaf of the same Nicotiana benthamiana. After injection, the samples were treated in the dark for 1 day and cultured under light for about 2 days, and then the luminescence was observed using a living imager (such as Figure 8 ).

[0106] We used dual luciferase assays to determine how BcWRKY1 regulates the activity level of BcCLH2. The results showed that BcWRKY1 negatively regulated the expression level of BcCLH2 ( Figure 8 ).

[0107] In summary, the application of a gene BcWRKY1 provided in the present application in regulating the yellow-green phenotype of Wucai leaves can be used in the genetic breeding of Wucai, providing a new idea for the cultivation of related varieties.

[0108] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Genes BcWRKY1 Application of the gene in regulating the yellow-green phenotype of Wucai leaves BcWRKY1 The nucleotide sequence is shown in SEQ ID NO.4, and the application includes overexpressing gene BcWRKY1 , suppressor gene BcCLH2 The expression of BcCLH2 The nucleotide sequence is as shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The overexpressed gene BcWRKY1 Including constructing genes BcWRKY1 overexpression vector.

3. The use as claimed in claim 1, wherein the Wucai variety is W7-2 or WS-1.

4. A method for regulating chlorophyllase activity in Wucai leaves, the method comprising: Overexpression of the gene of claim 1 BcWRKY1 , inhibiting the gene described in claim 1 BcCLH2 The expression of chlorophyllase in Wucai leaves decreased.

5. Genes BcWRKY1 ,Gene BcWRKY1 Encoded protein or containing gene BcWRKY1 Application of biological materials in the preparation of products for promoting the transformation of Wucai leaves from yellow to green; BcWRKY1 The nucleotide sequence is shown in SEQ ID NO.

4.

6. The use according to claim 5, wherein the biological material is a plasmid, an expression vector or a transgenic cell.

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