Application of BcPHYE gene in genetic breeding of Wucai

Abstract

The present invention provides an application of a BcPHYE gene in the genetic breeding of wucai, and relates to the technical field of genetic breeding. The nucleotide sequence table of the BcPHYE gene is shown in SEQ ID NO.1. By silencing the BcPHYE gene in wucai seedlings, the color of the heart leaves of wucai can be changed from chlorosis to yellow. The BcPHYE gene provided by the present invention provides a guiding idea for obtaining wucai seeds with yellow heart leaves, and is helpful for the genetic breeding of wucai.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding, and in particular to an application of a BcPHYE gene in the genetic breeding of Wucai. Background Art

[0002] Wucai, also known as collapsed cabbage, black cabbage, collapsed cabbage, collapsed pine, etc., is a variant of the Brassica rapa subspecies in the Cruciferae family. It is also called "vitamin cabbage" because it is rich in vitamins. It is the main seasonal leafy vegetable in the Jianghuai region of my country in autumn and winter. It has excellent characteristics such as wide adaptability, simple cultivation, and low production cost. Leaf color affects the commercial value and appearance quality of the commodity to a certain extent, so people have successively cultivated Wucai varieties with different leaf colors for consumers to choose from. Among them, "Huangxin Wu" is deeply loved by consumers because of its bright leaf color and sweet taste.

[0003] In the prior art, the methods for obtaining seeds of black cabbage with yellow heart leaves are mostly traditional planting methods, which have the disadvantages of long cycle and slow effect.

[0004] Therefore, from a genetic level, studying the mechanism and influencing factors of yellowing of the heart leaves of wucai is an important means to cultivate wucai varieties with yellow heart leaves. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies in the prior art, the present invention provides an application of the BcPHYE gene in the genetic breeding of wucai, so as to solve the technical problems such as the long cycle in obtaining wucai germplasm with yellow heart leaves by traditional planting methods in the prior art.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In a first aspect of the present invention, a BcPHYE gene is provided for use in genetic breeding of Wucai, wherein the nucleotide sequence of the BcPHYE gene is shown in SEQ ID NO.1.

[0010] Furthermore, the BcPHYE gene in the wucai seedlings was silenced to make its heart leaves fade.

[0011] Furthermore, after silencing the BcPHYE gene in the Wucai seedlings to make their heart leaves turn green, the method further includes: collecting the chlorotic parts of the leaves of the Wucai seedlings, cutting them into pieces, adding PBS buffer and fully grinding them to obtain grinding liquid, and sealing and storing them at -80°C.

[0012] Furthermore, the method also includes injecting the grinding liquid into the leaves of the wucai seedlings to make the heart leaves of the wucai turn green.

[0013] Furthermore, the silencing of the BcPHYE gene in the Wucai seedlings includes constructing a silencing vector;

[0014] The method for constructing the silencing vector comprises inserting a specific sequence into a linearized PTY vector to obtain a silencing vector; the nucleotide sequence of the specific sequence is shown in SEQ ID NO.2.

[0015] Furthermore, the silencing of the BcPHYE gene in the Wucai seedlings also includes:

[0016] The silencing vector is embedded with gold powder to obtain a plasmid wrapped with gold powder;

[0017] The plasmids wrapped in gold powder are blasted into the leaves of the black cabbage seedlings.

[0018] Furthermore, embedding the silencing vector with gold powder comprises:

[0019] To a 1.5mL centrifuge tube, add 8μL of liquid gold powder, 5μg of silencing vector, 50μL of 2.5mol / LCaCl2 and 20μL of 0.1mol / L spermidine in sequence; after adding the samples, place in an ice bath for 20min, vortex to mix; then continue vortexing for 10min; after vortexing, centrifuge at 8000rpm for 1min, discard the supernatant, add 10μL of anhydrous ethanol, and suspend for later use.

[0020] The second aspect of the present invention also provides an application of the aforementioned grinding liquid in the genetic breeding of Wucai.

[0021] Furthermore, the grinding liquid is injected into the Wucai seedlings to make the Wucai heart leaves turn green.

[0022] (III) Beneficial effects

[0023] The application of the BcPHYE gene provided by the present invention in the genetic breeding of Wucai has the following beneficial effects compared with the prior art:

[0024] There is a positive correlation between the BcPHYE gene and the yellowing or chlorosis of the heart leaves of Wucai. By silencing this gene in Wucai seedlings, the heart leaves of Wucai can turn from green to yellow, which is helpful for genetic breeding in related directions and shortens the required time, avoiding the defects of the existing technology such as the long cultivation cycle. 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 This is a comparison diagram of the results of the wucai seedlings being injected with plasmids in the embodiment;

[0027] Figure 2 This is a comparison chart of the fluorescence quantitative analysis results of the BcPHYE gene in the embodiment;

[0028] Figure 3 This is a comparison chart of the results after the Wucai seedlings in the embodiment were injected with grinding liquid;

[0029] Figure 4 This is a comparison chart of the results of fluorescence quantitative analysis of the BcPHYE gene after the Wucai seedlings were injected with grinding liquid in the example. DETAILED DESCRIPTION

[0030] 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.

[0031] In order to solve the defects of the prior art in obtaining black cabbage seeds with yellow heart leaves, such as a long period of time, the present application proposes a BcPHYE gene. By silencing the gene in black cabbage seedlings, the heart leaves of black cabbage can be made to turn green. After the grinding liquid obtained after collecting the chlorotic part is injected into the black cabbage seedlings, the heart leaves of black cabbage can be made to turn green and then turn yellow, thereby providing a new guiding idea for the genetic breeding of black cabbage.

[0032] In order to verify that the BcPHYE gene provided in the present application has relevant functions, the present application also provides the following examples.

[0033] Example 1

[0034] The following operations were performed using the Wucai variety W7-1 with green heart leaves as the material.

[0035] The seeds of W7-1 were planted in a plant growth room under the following conditions: 24±1°C during the day, 18±1°C at night, 14h / 10h photoperiod (light / dark), and 300μmol·m -2 ·s -1, relative humidity is 70%.

[0036] When the Wucai seedlings grow to 4-5 true leaves, the BcPHYE gene (shown in SEQ ID NO.1) is silenced.

[0037] The specific operations are:

[0038] 1. Construction of silencing vector:

[0039] A 40 bp specific sequence was selected from the coding region of the BcPHYE gene and combined with its antisense sequence to form an 80 bp specific sequence (SEQ ID NO. 2), which was submitted to GenScript Biotech (Nanjing) for synthesis to form a hairpin structure;

[0040] Then, the specific sequence was inserted into the linearized PTY vector (PTY vector was reported by J.YU in the foreign journal BIOLOGIA PLANTARUM, Issue 4, Pages 826-834; the PTY vector used in this example was purchased from GenScript Biotech (Nanjing)), and after sequencing verification, the silencing vector pTY-BcPHYE (completed by GenScript Biotech (Nanjing)) was obtained.

[0041] 2. Obtaining silent plants

[0042] The PTY empty plasmid and the silencing vector pTY-BcPHYE are respectively embedded with gold powder; wherein the steps of embedding the silencing vector pTY-BcPHYE with gold powder are as follows:

[0043] To a 1.5mL centrifuge tube, add 8μL of liquid gold powder, 5μg of silencing vector pTY-BcPHYE, 50μL of 2.5mol / L CaCl2 and 20μL of 0.1mol / L spermidine in sequence; after adding the samples, place in an ice bath for 20 minutes, vortex to mix; then continue vortexing for 10 minutes; after vortexing, centrifuge at 8000rpm for 1 minute, discard the supernatant, add 10μL of anhydrous ethanol, suspend for later use, and obtain the silencing vector pTY-BcPHYE wrapped with gold powder.

[0044] The steps of embedding the PTY empty plasmid with gold powder are the same as above, that is, 5 μg of the silencing vector pTY-BcPHYE is replaced with 5 μg of the PTY empty plasmid, and the obtained PTY empty plasmid is wrapped with gold powder.

[0045] Do the following:

[0046] The silencing vector pTY-BcPHYE wrapped with gold powder was synchronously injected into 5 Wucai seedlings obtained above using a desktop gene gun (PDS-1000 / He, USA);

[0047] The process was repeated 4 times to obtain 20 Wucai seedlings injected with the silencing vector pTY-BcPHYE wrapped in gold powder, which were used as seedling group 2.

[0048] The same operation as above was performed on the PTY empty plasmid wrapped with gold powder to obtain 20 Wucai seedlings bombarded with the PTY empty plasmid wrapped with gold powder, as seedling group 1.

[0049] Among them, seedling group 1 and seedling group 2 were obtained at the same time to maintain the same growth potential of the two, thereby ensuring the accuracy of the experiment.

[0050] About 28-32 days after the seedlings were bombarded, samples were taken from the diseased (chlorotic) parts of the two groups. Figure 1 As shown, A is a schematic diagram of the diseased parts of the plants in seedling group 1, and B is a schematic diagram of the diseased parts of the plants in seedling group 2. It can be seen that the heart leaves of the plants in seedling group 2 showed more significant chlorosis, and the expression level of the BcPHYE gene was determined by fluorescent quantitative PCR to detect the silencing efficiency.

[0051] The specific detection of silencing efficiency includes:

[0052] 1. Extract total RNA from the genomic DNA of Wucai according to the instructions of Takara RNA extraction kit. After quantification, the obtained RNA was added according to the system in Table 1 to synthesize cDNA, and the specific steps were carried out according to the instructions of Takara reverse transcription.

[0053] Table 1

[0054]

[0055] 2. According to the nucleotide sequence shown in SEQ ID NO.1, fluorescent primers for detecting the expression of BcPHYE gene were designed using Oligo 7.0 software, and the housekeeping gene Actin was used as a reference. The primer sequences were sent to General Biotechnology Company for synthesis (the fluorescent primers and housekeeping genes for detecting the expression of BcPHYE gene are shown in Table 2). After semi-quantitative PCR detection, the primers had no dimers and non-specific amplification, and NTC was set to detect whether the operation and primers met the requirements. After both were qualified, the samples were added on ice, and each sample was repeated 3 times and three biological replicates were performed. After the sample cover was closed, it was slightly centrifuged and the gene expression was detected in the CFX 96PCR instrument; wherein, the qRT-PCR system is shown in Table 3, and the qRT-PCR program is: 95℃30s, 95℃5s, 60℃15s, 72℃10s, image acquisition, 40 cycles, melting curve 58℃to 95℃5s.

[0056] Table 2

[0057]

[0058] Table 3

[0059]

[0060] The test results are as follows Figure 2 As shown, the results showed that compared with seedling group 1, the average expression level of the BcPHYE gene in seedling group 2 was significantly decreased, indicating that the silencing was successful and the silencing effect could be used in subsequent experiments.

[0061] At the same time, the leaf color comparison of the diseased parts of seedling group 1 and seedling group 2 showed that the red-green value a*, yellow-blue value b*, and lightness L* of group 2 were significantly higher than those of group 1 (Table 4).

[0062] Depend on Figure 2 As shown in Table 4, silencing of the BcPHYE gene can cause the corresponding diseased parts of Wucai to significantly turn green.

[0063] Table 4 Color difference values ​​of diseased parts

[0064] Onset site Brightness value L* Red and green value a* Yellow-blue value b* Group 1 47.14 -12.67 30.05 Group 2 67.22 -3.63 44.21

[0065] Among them, the color difference value is specifically determined as follows:

[0066] The leaf color parameters of the leaves were measured using a DS-700D colorimeter (Hangzhou Zhongse Technology Co., Ltd.). The diseased parts were measured separately, 3 positions were measured each time, and five biological replicates were performed at each position. The three main leaf color parameters are the brightness value L*, the red-green value a*, and the yellow-blue value b*. The brightness value L* represents the brightness of the test sample, and the brightness range of 0-100 represents from black to white. The red-green value a* represents the red and green colors of the sample, with positive values ​​representing red and negative values ​​representing green. The yellow-blue value b* represents the yellow and blue colors of the sample, with positive values ​​representing yellow and negative values ​​representing blue.

[0067] 3. Virus recovery and utilization

[0068] The chlorotic part of seedling group 2 was collected, 1 g was cut into pieces, mixed with 10 mL of PBS buffer, and then fully ground in a mortar. The ground liquid was collected in a 5 ml centrifuge tube to obtain the grinding liquid. The centrifuge tube was sealed again with a sealing film and stored in a -80 °C refrigerator.

[0069] When using, take out the grinding liquid and let it melt at room temperature, then inject it into the back of any green leaf of Wucai with 4-5 true leaves. 12-15 days after injection, the heart leaves of the plant injected with the grinding liquid will turn yellow.

[0070] Total RNA was extracted from the uninjected and injected plants. The concentration of RNA was detected using a spectrophotometer, and reverse transcription was performed based on the RNA concentration. The expression of the BcPHYE gene was detected in a CFX 96PCR instrument (using the same method as above), and the phenotype of the corresponding plants was observed and the color difference value was determined.

[0071] Phenotypic observations Figure 3 As shown, A is a control plant (not injected with grinding solution), and B is a seedling plant injected with grinding solution. Compared with the control plant (not injected with grinding solution), the heart leaves of the seedling plant injected with grinding solution are yellow.

[0072] Leaf color parameters were measured for the control plants and the seedling plants injected with the abrasive solution. The results showed that the red-green value a*, yellow-blue value b*, and lightness L* of the seedling plants injected with the abrasive solution were significantly higher than those of the control plants (Table 5), indicating that the heart leaves of the seedling plants injected with the abrasive solution were yellower and brighter.

[0073] Table 5 Seedling heart leaf color difference

[0074] Seedling heart leaf Brightness value L* Red and green value a* Yellow-blue value b* No injection of grinding fluid 45.09 -13.38 31.68 Injection grinding fluid 70.31 -2.09 45.74

[0075] Among them, the color difference value is specifically determined as follows:

[0076] The leaf color parameters were measured using a DS-700D colorimeter (Hangzhou Zhongse Technology Co., Ltd.). The central part of the heart leaf (3-4 leaves from the inside out) was measured, avoiding the veins. Three positions were measured each time, and five biological replicates were performed for each position.

[0077] Example 2

[0078] The same operation was performed on the green leaf germplasm materials of W15-7-9-1, W19-21-3, W20-14-1, W18-1-2-4, and W20-21-2. After 28-32 days of bombardment, the red-green value a*, yellow-blue value b*, and lightness L* of the diseased parts of these varieties were significantly higher than those of Group 1 (as shown in Table 6), and a chlorotic phenotype appeared; about 12-15 days after the injection of the grinding liquid obtained in Example 1, the red-green value a*, yellow-blue value b*, and lightness L* of the heart leaves of the plants after injection were significantly higher than those before injection (as shown in Table 7), indicating that the heart leaves of the plants injected with the grinding liquid turned yellow in color at the seedling stage. It can be obtained that silencing the BcPHYE gene can make the heart leaves of the Wucai germplasm with evergreen heart leaves turn green at the seedling stage, and the heart leaves of the Wucai germplasm with evergreen heart leaves injected with the grinding liquid can turn yellow rapidly at the seedling stage.

[0079] Table 6 Statistical color difference values ​​of diseased parts of green leaf varieties

[0080] Onset site Brightness value L* Red and green value a* Yellow-blue value b* Group 1 47.14 -12.67 30.05 W15-7-9-1 55.72 -3.39 40.25 W19-21-3 56.09 -2.63 38.05 W20-14-1 53.42 -3.36 39.75 W18-1-2-4 57.18 -2.03 39.13 W20-21-2 54.39 -4.82 40.87

[0081] Table 7 Color difference of green leaf varieties before and after injection

[0082]

[0083] In summary, the BcPHYE gene proposed in this application can be used for the genetic breeding of Wucai and plays a guiding role in the genetic breeding of Wucai. After silencing the gene, the heart leaves of Wucai can turn green, and the corresponding virus injected into Wucai seedlings can turn the heart leaves of Wucai yellow, which is of great significance to the genetic breeding of Wucai.

[0084] 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.

[0085] 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. A Bc Application of gene in making the leaves of wucai turn green, the Bc The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method according to claim 1 Bc The application of the gene in causing the leaves of Wucai to turn green is characterized in that: On the seedlings of Wucai Bc The gene was silenced to cause the heart leaves to turn green.

3. A method according to claim 2 Bc The application of the gene in causing the leaves of Wucai to turn green is characterized in that: The Bc After the gene is silenced to make the heart leaves turn green, the method further comprises: collecting the chlorotic parts of the leaves of the Wucai seedlings, cutting them into pieces, adding PBS buffer and grinding them fully to obtain grinding liquid, and sealing and storing them at -80°C; The Bc The gene silencing comprises: constructing a silencing vector, embedding the silencing vector with gold powder to obtain a plasmid wrapped with gold powder, and injecting the plasmid wrapped with gold powder into leaves of Wucai seedlings; The method for constructing the silencing vector comprises inserting a specific sequence into a linearized PTY vector to obtain a silencing vector; the nucleotide sequence of the specific sequence is shown in SEQ ID NO.

2.

4. A method according to claim 3 Bc The application of the gene in causing the leaves of Wucai to turn green is characterized in that: The method also includes injecting the grinding liquid into the leaves of the wucai seedlings to make the heart leaves of the wucai seedlings turn green.

5. A method according to claim 3 Bc The application of the gene in causing the leaves of Wucai to turn green is characterized in that: Embedding the silent vector with gold powder comprises: To a 1.5mL centrifuge tube, add 8μL of liquid gold powder, 5μg of silencing vector, 50μL of 2.5 mol / LCaCl2 and 20μL of 0.1mol / L spermidine in sequence; after adding the samples, place in an ice bath for 20 minutes, vortex to mix; then continue vortexing for 10 minutes; after vortexing, centrifuge at 8000rpm for 1 minute, discard the supernatant, add 10μL of anhydrous ethanol, and suspend for later use.

6. Use of the grinding liquid described in claim 3 in making the leaves of Wucai turn green.

Images