Application and method of BcCGA1. A gene in regulation and control of ascorbic acid content of plants

Abstract

The invention discloses an application and a method of a BcCGA1. A gene in regulating and controlling the content of ascorbic acid in a plant. A large number of experimental studies find that the BcCGA1. A gene plays an important role in regulating and controlling the ascorbic acid content of the plant, specifically, the ascorbic acid content of the plant can be increased by reducing the expression quantity of the BcCGA1. A gene, and the expression quantity of the BcCGA1. A gene is influenced by the gibberellin content. On the basis, the invention provides a reagent, namely paclobutrazol, for increasing the ascorbic acid content of the plants, and an application method is designed according to the reagent, the method can effectively increase the ascorbic acid content of the plants under the weak light condition, and is simple, convenient, wide in application range, low in applied paclobutrazol content and low in influence on the environment; popularization and application are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an application and method of a BcCGA1.a gene in regulating plant ascorbic acid content. Background Art

[0002] Ascorbic acid is one of the essential nutrients for humans. Lack of ascorbic acid can lead to decreased immunity and, in severe cases, scurvy. Humans cannot synthesize ascorbic acid themselves and rely mainly on foods such as vegetables and fruits. Non-heading cabbage is a common leaf vegetable in my country, occupying an important market share in my country's vegetable industry. Ascorbic acid is a common nutrient in vegetables. With the improvement of people's living standards, the requirements for the nutritional quality of vegetables are also getting higher and higher. Improving the ascorbic acid content in non-heading cabbage is an important part of the cultivation of high-quality non-heading cabbage.

[0003] Light is an important factor affecting plant growth. In previous studies, our research group found that when tomatoes and pineapples were exposed to light and darkness, the ascorbic acid content was significantly higher under light than under darkness; weak light treatment during the ripening period of tomato fruits would lead to a decrease in the ascorbic acid content of tomato fruits, especially when weak light treatment was applied during the fruit harvest period, the ascorbic acid content concentration dropped to the lowest. Non-heading cabbage is a cold-loving vegetable. Traditional land cultivation often starts sowing in late August. The light intensity changes from strong to weak, which will have a greater impact on the quality of the cabbage, especially in areas with more rainy weather. In addition, in the process of facility cultivation of non-heading cabbage, the light intensity in the facility greenhouse is lower than that of land cultivation. The aging of the covering material on the top of the greenhouse, dust pollution, and refraction of water droplets will affect the transmittance of the sun. In addition, the shading of the frame material and changes in the building orientation will affect the light intensity inside the greenhouse. In order to pursue higher quality non-heading cabbage, artificial lighting is often used, but the use of this method will increase production costs. The installation and maintenance of the fill light also requires professional technical support, which is not suitable for people without relevant technical conditions. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an application and method of the BcCGA1.a gene in regulating the ascorbic acid content of plants, which can effectively increase the ascorbic acid content of plants under weak light conditions, and has the advantages of low cost, simplicity, ease of operation, and wide applicability.

[0005] The present invention is achieved through the following technical solutions:

[0006] Application of BcCGA1.a gene in regulating plant ascorbic acid content: Reducing the expression level of BcCGA1.a gene can increase the ascorbic acid content of plants.

[0007] Application of agents for inhibiting the expression of BcCGA1.a gene in increasing the ascorbic acid content of plants.

[0008] Preferably, the agent that inhibits the expression of the BcCGA1.a gene is a gibberellin biosynthesis inhibitor.

[0009] Preferably, the gibberellin biosynthesis inhibitor is paclobutrazol.

[0010] Preferably, the gibberellin content of the plant is reduced by applying paclobutrazol, thereby reducing the expression level of the BcCGA1.a gene, thereby increasing the ascorbic acid content of the plant.

[0011] Preferably, the plant is non-heading cabbage.

[0012] A method for increasing the ascorbic acid content of plants comprises the following steps:

[0013] Step 1) dissolving paclobutrazol in anhydrous ethanol to prepare a paclobutrazol mother solution;

[0014] Step 2) The paclobutrazol mother solution is prepared with water to prepare a paclobutrazol spraying solution, which is evenly sprayed on the leaves of the plants.

[0015] Preferably, the concentration of the paclobutrazol mother solution in step 1) is 50 mg / mL.

[0016] Preferably, the final concentration of the paclobutrazol spraying solution in step 2) is 50 mg / L.

[0017] Preferably, the plants in step 2) are cultivated under weak light.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention has found through a large number of experimental studies that the BcCGA1.a gene plays an important role in regulating the ascorbic acid content of plants. According to the association between the BcCGA1.a gene and the ascorbic acid content, a new germplasm of non-heading cabbage with a high ascorbic acid content is obtained by CRISPR knockout. At the same time, according to the relationship between the BcCGA1.a gene and the gibberellin content and the weak light environment, a reagent for increasing the ascorbic acid content of non-heading cabbage under weak light conditions and a method for using the reagent have been discovered. The reagent is easy to purchase and the method is simple and easy to use. The reagent can effectively increase the ascorbic acid of non-heading cabbage by more than 50% compared with the control group under weak light conditions. In addition, the reagent and method have a low impact on the environment and can be applied to the large-scale production of non-heading cabbage with a high ascorbic acid content. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The graphs are the changes of ascorbic acid content (A), total ascorbic acid content (B) and expression level of BcCGA1.a gene (C) under different shading levels in Example 1;

[0021] Figure 2 This is a comparison diagram of the expression level (A), ascorbic acid content (B) and total ascorbic acid content (C) of the BcCGA1.a gene after virus-induced silencing in Example 1;

[0022] Figure 3 It is a specific flow chart of the application of paclobutrazol reagent in Example 2;

[0023] Figure 4 This is a comparison chart of the ascorbic acid content of non-heading cabbages in each experimental group in Example 2. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] The non-heading cabbage variety “49 caixin” used in the following examples was from the Cabbage Systems Biology Laboratory of the College of Horticulture, Nanjing Agricultural University.

[0028] Example 1 Screening of agents for increasing the ascorbic acid content of non-heading Chinese cabbage under weak light conditions

[0029] 1. Experimental steps

[0030] (1) Based on the constructed high-density genetic linkage map and the ascorbic acid content measured at multiple points over many years, QTL positioning was performed, and a total of 19 QTL loci were obtained. The 19 QTL loci were annotated and 3367 candidate genes were obtained. Then, transcriptome analysis was performed on the 3367 candidate genes. Based on the co-expression of candidate genes in varieties with high ascorbic acid content, 524 genes were screened. GO analysis of the 524 candidate genes showed that these 524 candidate genes mainly responded to the environment. Transcription factors are important factors for plants to respond to the environment. 23 transcription factors were extracted from the 524 candidate genes. Promoter analysis showed that these 23 transcription factors mainly responded to light.

[0031] (2) "Wutacai" with high ascorbic acid content was selected and subjected to normal light, 20% shading, 40% shading and 90% shading treatments. The ascorbic acid content was measured on the third day of treatment. In order to ensure the stability of the effect of shading on the ascorbic acid content, the ascorbic acid content and the expression level of the BcCGA1.a gene were measured again 24 days after treatment.

[0032] (3) The BcCGA1.a gene was silenced in "49 Chinese cabbage" using virus-induced silencing technology. A 40 bp highly specific fragment was designed in the gene domain region and sent to GenScript for synthesis of the vector pTY-BcCGA1.a. The pTY empty vector was used as a control. The "49 Chinese cabbage" seedlings at one month old were bombarded by gene gun to obtain a silent strain. The expression level of the BcCGA1.a gene in the strain was then identified by fluorescent quantitative PCR, and the ascorbic acid content was measured.

[0033] 2. Experimental results

[0034] like Figure 1 As shown in the figure, after 24 days of shading treatment at different degrees, the ascorbic acid content was determined and it was found that the ascorbic acid content ( Figure 1 A) and total ascorbic acid content ( Figure 1 B) gradually decreased with the increase of shading degree, and the expression level of BcCGA1.a gene was found ( Figure 1 Middle C) gradually increases with the increase of shading degree and is negatively correlated with ascorbic acid content.

[0035] The BcCGA1.a gene is a member of the GATA transcription factor family and a cytokinin response factor. Its NCBI number is NC_003075.

[0036] Previous studies (Richter R, Behringer C, Müller IK, Schwechheimer C. The GATA-type transcription factors GNC and GNL / CGA1 repress gibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS. Genes Dev. 2010 Sep 15; 24 (18): 2093-104. doi: 10.1101 / gad.594910.) have shown that the expression of BcCGA1.a is affected by the gibberellin content. Based on this, we measured the endogenous gibberellin content in non-heading cabbage after 24 days of shading treatment, and the gibberellin content was significantly increased after shading.

[0037] At the same time, we also silenced the expression of BcCGA1.a gene in "49 choy sum" by virus-induced silencing technology. Figure 2 As shown, the expression level of BcCGA1.a gene in the two strains was significantly decreased by fluorescence quantitative PCR. Figure 2 A), further determination of ascorbic acid content found that the ascorbic acid content ( Figure 2 B) and total ascorbic acid content ( Figure 2 C) were significantly increased (P < 0.05), which further illustrates that the BcCGA1.a gene is related to the ascorbic acid content in non-heading cabbage.

[0038] Based on the above experimental data, we speculate that the gibberellin content of non-heading cabbage increased after shading, resulting in an increase in the expression of the BcCGA1.a gene, which in turn led to a decrease in ascorbic acid content. Paclobutrazol, as a gibberellin biosynthesis inhibitor, may increase the ascorbic acid content of non-heading cabbage under low light conditions.

[0039] Example 2 Effect of Paclobutrazol on the Ascorbic Acid Content of Non-heading Cabbage Grown under Low Light Conditions

[0040] 1. Experimental steps (such as Figure 3 (shown)

[0041] (1) Cultivated 35-day-old "49 Chinese cabbage" seedlings were subjected to 90% shading for 3 days, and a normal light treatment was set as a control group.

[0042] (2) Paclobutrazol was dissolved in anhydrous ethanol to prepare a 50 mg / mL paclobutrazol stock solution.

[0043] (3) The paclobutrazol mother solution was prepared with water to prepare a paclobutrazol spray solution with a final concentration of 50 mg / L, and the solution was evenly sprayed on the leaves of "49 Chinese cabbage heart" treated with weak light. Clean water was used as a blank control. The spray droplets were evenly distributed on the leaf surface without dripping in strands. At the same time, blank treatment and gibberellin (GA) treatment were carried out under normal light.

[0044] (4) The ascorbic acid content was measured 8 days after spraying.

[0045] 2. Experimental results

[0046] like Figure 4As shown, according to the measured ascorbic acid content, it was found that under weak light conditions, the ascorbic acid content of non-heading cabbage sprayed with paclobutrazol solution (Shading-Paclobutrazol group) was significantly increased by 50% compared with the clear water treatment (Shading-CK group) (P < 0.05); while under normal light, the ascorbic acid content of non-heading cabbage treated with gibberellin (Light-GA group) was significantly decreased compared with the clear water treatment (Light-CK group) (P < 0.05).

[0047] The experimental results of this embodiment show that under normal light, gibberellins reduce the ascorbic acid content, which is similar to the reduction of ascorbic acid content under weak light conditions. Gibberellins may be an important endogenous regulatory hormone for reducing ascorbic acid content under weak light. Therefore, when paclobutrazol is applied under weak light cultivation, the ascorbic acid content of plants can be effectively increased.

[0048] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. Application of the BcCGA1.a gene in regulating the ascorbic acid content of plants, characterized in that: Reducing the expression level of the BcCGA1.a gene can increase the ascorbic acid content in plants.

2. Application of agents that inhibit the expression of the BcCGA1.a gene in increasing the ascorbic acid content in plants.

3. The use according to claim 2, characterized in that: The agent for inhibiting the expression of the BcCGA1.a gene is a gibberellin biosynthesis inhibitor.

4. The use according to claim 3, characterized in that: The gibberellin biosynthesis inhibitor is paclobutrazol.

5. The use according to claim 4, characterized in that: The gibberellin content of the plant is reduced by applying paclobutrazol, thereby reducing the expression level of the BcCGA1.a gene, thereby increasing the ascorbic acid content of the plant.

6. The use according to any one of claims 1 to 5, characterized in that: The plant is non-heading cabbage.

7. A method for increasing the ascorbic acid content of plants, characterized in that: The following steps are involved: Step 1) dissolving paclobutrazol in anhydrous ethanol to prepare a paclobutrazol mother solution; Step 2) The paclobutrazol mother solution is prepared with water to prepare a paclobutrazol spraying solution, which is evenly sprayed on the leaves of the plants.

8. A method for increasing the ascorbic acid content of plants according to claim 7, characterized in that: The concentration of the paclobutrazol mother solution in step 1) is 50 mg / mL.

9. A method for increasing the ascorbic acid content of plants according to claim 7, characterized in that: Step 2) The final concentration of the paclobutrazol spraying solution is 50 mg / L.

10. A method for increasing the ascorbic acid content of plants according to claim 7, characterized in that: Step 2) The plants are cultivated under weak light.

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