BcRPGE1 gene for regulating petiole color differentiation of non-heading Chinese cabbage and application of BcRPGE1 gene
By cloning and validating the BcRPGE1 gene and regulating its expression level to change the petiole color of non-heading Chinese cabbage, the problem of insufficient research on petiole color differentiation genes has been solved, and efficient breeding improvement has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-03-27
- Publication Date
- 2026-05-01
AI Technical Summary
Current technologies lack research on key regulatory genes for petiole color differentiation in non-heading Chinese cabbage, making it difficult to meet the industrial needs of targeted breeding.
The BcRPGE1 gene was cloned and validated. By regulating its expression level, the color differentiation of petioles in non-heading Chinese cabbage was controlled, including silencing or overexpressing the gene to alter chlorophyll accumulation in the petioles, thereby achieving targeted regulation of petiole color.
It provides clearly defined target genes for the targeted improvement of petiole color in non-heading Chinese cabbage, significantly shortening the breeding cycle and improving breeding efficiency. It is highly stable and has strong targeting, making it suitable for molecular marker-assisted breeding and gene editing breeding.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of plant genetic engineering and molecular biology, and in particular to a method for regulating the color differentiation of petioles in non-heading Chinese cabbage. BcRPGE1 Genes and their applications. Background Technology
[0002] Non-heading cabbage ( Brassicacampestris L. ssp. chinensis Makino (Brassica rapa) is a subspecies of Chinese cabbage belonging to the Brassicaceae family. It includes varieties such as green-stemmed cabbage, black cabbage, and choy sum. It is the third largest vegetable crop in my country in terms of planting area and consumer demand, possessing both high nutritional and economic value. As a leafy vegetable, the agronomical characteristics of its leaves, such as color and morphology, directly determine its market value. Current research on non-heading Chinese cabbage focuses primarily on the leaves, resulting in the cultivation of varieties with various leaf colors, including green, purple, and yellow. Extensive research has been conducted on leaf color. The petiole is also a core edible organ of non-heading Chinese cabbage, and its color is a key indicator for evaluating the commercial quality of this crop.
[0003] Different regional markets exhibit significant preferences for the petiole color of non-heading Chinese cabbage. In North China, the dark green petioles of the Qinggeng variety are preferred, while in the Yangtze-Huaihe River Basin, the white petioles and dark green leaves of the Wucai variety are the mainstream cultivation. Research on the regulatory mechanism of petiole color differentiation is of significant industrial value for the targeted breeding of non-heading Chinese cabbage varieties that meet market demands. However, current research on the discovery and functional verification of key regulatory genes for petiole color differentiation is still relatively scarce, and its molecular regulatory mechanism remains unclear, making it difficult to meet the industrial needs of targeted breeding for non-heading Chinese cabbage with well-defined petiole colors.
[0004] Gene mapping and functional verification are the core methods for elucidating the molecular mechanisms of plant trait differentiation. By identifying key functional genes that regulate the differentiation of petiole color in non-heading Chinese cabbage and clarifying their roles in color trait regulation, we can provide core gene resources and theoretical basis for molecular breeding of petiole color in non-heading Chinese cabbage.
[0005] Our laboratory previously identified the gene locus numbered Bc03G00770.1 in non-heading Chinese cabbage through gene mapping. The genome sequence of this locus has been described in the literature "Lingyun Yuan, Jian Wang, Haoying Zhang, et al. The wucai genome and DNA methylation regulation on the inner leaves'yellowing response to low temperature [J]". Horticulture Research,The study disclosed in "2025, 12:uhaf231" that the gene's specific function, whether it is involved in the regulation of petiole color differentiation, and its application value in improving the color trait of non-heading Chinese cabbage have not been verified.
[0006] Based on this, the present invention addresses the above-mentioned... Bc03G00770.1 Gene cloning and functional verification studies were conducted, and it was named after sequence alignment. BcRPGE1 The gene was identified and confirmed to regulate the color differentiation of petioles in non-heading Chinese cabbage, providing new genetic resources and technical support for the molecular regulation and breeding application of petiole color in non-heading Chinese cabbage. Summary of the Invention
[0007] The technical problem to be solved by this invention is to provide a method for regulating the color differentiation of petioles in non-heading Chinese cabbage. BcRPGE1 Genes and their applications.
[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A method for regulating the color differentiation of petioles in non-heading Chinese cabbage BcRPGE1 Genes, the ones mentioned BcRPGE1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0009] As one of the preferred embodiments of the present invention, the BcRPGE1 The expression level of the gene is negatively correlated with the accumulation of chlorophyll in the petioles of non-heading Chinese cabbage; by adjusting the expression of this gene in non-heading Chinese cabbage plants, the differentiation process of petiole color can be directly regulated.
[0010] One of the above BcRPGE1 Application of genes in regulating the color differentiation of petioles in non-heading Chinese cabbage.
[0011] A method for regulating the color of petioles in non-heading Chinese cabbage, by controlling the internal structure of the non-heading Chinese cabbage plant. BcRPGE1 The gene expression level alters the amount of chlorophyll accumulation in the petiole, thereby achieving targeted regulation of petiole color; BcRPGE1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0012] As one of the preferred embodiments of the present invention, the aforementioned [factors] within the non-heading cabbage are silenced or knocked out. BcRPGE1 The gene is reduced, which increases the accumulation of chlorophyll in the plant's petiole and deepens the green color of the petiole.
[0013] As one of the preferred embodiments of the present invention, by overexpressing the BcRPGE1 The gene was increased to improve its expression level in non-heading Chinese cabbage plants, resulting in reduced chlorophyll accumulation in the petioles and a paler green color in the petioles.
[0014] The above method is applied to the directional improvement of petiole color in non-heading Chinese cabbage and the cultivation of non-heading Chinese cabbage varieties with different petiole colors.
[0015] The advantages of this invention compared to the prior art are: This invention is the first to clone a substance that regulates the color differentiation of petioles in non-heading Chinese cabbage. BcRPGE1 This gene fills the technological gap in the existing technology regarding the lack of research on key genes for petiole color differentiation in non-heading Chinese cabbage. The BcRPGE1 The expression level of the gene is negatively correlated with the accumulation of chlorophyll in the petioles of non-heading Chinese cabbage. The change of petiole color between green and white can be precisely regulated by silencing or overexpressing the gene, providing a clear target gene for the targeted improvement of petiole color in non-heading Chinese cabbage. Meanwhile, the genes and regulation methods provided by this invention have high stability and strong targeting, and can be applied to molecular marker-assisted breeding and gene editing breeding of non-heading Chinese cabbage. They can significantly shorten the breeding cycle and improve the breeding efficiency of varieties with different petiole colors, and have important theoretical value and industrial application prospects. Attached Figure Description
[0016] Figure 1 This is the pEarleyGate202 vector map in Example 2 (including...) BcRPGE1 (Gene insertion site and related element annotation); Figure 2 In Example 2 BcRPGE1 Comparison of electrophoresis results for genes and recombinant vectors; Figure 3 This is a graph showing the results of Western blot analysis of BcRPGE1 protein expression levels using Anti-FLAG antibody in Example 3 (in the graph, Col-0 represents wild-type Arabidopsis thaliana, and OE-1, OE-2, and OE-3 represent OE-...). BcRPGE1 (transgenic Arabidopsis thaliana plants) Figure 4 It is the wild-type Arabidopsis thaliana and OE- in Example 3 BcRPGE1 Phenotypic diagram of transgenic Arabidopsis plants (in the diagram, Col-0 represents wild-type Arabidopsis, OE-1, OE-2, and OE-3 represent OE-type Arabidopsis). BcRPGE1 (transgenic Arabidopsis thaliana plants) Figure 5 This is a comparison of petiole color phenotypes in VIGS-silenced plants of non-heading Chinese cabbage in Example 4 (in the figure, pCVA::00 / W16-18 is the empty vector control plant, pCVA:: BcRPGE1 #1 / W16-18 and pCVA:: BcRPGE1 #2 / W16-18 is BcRPGE1 Gene-silenced lines (the petioles of the silenced lines were significantly darker in color than the control). Detailed Implementation
[0017] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0018] Example 1 BcRPGE1 Gene cloning and purification: (1) Total RNA extraction and cDNA reverse transcription Total RNA was extracted from non-heading Chinese cabbage plants using the Trizol method in combination with chloroform and isopropanol reagents. The extracted total RNA was then reverse transcribed to synthesize cDNA, which served as a template for subsequent gene amplification and was stored at low temperature for later use.
[0019] (2) Specific primer design and PCR amplification according to BcRPGE1 Specific homologous recombination primers BcRPGE1-F (SEQ ID NO.2) and BcRPGE1-R (SEQ ID NO.3) were designed based on the gene sequence (SEQ ID NO.1). Using the non-heading Chinese cabbage cDNA obtained in step (1) as a template, a PCR reaction system was configured and amplified. The PCR reaction system is shown in Table 1. The PCR amplification program was as follows: 98 ℃ pre-denaturation for 30 s; 98 ℃ denaturation for 15 s, 58 ℃ annealing for 15 s, 68 ℃ extension for 10 s, for a total of 32 cycles; 68 ℃ complete extension for 5 min, and cooling at 8 ℃ for 10 min.
[0020] Table 1 PCR reaction system
[0021] (3) Identification and purification of PCR products After amplification, the PCR products were identified by 1.5% agarose gel electrophoresis to confirm the acquisition of the target-specific band. Subsequently, the target gene fragment was purified using a product purification kit (Beijing TransGen Biotech Co., Ltd.), and the concentration of the recovered product was determined using a nucleic acid quantification instrument. BcRPGE1 The gene fragments were used in subsequent experiments.
[0022] Example 2 BcRPGE1 Construction of gene overexpression vectors and Agrobacterium-positive strains: (1) Vector plasmid extraction and double enzyme digestion The plasmid of the plant overexpression vector pEarleyGate202 was extracted using a plasmid extraction kit (Beijing TransGen Biotech Co., Ltd.) following the instructions. The extracted vector plasmid was then digested with Kpn I and Sal I, and the linearized vector fragment was recovered after complete digestion for later use.
[0023] The plant overexpression vector pEarleyGate202 is a well-known plant binary expression vector in the art. It carries a multiple cloning site and a natural 3×FLAG tag sequence downstream of the CaMV 35S promoter, which facilitates subsequent protein detection.
[0024] (2) Construction of recombinant carrier Based on the principle of homologous recombination, the purified product from Example 1... BcRPGE1 The target gene fragment was ligated with the linearized pEarleyGate202 vector fragment obtained in step (1) to construct the recombinant overexpression vector pEarleyGate202-35S- BcRPGE1 -3×FLAG, its insertion position and structure are shown in [reference]. Figure 1 The constructed recombinant vector was transferred into DH5α Escherichia coli competent cells, plated on resistant plates, and single-clone bacterial cultures were picked and sent to a sequencing company for sequencing.
[0025] Sequencing results showed that the obtained sequence was completely identical to the reference genome sequence, indicating that the recombinant overexpression vector was successfully constructed. Further PCR verification of the recombinant vector showed that the amplified vector fragment was longer than [a certain length]. BcRPGE1 Single gene segment ( Figure 2 ), directly prove BcRPGE1 The gene has been successfully inserted into the overexpression vector and can be used for subsequent Agrobacterium-mediated transformation.
[0026] (4) Preparation of Agrobacterium-positive strains The successfully constructed and verified pEarleyGate202-35S- BcRPGE1 The 3×FLAG recombinant vector plasmid was transformed into GV3101 Agrobacterium competent cells. After PCR amplification and verification that it was a positive strain, the bacterial culture was preserved for subsequent Arabidopsis genetic transformation experiments.
[0027] Example 3: Heterologous transformation of Arabidopsis thaliana BcRPGE1 Gene function verification: (1) Agrobacterium-mediated transformation of Arabidopsis thaliana Healthy wild-type Arabidopsis thaliana Col-0 plants were selected, and the inflorescences were inoculated with the GV3101-positive Agrobacterium tumefaciens obtained in Example 2. The inoculated plants were cultured in the dark for 24 hours, and then cultured under normal conditions until the plants matured and the seeds were harvested, which were the T0 generation transgenic Arabidopsis thaliana seeds.
[0028] (2) Screening of transgenic Arabidopsis plants T0 generation transgenic Arabidopsis seeds were sown on 1 / 2 MS agar selection medium supplemented with 50 µg / mL hygromycin B (HYG, Invitrogen, Carlsbad, California). Positive overexpressing Arabidopsis plants were obtained through resistance selection. Leaves from T1 generation transgenic plants were collected, and transgenic protein expression levels were detected by Western blotting (WB). Subsequent experiments used genetically stable T2 generation transgenic plants to ensure the reliability of the experimental results.
[0029] (3) Arabidopsis thaliana plant culture Wild-type Arabidopsis thaliana Col-0 was planted on ordinary 1 / 2 MS plates. BcRPGE1 Overexpression of transgenic Arabidopsis thaliana plants (OE- BcRPGE1 The samples were cultured on 1 / 2 MS selection plates containing 50 µg / mL hygromycin and cultured under conditions of 24 °C / 18 °C day / night temperature, 16 h light / 8 h dark, 75% relative humidity, and 200 mmol / L light intensity. -2 s -1 The seedlings were cultured under specific conditions, and after their growth stabilized, they were transplanted into nutrient pots for further cultivation for three weeks. The experiment was conducted in three biological replicates to reduce error.
[0030] (4) Protein expression level detection OE- was detected by Western blotting using FLAG antibody. BcRPGE1 The expression level of BcRPGE1 protein in plants was investigated, and the results showed that no BcRPGE1 protein was expressed in wild-type Col-0 plants, while OE- BcRPGE1 The accumulation of BcRPGE1 protein was significant in transgenic plants. Figure 3 This demonstrates that the genetic transformation of Arabidopsis thaliana was completely successful and can be used for subsequent phenotypic function verification.
[0031] (5) Phenotypic observation and gene function verification Phenotypic differences between the two groups of plants were observed under natural growth conditions: the leaves of wild-type Col-0 plants were normal green, while those of OE- BcRPGE1 The leaves of transgenic Arabidopsis plants exhibited a yellowish-green phenotype with a significantly lighter green color. Figure 4This result directly indicates that, BcRPGE1 The gene can regulate the leaf color differentiation of Arabidopsis thaliana. Combined with the previous gene localization and petiole color trait association analysis, it can be determined that the gene has a key regulatory function in the color trait differentiation of non-heading Chinese cabbage. This provides a solid core basis for subsequent verification of gene silencing and targeted regulation of petiole color in non-heading Chinese cabbage.
[0032] Example 4 BcRPGE1 Construction of gene silencing vector and functional verification of non-heading Chinese cabbage: To further explain BcRPGE1 In this embodiment, the regulatory role of white-stalked non-heading Chinese cabbage petioles was investigated using the VIGS gene silencing system to silence genes in the white-stalked non-heading Chinese cabbage variety 'W16-18'. BcRPGE1 The expression of genes was investigated to explore their regulatory function on petiole color differentiation. Among them, the non-heading Chinese cabbage variety 'W16-18' was described in the literature "Shuangshuang Wang. QTLmapping analysis of leaf size and wrinkle bubbles in Wucai ( Brassica campestris L.)[D]. Hefei: Anhui Agricultural University, 2022.; and this embodiment's 'W16-18' corresponds to material W16-18-1-2 in the literature.
[0033] (1) Silent fragment amplification according to BcRPGE1 Gene-specific sequence-specific primers for silencing fragment amplification, pCVA-BcRPGE1-F (SEQ ID NO. 4) and pCVA-BcRPGE1-R (SEQ ID NO. 5), were designed. Using non-heading Chinese cabbage cDNA as a template, amplification was performed according to the PCR amplification system and procedure in Example 1 to obtain pCVA. -BcRPGE1 The target fragment was obtained; the PCR amplification product was purified by gel extraction and the product concentration was determined using a nucleic acid quantification instrument for later use.
[0034] (2) Silent vector single enzyme digestion The plasmid of the silencing vector pCVA was extracted using a plasmid extraction kit. The vector plasmid was then digested with Kpn I rapid digestion enzyme. The digestion system (total volume 50 μL) consisted of 5 μL of 10×Quiekcut Buffer, 3 μL of Kpn I rapid digestion enzyme, 15 μL of vector plasmid, and 27 μL of ddH2O. After complete digestion, the linearized vector fragment was purified using a gel extraction kit, and the concentration of the recovered product was determined using a nucleic acid quantification instrument for later use.
[0035] (3) Construction and validation of recombinant silencing vectors Using a gene recombination kit, the purified linearized pCVA vector was combined with pCVA- BcRPGE1 The target fragment was ligated to construct the recombination silencing vector pCVA- BcRPGE1 The recombinant vector was transformed into DH5α competent E. coli cells, and single-clone bacterial cultures were picked and sent to a sequencing company for sequencing. The sequencing results were completely consistent with the reference genome sequence, indicating that pCVA- BcRPGE1 The silent carrier has been successfully constructed.
[0036] (4) Preparation of Agrobacterium-positive strains The correct pCVA will be verified. BcRPGE The silencing vector plasmid was transformed into GV3101 Agrobacterium competent cells. After being verified as a positive clone by PCR amplification, the bacterial culture was preserved for subsequent infection experiments on non-heading Chinese cabbage.
[0037] (5) Agrobacterium infection and phenotypic identification pCVA- The pCVB Agrobacterium-positive bacterial suspension was resuscitated separately, resuspended in injection buffer, and prepared as OD. 560 =0.8~1.0 bacterial suspension; after the two suspensions were left to stand in the dark for 3 hours, they were mixed at a volume ratio of 1:1 and injected to infect the cotyledons of 'W16-18' variety non-heading Chinese cabbage seedlings that were 10 days old.
[0038] The injection buffer preparation system consisted of: ddH2O 95 mL, glucose 0.5 g, 0.2 M MES-KOH 5 mL, 100 mM ACE 100 μL, and 0.2 M Na3PO4 1 mL.
[0039] The results showed that the white-stemmed, non-heading Chinese cabbage variety 'W16-18' was silent. After gene expression, the plant's white petioles turned green, demonstrating a significant phenotypic change and fully proving... Genes can effectively regulate the color differentiation of petioles in non-heading Chinese cabbage. ).
[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for regulating the color differentiation of petioles in non-heading Chinese cabbage BcRPGE1 Genes, characterized by, The BcRPGE1 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. As described in claim 1 BcRPGE1 Genes, characterized by, The BcRPGE1 The expression level of the gene is negatively correlated with the accumulation of chlorophyll in the petioles of non-heading Chinese cabbage; by adjusting the expression of this gene in non-heading Chinese cabbage plants, the differentiation process of petiole color can be directly regulated.
3. A device as described in claim 1 or 2 BcRPGE1 Application of genes in regulating the color differentiation of petioles in non-heading Chinese cabbage.
4. A method for adjusting the color of the petioles of non-heading Chinese cabbage, characterized in that, By regulating the internal structure of non-heading Chinese cabbage plants BcRPGE1 Gene expression levels can alter the amount of chlorophyll accumulation in petioles, thereby achieving targeted regulation of petiole color. The BcRPGE1 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
5. The method according to claim 4, characterized in that, By silencing or knocking out the aforementioned substances in non-heading cabbage... BcRPGE1 The gene is reduced, which increases the accumulation of chlorophyll in the plant's petiole and deepens the green color of the petiole.
6. The method according to claim 4, characterized in that, By overexpressing the above BcRPGE1 The gene was increased to improve its expression level in non-heading Chinese cabbage plants, resulting in reduced chlorophyll accumulation in the petioles and a paler green color in the petioles.
7. The application of the method as described in any one of claims 4 to 6 in the directional improvement of petiole color in non-heading Chinese cabbage and the cultivation of non-heading Chinese cabbage varieties with different petiole colors.