A CgFAD gene regulating fruit base length and its application
By identifying and regulating the CgFAD gene, the lack of genes related to the length of citrus fruit base was solved, resulting in a significant reduction in fruit base length and improved fruit appearance quality and shelf life.
Patent Information
- Application Number
- CN202510402688.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-01
- Publication Date
- 2025-11-14
- Estimated Expiration
- 2045-04-01
AI Technical Summary
Currently, no research has reported on genes related to the length of the fruit base in citrus fruits, which leads to a longer fruit base and reduces the appearance quality, edibility, and shelf life of grapefruits.
The CgFAD gene, which regulates the length of the fruit base, was discovered and identified. By overexpressing or reducing its expression level, the length of the fruit base can be regulated, providing a new gene resource.
By regulating the expression level of the CgFAD gene, the length of the fruit base was significantly reduced, the appearance quality of the fruit was improved, and the shelf life was extended.
Smart Images
Figure CN120210222B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a CgFAD gene that regulates the length of the fruit base and its application. Background Technology
[0002] Grapefruit (Citrus maxima (Burm.) Merr.) is an important citrus fruit. The fruit base, also called the fruit neck, fruit stem, or fruit stalk, refers to the distance from the stem end of the grapefruit to the flesh. Figure 1 Currently, the widely cultivated Shatin pomelo, Guanxi honey pomelo, and Dongshi early pomelo all have relatively long fruit bases. A longer fruit base significantly reduces the appearance quality, edible rate, and shelf life of the pomelo fruit.
[0003] Currently, no research reports have been found on genes related to the fruit base length trait of citrus fruits. Therefore, identifying and regulating genes that regulate the fruit base length of citrus fruits is of great significance for improving the fruit shape of citrus fruits using genetic engineering techniques. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a CgFAD gene that regulates fruit base length and its application. This invention discovers a CgFAD gene that can regulate fruit base length; overexpression of the CgFAD gene can reduce fruit base length, providing a new gene resource for fruit shape improvement.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] This invention provides a CgFAD gene that regulates the length of the fruit base, and the amino acid sequence of the protein encoded by the CgFAD gene is shown in SEQ ID NO.1.
[0007] Preferably, the nucleotide sequence of the CgFAD gene is shown in SEQ ID NO.2.
[0008] This invention provides the application of the CgFAD gene described in the above technical solution in regulating the length of the fruit base, wherein the fruit includes citrus fruits and / or tomato fruits.
[0009] Preferably, the regulation includes: increasing the expression level of the CgFAD gene and reducing the length of the fruit base.
[0010] Preferably, the citrus genus includes grapefruit (Citrus maxima (Burm.) Merr.); the tomato genus includes tomato (Solanum lycopersicum L.).
[0011] Preferably, the tomato includes the yellow pear-shaped tomato (Lycopersicon esculentum).
[0012] The present invention provides a primer pair for amplifying the CgFAD gene described in the above technical solution, wherein the primer pair includes primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4.
[0013] This invention provides a biomaterial for reducing the length of a fruit base, the biomaterial comprising: a recombinant vector and / or engineered bacteria; the engineered bacteria being Agrobacterium tumefaciens transformed into the recombinant vector; the recombinant vector comprising the CgFAD gene described in the above technical solution.
[0014] Preferably, the backbone vector of the recombinant vector includes the PBI121 vector; the Agrobacterium includes GV3101 Agrobacterium.
[0015] The present invention provides the application of the biomaterials described in the above technical solution in reducing the length of fruit base and / or cultivating transgenic plants, wherein the fruit includes citrus fruits and / or tomato fruits, and the plant includes citrus plants and / or tomato plants.
[0016] Beneficial effects:
[0017] This invention provides a CgFAD gene that regulates fruit base length, the amino acid sequence of which is shown in SEQ ID NO.1. By analyzing transcriptome data (RNA-seq) of the fruit base development process of Guanxi Honey Pomelo (long-base pomelo variety) and Pingshan Pomelo (short-base pomelo variety), this invention discovered a CgFAD gene that can regulate fruit base length. This CgFAD gene negatively regulates fruit base length; that is, the higher the expression level, the shorter the fruit base length. Experiments have confirmed that the CgFAD gene can negatively regulate fruit base length, providing a new gene resource for fruit shape improvement. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0019] Figure 1 A schematic diagram of the base of a pomelo fruit;
[0020] Figure 2 The results show the gene expression of CgFAD during the development of the fruit base of Guanxi honey pomelo and Pingshan pomelo; among them, 10DAF: 10 days after flowering; 20DAF: 20 days after flowering; 30DAF: 30 days after flowering; 40DAF: 40 days after flowering.
[0021] Figure 3 Images and measurement results of fruit base length of T2 generation yellow pear-shaped tomato overexpressing the CgFAD gene; where #OE1-#OE3: tomato lines overexpressing the CgFAD gene; WT: wild-type yellow pear-shaped tomato (Lycopersiconesculentum); dashed boxes indicate fruit base length of yellow pear-shaped tomato; scale bar is 1 cm; ** indicates extremely significant difference compared with WT (P<0.01). Detailed Implementation
[0022] This invention provides a CgFAD gene for regulating fruit base length. The amino acid sequence of the protein encoded by the CgFAD gene is shown in SEQ ID NO.1, and is as follows:
[0023] METVEKDVVIIGAGIAGLATALALKRLGIKPLVLEKSDGLRGTGAAINFAPNAWLALDALGVSHKLASIYDPVKRLFVTNLRTGATQETSLAGKSENGSGIRYIHRKKLLETLADELPNDTIHFSSKIAAINSETHDALSPVIIHLADGTIVKAKVLIGCDGIHSTVARWLGLSEPLNAGRSAGLGLAVFPEGHGLNKEVRLFVDAGMR AGYVPLNDKEIYWFLVCNCSAEGENKAGNPELIQKEVLEKYAKVLPPFYSVIVRRSDASTLHWAPLMFRHPWNVFFGNLSKGNVTVAGDAMHPMTPDLGQGGCQALED AVVLGRHIGNLLIKTKGHIATTGDNNVAQAIDGYVKERKWRVTGLVIGSYLSGWVQDGGSNWWMRFLRDVIFYRFLVGGVLGNKVTGYDCGKLPDVSLGEMDNPCKID;
[0024] As one implementation method, the nucleotide sequence of the CgFAD gene is shown in SEQ ID NO.2, specifically as follows:
[0025]
[0026] This invention, through analysis of transcriptomic data (RNA-seq) of the fruit base development process of Guanxi Honey Pomelo (long-base pomelo germplasm) and Pingshan Pomelo (short-base pomelo germplasm), discovered a CgFAD gene that can regulate fruit base length. This CgFAD gene negatively regulates fruit base length; that is, the higher the expression level, the shorter the fruit base. Overexpression experiments confirmed that the CgFAD gene can negatively regulate fruit base length (particularly applicable to citrus fruits and / or tomato fruits), providing a new gene resource for fruit shape improvement.
[0027] Based on the above advantages, the present invention provides the application of the CgFAD gene described in the above technical solution in regulating the length of the fruit base, wherein the fruit includes citrus fruits and / or tomato fruits.
[0028] As one implementation, the regulation includes: increasing the expression level of the CgFAD gene to decrease the fruit base length, or decreasing the expression level of the CgFAD gene to increase the fruit base length. As one implementation, the citrus genus includes pomelo (Citrus maxima (Burm.) Merr.); the tomato genus includes tomato (Solanum lycopersicum L.). As one implementation, the tomato includes yellow pear-shaped tomato (Lycopersicon esculentum).
[0029] Based on the above advantages, this invention provides a primer pair for amplifying the CgFAD gene described in the above-mentioned technical solution. The primer pair includes primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4. This invention provides a primer pair that can specifically amplify the CgFAD gene described in the above-mentioned technical solution.
[0030] This invention provides a biomaterial for reducing fruit base length. The biomaterial comprises a recombinant vector and / or engineered bacteria; the engineered bacteria is Agrobacterium tumefaciens transformed into the recombinant vector; the recombinant vector includes the CgFAD gene described in the above-mentioned technical solution. The biomaterial provided by this invention can increase the expression level of the CgFAD gene, thereby reducing fruit base length.
[0031] In one embodiment, the backbone vector of the recombinant vector includes the PBI121 vector; the Agrobacterium includes GV3101 Agrobacterium.
[0032] Based on the above advantages, the present invention provides the application of the biomaterials described in the above technical solution in reducing the length of fruit base and / or cultivating transgenic plants, wherein the fruit includes citrus fruits and / or tomato fruits, and the plant includes citrus plants and / or tomato plants.
[0033] In one embodiment, the citrus genus includes grapefruit; the tomato genus includes tomato. In one embodiment, the tomato includes yellow pear-shaped tomato.
[0034] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a CgFAD gene for regulating fruit base length and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.
[0035] Example 1
[0036] This invention provides a CgFAD gene that regulates the length of the fruit base of Citrus fruits, the nucleotide sequence of which is shown in SEQ ID NO.2.
[0037] This invention analyzed the gene expression of the CgFAD gene during the development of the fruit base of Guanxi Honey Pomelo (long-base pomelo germplasm) and Pingshan Pomelo (short-base pomelo germplasm) using transcriptome data (RNA-seq). The results are shown in […]. Figure 2 .
[0038] The results showed that the CgFAD gene was upregulated during the development of the fruit base of both Guanxi honey pomelo and Pingshan pomelo. However, the CgFAD gene expression level in Pingshan pomelo was significantly higher than that in Guanxi honey pomelo, indicating that the CgFAD gene negatively regulates the length of the pomelo fruit base, that is, the higher the expression level, the shorter the fruit base length.
[0039] Example 2: CgFAD overexpression in Lycopersicon esculentum (a pear-shaped tomato).
[0040] This invention uses PBI121 as the expression vector, and constructs an overexpression vector by fusing the full-length CgFAD gene (with the stop codon TGA removed) with the vector using homologous recombination. The resulting vector is then used to genetically transform tomatoes using Agrobacterium tumefaciens GV3101. The specific method is as follows:
[0041] 1. The PBI121 vector was double-digested with XbaⅠ and XhoⅠ. After digestion at 37℃ for 2 h, the linearized PBI121 vector was recovered.
[0042] 2. Using total cDNA obtained by reverse transcription of RNA from the basal part of Pingshan pomelo fruit as a template, PCR amplification was performed to obtain the CDS region clone product of the CgFAD gene; the primers used for the PCR amplification are as follows:
[0043] Forward primer (SEQ ID NO.3): 5'-ATGGAAACGGTAGAAAAAGATG-3';
[0044] Reverse primer (SEQ ID NO.4): 5'-CTAATCTATTTTGCATGGGTTG-3';
[0045] The PCR amplification reaction system consisted of: 10 μL of 2×TaqPCRMix (Takara), 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of template, and 8 μL of ddH2O.
[0046] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 3 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ extension for 5 min.
[0047] 3. Using the CDS region cloning product of the CgFAD gene obtained in step 2 as a template, PCR amplification was performed using the recombinant primer pair. After PCR amplification, the recombinant product obtained was recovered. The reaction system and procedure for the PCR amplification are described in step 2. The nucleotide sequence of the recombinant primer pair is as follows:
[0048] Forward primer (SEQ ID NO.5):
[0049] 5'-gagaacacgggggac tctaga ATGGAAACGGTAGAAAAAGATG-3';
[0050] Reverse primer (SEQ ID NO.6):
[0051] 5'-ggggactcgcggccg ctcgag ATCTATTTTGCATGG-3';
[0052] Among them, the 5'-gagaacacgggggac-3' (SEQ ID NO.7) in the forward primer is the homologous arm of the PBI121 vector, and the 5'- tctaga -3' is the XbaⅠ restriction site sequence, and the thickened 5'-ATGGAAACGGTAGAAAAAGATG-3' (SEQ ID NO. 3) is the amplification primer for the CgFAD gene; the 5'-ggggactcgcggccg-3' (SEQ ID NO. 8) in the reverse primer is the homologous arm of the PBI121 vector, 5'- ctcgag -3' is the XhoⅠ restriction site sequence, and the bolded 5'-ATCTATTTTGCATGG-3' (SEQ ID NO.4) is the amplification primer for the CgFAD gene (with the stop codon TGA removed).
[0053] 4. The recovered linearized PBI121 vector and recombinant product were subjected to a recombination ligation reaction at 37°C. The specific recombination ligation reaction system was as follows: 1.5 μL of linearized PBI121 vector, 1.5 μL of recombinant product, 1 μL of recombinase (Exnase II), 2 μL of 5×CEII buffer, and 4 μL of ddH2O. After the reaction, the ligation product was added to DH5α competent cells and mixed well. After mixing, the cells were first incubated on ice for 30 min, then heat-shocked at 42°C for 90 s, and then incubated on ice for 1 min. Subsequently, 500 mL of fresh LB liquid medium was added, and the cells were incubated on a shaker at 37°C at 220 r / min for 2 h. After centrifugation at 12000 rpm for 2 min, the supernatant was discarded, and 200 μL of fresh LB liquid medium was added to the centrifuge tube and mixed well. The mixture was then spread onto a culture dish containing LB solid medium supplemented with kanamycin and incubated upside down at 37°C overnight. After selecting single clones for plaque PCR detection, positive clones were sent to the company for sequencing. After comparing and analyzing the sequencing results, the vector construction was finally confirmed to be successful.
[0054] 5. Use the Plasmid Extraction Mini Kit (Solarbio LifeSciences, China) to extract plasmids from the correctly sequenced E. coli culture. Refer to the kit instructions for specific methods.
[0055] 6. Transform the plasmid extracted in step 5 into GV3101 Agrobacterium competent cells using a freeze-thaw method. The specific procedure is as follows: Remove the Agrobacterium competent cells GV3101 from the -80℃ freezer and freeze-thaw them on ice. Add 1 μg of plasmid, place on ice for 5 min, then flash-freeze in liquid nitrogen for 5 min. Quickly remove and incubate at 37℃ for 5 min, then place on ice for 5 min. Add 1 mL of fresh LB liquid medium to the competent cells and shake at 220 rpm for 10 h at 28℃. Centrifuge at 12000 rpm for 2 min, discard the supernatant, and add 200 μL of fresh LB liquid medium to the centrifuge tube. Mix well, then spread on a culture dish containing LB solid medium with kanamycin and rifampin. Incubate upside down in a 28℃ incubator overnight. After selecting single clones for plaque PCR detection, positive clones were sent to the company for sequencing. After comparing and analyzing the sequencing results, the positive clone bacterial solution was finally determined. The positive clone bacterial solution was then mixed with 50% glycerol in a 1:1 ratio and stored at -80℃ for later use. After activation, it can be used for tomato genetic transformation.
[0056] 7. CgFAD overexpression in yellow pear-shaped tomatoes:
[0057] 1) Explant culture and tomato genetic transformation were completed by Wuhan Boyuan Biotechnology Co., Ltd.
[0058] 2) Screening of positive transgenic plants: When the transgenic seedlings have grown 5-8 tender leaves, randomly select 2-3 leaves and extract genomic DNA from the tomato using the CTAB method. Using the extracted genomic DNA as a template, screen for T0 generation positive plants using RT-PCR. The RT-PCR reaction procedure and reaction system are described in step 2. The primers used in the RT-PCR method are as follows:
[0059] Forward primer (SEQ ID NO.9): 5'-ACTGGGCACCATTGATGTTTAG-3';
[0060] Reverse primer sequence (SEQ ID NO.10): 5'-CTCATCCACCAATTTGACCCTC-3'.
[0061] After the reaction, positive plants were detected by electrophoresis using 2% agarose gel. When the amplified band was 334 bp, it indicated that the plant was positive. If no band was amplified, it indicated that the plant was not positive.
[0062] 8. Evaluation of fruit base length of T2 generation transgenic plants:
[0063] After self-pollination, T0 generation positive plants produced T1 generation seeds. After sowing, when the plants had 5-8 young leaves, T1 generation positive transgenic plants were further screened using the RT-PCR method described in step 7. These T1 generation positive plants then self-pollinated to produce T2 generation seeds. After sowing, when the T2 generation seeds had 5-8 young leaves, T2 generation positive transgenic plants were further screened using the RT-PCR method described in step 7. After the T2 generation positive plants produced fruit, the fruit base length of the T2 generation positive plants was evaluated. The results are shown in […]. Figure 3 .
[0064] The results showed that the fruit base length of T2 generation positive plants (#OE1~#OE3) overexpressing the CgFAD gene was significantly shorter than that of wild-type (WT) plants.
[0065] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. Application of CgFAD gene in regulating fruit base length, wherein the fruit is a yellow pear-shaped tomato (Lycopersiconesculentum) or Pingshan pomelo; the regulation is: increasing the expression level of CgFAD gene to reduce fruit base length; the amino acid sequence of the protein encoded by the CgFAD gene is shown in SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, The nucleotide sequence of the CgFAD gene is shown in SEQ ID NO.
2.
3. Application of biological materials in reducing fruit base length and / or cultivating transgenic plants, wherein the fruit is a yellow pear-shaped tomato (Lycopersicon esculentum) or a Pingshan pomelo; the biological materials include: Recombinant vector and / or engineered bacteria; the engineered bacteria is Agrobacterium tumefaciens transformed into the recombinant vector; the recombinant vector includes the CgFAD gene; The amino acid sequence of the protein encoded by the CgFAD gene is shown in SEQ ID NO.
1.
4. The application according to claim 3, characterized in that, The recombinant vector's backbone vector includes the PBI121 vector; the Agrobacterium includes GV3101 Agrobacterium.
5. The application according to claim 3, characterized in that, The nucleotide sequence of the CgFAD gene is shown in SEQ ID NO.2.