CgFAD gene for regulating fruit base length and application of CgFAD gene

Abstract

The invention relates to the technical field of gene engineering, in particular to a CgFAD gene for regulating and controlling the length of a fruit base and application of the CgFAD gene. The amino acid sequence of the protein encoded by the CgFAD gene is as shown in SEQ ID NO. 1. By analyzing transcriptome data (RNA-seq) in the fruit base development process of Guanxi honey pomelo (long fruit base pomelo germplasm) and Pingshan pomelo (short fruit base pomelo germplasm), the CgFAD gene capable of regulating and controlling the fruit base length is found, and the CgFAD gene negatively regulates and controls the fruit base length of the pomelo fruit, that is, the higher the expression quantity is, the shorter the fruit base length is. The experiment of overexpressing the yellow pear-shaped tomato shows that the CgFAD gene can negatively regulate and control the length of the fruit base, and a new gene resource is provided for improving the fruit shape of the fruit.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a CgFAD gene for regulating the length of the fruit stalk of a fruit and its application. Background Art

[0002] Pomelo (Citrus maxima (Burm.) Merr.) is an important fruit of the genus Citrus. The fruit stalk, also known as the fruit neck, fruit collar or fruit stem, refers to the distance from the fruit pedicel of the pomelo fruit to the pulp ( Figure 1 ). At present, the Shatian pomelo, Guanxi honey pomelo and Dongshi early pomelo that are widely planted all have relatively long fruit stalks. The relatively long fruit stalk significantly reduces the appearance quality, edible rate and shelf life of the pomelo fruit.

[0003] At present, there are still no research reports on genes related to the fruit stalk length trait of fruits of the genus Citrus. Therefore, excavating and identifying genes that regulate the fruit stalk length of fruits of the genus Citrus is of great significance for improving the fruit shape of fruits of the genus Citrus by means of genetic engineering. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a CgFAD gene for regulating the length of the fruit stalk of a fruit and its application. The present invention has discovered a CgFAD gene that can regulate the length of the fruit stalk of a fruit. After overexpressing the CgFAD gene, the length of the fruit stalk of the fruit can be reduced, providing a new gene resource for improving the fruit shape of the fruit.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a CgFAD gene for regulating the length of the fruit stalk of a fruit, and the amino acid sequence of the protein encoded by the CgFAD gene is as shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the CgFAD gene is as shown in SEQ ID NO.2.

[0008] The present invention provides the application of the CgFAD gene described in the above technical solution in regulating the length of the fruit stalk of a fruit, and the fruit includes fruits of the genus Citrus and / or fruits of the genus Solanum.

[0009] Preferably, the regulation includes: increasing the expression level of the CgFAD gene to reduce the length of the fruit stalk of the fruit.

[0010] Preferably, the genus Citrus includes pomelo (Citrus maxima (Burm.) Merr.); the genus Solanum includes tomato (Solanum lycopersicum L.).

[0011] Preferably, the tomato includes 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, and the primer pair includes primers with nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] The present invention provides a biological material for reducing the length of the fruit base, and the biological material includes: a recombinant vector and / or an engineered bacterium; the engineered bacterium is Agrobacterium tumefaciens into which the recombinant vector is transferred; the recombinant vector includes the CgFAD gene described in the above technical solution.

[0014] Preferably, the backbone vector of the recombinant vector includes the PBI121 vector; the Agrobacterium tumefaciens includes GV3101 Agrobacterium tumefaciens.

[0015] The present invention provides the application of the biological material described in the above technical solution in reducing the length of the fruit base and / or cultivating transgenic plants. The fruit includes citrus fruits and / or tomato fruits, and the plants include citrus plants and / or tomato plants.

[0016] Beneficial effects:

[0017] The present invention provides a CgFAD gene for regulating the length of the fruit base. The amino acid sequence of the protein encoded by the CgFAD gene is shown in SEQ ID NO.1. By analyzing the transcriptome data (RNA-seq) of the fruit base development process of Guanxi honey pomelo (long fruit base pomelo germplasm) and Pingshan pomelo (short fruit base pomelo germplasm), the present invention discovers a CgFAD gene that can regulate the length of the fruit base. The CgFAD gene negatively regulates the length of the fruit base, that is, the higher the expression level, the shorter the length of the fruit base. Through experiments, it is determined that the CgFAD gene can negatively regulate the length of the fruit base, providing a new gene resource for fruit shape improvement. Description of the drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0019] Figure 1 It is a schematic diagram of the fruit base of pomelo;

[0020] Figure 2 It is the analysis result of the gene expression of the CgFAD gene during the fruit base development process of Guanxi honey pomelo and Pingshan pomelo; among them, 10 DAF: 10 days after flowering; 20 DAF: 20 days after flowering; 30 DAF: 30 days after flowering; 40 DAF: 40 days after flowering;

[0021] Figure 3 Figure showing the physical map and measurement results of the fruit stalk length of T2-generation yellow pear-shaped tomatoes overexpressing the CgFAD gene; among them, #OE1-#OE3: tomato lines overexpressing the CgFAD gene; WT: wild-type yellow pear-shaped tomato (Lycopersicon esculentum); the dashed box indicates the fruit stalk length of the yellow pear-shaped tomato; the scale bar is 1 cm for all; ** indicates extremely significant difference compared with WT (P < 0.01). Detailed implementation manners

[0022] The present invention provides a CgFAD gene for regulating fruit stalk length. The amino acid sequence of the protein encoded by the CgFAD gene is as shown in SEQ ID NO.1, specifically as follows:

[0023] METVEKDVVIIGAGIAGLATALALKRLGIKPLVLEKSDGLRGTGAAINFAPNAWLALDALGVSHKLASIYDPVKRLFVTNLRTGATQETSLAGKSENGSGIRYIHRKKLLETLADELPNDTIHFSSKIAAINSETHDALSPVIIHLADGTIVKAKVLIGCDGIHSTVARWLGLSEPLNAGRSAGLGLAVFPEGHGLNKEVRLFVDAGMRAGYVPLNDKEIYWFLVCNCSAEGENKAGNPELIQKEVLEKYAKVLPPFYSVIVRRSDASTLHWAPLMFRHPWNVFFGNLSKGNVTVAGDAMHPMTPDLGQGGCQALEDAVVLGRHIGNLLIKTKGHIATTGDNNVAQAIDGYVKERKWRVTGLVIGSYLSGWVQDGGSNWWMRFLRDVIFYRFLVGGVLGNKVTGYDCGKLPDVSLGEMDNPCKID;

[0024] As an implementation manner, the nucleotide sequence of the CgFAD gene is as shown in SEQ ID NO.2, specifically as follows:

[0025]

[0026] By analyzing the transcriptome data (RNA-seq) of the fruit stalk development process of Guanxi honey pomelo (long fruit stalk pomelo germplasm) and Pingshan pomelo (short fruit stalk pomelo germplasm), the present invention discovered a CgFAD gene that can regulate the length of the fruit stalk. The CgFAD gene negatively regulates the length of the fruit stalk, that is, the higher the expression level, the shorter the fruit stalk length. And through overexpression experiments, it was determined that the CgFAD gene can negatively regulate the length of the fruit stalk (especially applicable to fruits of the genus Citrus and / or fruits of the genus Solanum), 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 stalk, and the fruits include fruits of the genus Citrus and / or fruits of the genus Solanum.

[0028] As an implementation manner, the regulation includes: increasing the expression level of the CgFAD gene to reduce the length of the fruit stalk, or decreasing the expression level of the CgFAD gene to increase the length of the fruit stalk. As an implementation manner, the genus Citrus includes pomelo (Citrus maxima (Burm.) Merr.); the genus Solanum includes tomato (Solanum lycopersicum L.). As an implementation manner, the tomato includes yellow pear-shaped tomato (Lycopersicon esculentum).

[0029] Based on the above advantages, the present invention provides a primer pair for amplifying the CgFAD gene described in the above technical solution, and the primer pair includes primers with nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4. The present invention provides a primer pair that can specifically amplify the CgFAD gene described in the above technical solution.

[0030] The present invention provides a biological material for reducing the length of the fruit stalk, and the biological material includes: a recombinant vector and / or an engineered bacterium; the engineered bacterium is Agrobacterium tumefaciens into which the recombinant vector is transferred; the recombinant vector includes the CgFAD gene described in the above technical solution. The biological material provided by the present invention can increase the expression level of the CgFAD gene, thereby reducing the length of the fruit stalk.

[0031] As an implementation manner, the backbone vector of the recombinant vector includes the PBI121 vector; the Agrobacterium tumefaciens includes GV3101 Agrobacterium tumefaciens.

[0032] Based on the above advantages, the present invention provides the application of the biological material described in the above technical solution in reducing the length of the fruit stalk and / or cultivating transgenic plants, the fruits include fruits of the genus Citrus and / or fruits of the genus Solanum, and the plants include plants of the genus Citrus and / or plants of the genus Solanum.

[0033] As an implementation manner, the citrus genus includes pomelo; the tomato genus includes tomato. As an implementation manner, the tomato includes yellow pear-shaped tomato.

[0034] To further illustrate the present invention, a CgFAD gene for regulating the fruit stalk length and its application provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] The present invention provides a CgFAD gene for regulating the fruit stalk length of the citrus genus, and its nucleotide sequence is shown in SEQ ID NO.2.

[0037] Based on the transcriptome data (RNA-seq) of the fruit stalk development process of Guanxi pomelo (long fruit stalk pomelo germplasm) and Pingshan pomelo (short fruit stalk pomelo germplasm), the present invention analyzed the gene expression of the CgFAD gene in the fruit stalk development process of Guanxi pomelo (long fruit stalk pomelo germplasm) and Pingshan pomelo (short fruit stalk pomelo germplasm). The results are shown in Figure 2 .

[0038] The results show that the CgFAD gene is up-regulated in the fruit stalk development process of both Guanxi pomelo and Pingshan pomelo, but the expression level of the CgFAD gene in Pingshan pomelo is significantly higher than that in Guanxi pomelo, indicating that the CgFAD gene negatively regulates the fruit stalk length of pomelo, that is, the higher the expression level, the shorter the fruit stalk length.

[0039] Example 2 Overexpression of CgFAD in yellow pear-shaped tomato (Lycopersicon esculentum)

[0040] The present invention uses PBI121 as an expression vector, and constructs an overexpression vector by fusing the full length of the CgFAD gene (removing the stop codon TGA) with the vector by homologous recombination method, and uses Agrobacterium tumefaciens GV3101-mediated genetic transformation of tomato. The specific method is as follows:

[0041] 1. Use XbaⅠ and XhoⅠ to double digest the PBI121 vector. After digesting at 37°C for 2 h, recover the linearized PBI121 vector.

[0042] 2. Using the total cDNA reverse transcribed from the RNA of the fruit stalk part of Pingshan pomelo as a template, perform PCR amplification to obtain the CDS region cloning 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 reaction system for the PCR amplification is as follows: 10 μL of 2× Taq PCR Mix (Takara), 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of template, and 8 μL of ddH2O.

[0046] The reaction conditions for the PCR amplification are: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; extension at 72°C for 5 min.

[0047] 3. Using the CDS region cloning product of the CgFAD gene obtained in step 2 as a template, perform PCR amplification with the recombinant primer pair. After the PCR amplification is completed, recover the amplified recombinant product; the reaction system and reaction program for the PCR amplification refer to step 2. The nucleotide sequences of the recombinant primer pair are 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, 5'-gagaacacgggggac-3' (SEQ ID NO.7) in the forward primer is the homologous arm of the PBI121 vector, 5'- tctaga -3' is the XbaⅠ restriction site sequence, and the bold 5'-ATGGAAACGGTAGAAAAAGATG-3' (SEQ ID NO.3) is the amplification primer for the CgFAD gene; 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 bold 5'-ATCTATTTTGCATGG-3' (SEQ ID NO.4) is the amplification primer for the CgFAD gene (removing the stop codon TGA).

[0053] 4. Recombinant ligation reaction was carried out between the recovered linearized PBI121 vector and the recombinant product at 37°C. The specific recombinant ligation reaction system was as follows: 1.5 μL of linearized PBI121 vector, 1.5 μL of recombinant product, 1 μL of recombinase (ExnaseⅡ), 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 evenly. After mixing, it was first ice-bathed for 30 min and then heat-shocked at 42°C for 90 s, and then left standing on ice for 1 min. Subsequently, 500 mL of fresh LB liquid medium was added, and it was incubated at 220 r / min on a shaker at 37°C for 2 h. After centrifuging at 12000 rpm for 2 min, the supernatant was discarded, and 200 μL of fresh LB liquid medium was re-aspirated and added to the above centrifuge tube and mixed evenly. Then it was spread on a culture dish of LB solid medium supplemented with kanamycin and incubated overnight in an inverted position in an incubator at 37°C. After picking monoclonal colonies for PCR detection, the positive clones were sent to the company for sequencing. After comparing and analyzing the sequencing results, the successful construction of the vector was finally determined.

[0054] 5. The Escherichia coli solution with correct sequencing was used for plasmid extraction using the Plasmid Extraction Mini Kit (Solarbio Life Sciences, China). The specific method was carried out according to the kit instructions.

[0055] 6. The plasmid extracted in step 5 was transferred into GV3101 Agrobacterium competent cells by the freeze-thaw method. The specific operation was as follows: The Agrobacterium competent GV3101 was taken out from the -80°C refrigerator and freeze-thawed on ice. 1 μg of plasmid was added, and it was placed on ice for 5 min and then quickly frozen in liquid nitrogen for 5 min. After quickly taking it out, it was incubated in a constant temperature instrument at 37°C for 5 min, and then placed on ice for 5 min. Then 1 mL of fresh LB liquid medium was added to the competent cells, and it was shaken at 220 r / min on a shaker at 28°C for 10 h. After centrifuging at 12000 rpm for 2 min, the supernatant was discarded, and 200 μL of fresh LB liquid medium was re-aspirated and added to the above centrifuge tube and mixed evenly. Then it was spread on a culture dish of LB solid medium supplemented with kanamycin and rifampicin and incubated overnight in an inverted position in an incubator at 28°C. After picking monoclonal colonies for PCR detection, the positive clones were sent to the company for sequencing. After comparing and analyzing the sequencing results, the positive clone bacterial solution was finally determined. Finally, the positive clone bacterial solution was mixed evenly with 50% glycerol in an equal volume ratio of 1:1 and stored in a -80°C refrigerator for later use. It can be used for tomato genetic transformation after activation.

[0056] 7. Overexpression of CgFAD in yellow pear-shaped tomatoes:

[0057] 1) The culture of explants and tomato genetic transformation were completed by Wuhan Boyuan Biotechnology Co., Ltd.

[0058] 2) Screening of positive transgenic plants: When the transgenic seedlings grow 5 - 8 young leaves, randomly pick 2 - 3 of them, extract the genomic DNA of tomatoes by the CTAB method. Using the extracted genomic DNA as a template, screen out the T0 generation positive plants by RT-PCR method; the reaction program and reaction system of the RT-PCR are shown in step 2, and 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, use 2% agarose gel to detect the electrophoresis of positive plants. When the amplified band is 334bp, it indicates a positive plant. If no band is amplified, it indicates a non-positive plant.

[0062] 8. Evaluation of the fruit stalk base length of T2 generation transgenic plants:

[0063] The T0 generation positive plants self-cross to produce T1 generation seeds; after sowing the positive T1 generation seeds, when the plants grow 5 - 8 young leaves, further screen out the T1 generation positive transgenic plants by the RT-PCR method in step 7. The T1 generation positive plants self-cross to produce T2 generation seeds; after sowing the T2 generation seeds, when the plants grow 5 - 8 young leaves, further screen out the T2 generation positive transgenic plants by the RT-PCR method in step 7. After the T2 generation positive plants bear fruits, conduct an evaluation of the fruit stalk base length of the T2 generation positive plant fruits. The results are shown in Figure 3 .

[0064] The results show that: compared with the fruit stalk base length of wild type (WT) plants, the fruit stalk base length of the T2 generation positive plants (#OE1 - #OE3) overexpressing the CgFAD gene is significantly shorter.

[0065] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A CgFAD gene for regulating fruit base length, characterized in that: The amino acid sequence of the protein encoded by the CgFAD gene is shown in SEQ ID NO.

1.

2. The CgFAD gene according to claim 1, characterized in that The nucleotide sequence of the CgFAD gene is shown as SEQ ID NO.

2.

3. Use of the CgFAD gene according to claim 1 or 2 in regulating the length of the fruit base, wherein the fruit comprises citrus fruit and / or tomato fruit.

4. The use according to claim 3, characterized in that: The regulation includes: increasing the expression level of CgFAD gene and reducing the length of fruit base.

5. The use according to claim 3, characterized in that: The genus Citrus includes Citrus maxima (Burm.) Merr.; the genus Solanum includes Solanum lycopersicum L.

6. The use according to claim 5, characterized in that: The tomatoes include Lycopersicon esculentum.

7. A primer pair for amplifying the CgFAD gene according to claim 1 or 2, characterized in that: The primer pair includes primers whose nucleotide sequences are shown as SEQ ID NO.3 and SEQ ID NO.

4.

8. A biological material for reducing the length of the fruit base, characterized in that: The biological material comprises: a recombinant vector and / or an engineered bacterium; the engineered bacterium is Agrobacterium into which the recombinant vector is transferred; and the recombinant vector comprises the CgFAD gene according to claim 1 or 2.

9. The biomaterial according to claim 8, characterized in that The backbone vector of the recombinant vector includes the PBI121 vector; the Agrobacterium includes the GV3101 Agrobacterium.

10. Use of the biological material according to claim 8 or 9 in reducing the fruit base length and / or cultivating transgenic plants, wherein the fruit comprises citrus fruit and / or tomato fruit, and the plant comprises citrus plant and / or tomato plant.

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