Application of citrus CgABR gene in regulating fruit base length of pomelo fruit

Abstract

The application discloses application of a citrus CgABR gene in regulating fruit base length of a grapefruit. The application proves for the first time that the CgABR gene negatively regulates the fruit base length of the grapefruit. The application further takes tomatoes as a model to prove through experiments that, compared with the fruit base length of a wild type plant, the fruit base length of a T2 generation positive plant fruit overexpressing the CgABR is significantly shortened, which lays a foundation for improving citrus plants, especially grapefruit plants, in fruit shape by using genetic engineering and provides a new gene resource.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the citrus CgABR gene in regulating the length of grapefruit fruit base. 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 Varieties such as Shatang pomelo and Guanxi honey pomelo are characterized by long fruit bases. A longer fruit base significantly reduces the economic benefits of the fruit. This is mainly manifested in: (1) a longer fruit base significantly reduces the edible rate and appearance quality of the fruit. (2) during harvesting and storage, the fruit base of the pomelo fruit, due to its special structure, is easily damaged by external forces, leading to a significant reduction in the fruit's shelf life and marketability. (3) pomelo fruits with longer fruit bases occupy a large amount of transportation space during storage and transportation, increasing transportation costs. In conclusion, the length of the pomelo fruit base has become a bottleneck affecting the development of the pomelo industry.

[0003] Currently, no research has been reported on genes related to the regulation of pomelo fruit base length. Therefore, identifying and utilizing genes that regulate pomelo fruit base length is of great significance for improving pomelo fruit shape through genetic engineering. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide the application of the citrus CgABR gene in regulating the length of grapefruit base.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A citrus CgABR gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The above-mentioned citrus CgABR gene-related biological materials include at least one of the following biological materials:

[0008] A. Homologous nucleic acid molecules of the CgABR gene in citrus;

[0009] B. The protein encoded by the citrus CgABR gene or its homologous amino acid sequence;

[0010] C. Recombinant vectors, expression cassettes, transgenic plant tissues, or recombinant bacteria containing the citrus CgABR gene or its homologous nucleic acid molecules.

[0011] Furthermore, the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0012] The above-mentioned application of the citrus CgABR gene refers to any one or more of the following applications: 1), 2), and 3).

[0013] 1) Application of negative regulation of fruit base length in plants;

[0014] 2) Application in improving the shape of plant fruits;

[0015] 3) Application of genetically modified plants.

[0016] Furthermore, the plant mentioned is either pomelo or tomato.

[0017] Furthermore, the pomelo mentioned includes long-based varieties such as Guanxi Honey Pomelo and Shatin Pomelo; the tomato mentioned includes the yellow pear-shaped tomato (Lycopersicon esculentum).

[0018] Furthermore, the transgenic process involves introducing a recombinant expression vector containing the citrus CgABR gene into a recipient plant using an Agrobacterium-mediated method to obtain a transgenic plant line; the fruit base of the transgenic plant line is shorter than that of the recipient plant.

[0019] Furthermore, the plant expression vector includes the plant binary expression vector PBI121.

[0020] Furthermore, the Agrobacterium includes Agrobacterium GV3101.

[0021] The present invention has the following advantages and effects compared with the prior art:

[0022] This invention is the first to demonstrate that the CgABR gene negatively regulates the fruit base length of grapefruit. Further experiments using tomato as a model plant showed that, compared to wild-type plants, the fruit base length of T2 generation positive plants overexpressing CgABR was significantly shorter. This lays the foundation for using genetic engineering to improve the fruit shape of citrus plants, especially grapefruits. Attached Figure Description

[0023] Figure 1 Example image of a pomelo fruit base.

[0024] Figure 2 This is a graph showing the gene expression of CgABR during the fruit basal development of Guanxi Honey Pomelo (GX) and Pingshan Pomelo (PS); where 10DAF: 10 days after flowering; 20DAF: 20 days after flowering; 30DAF: 30 days after flowering; 40DAF: 40 days after flowering.

[0025] Figure 3The image shows the results of measuring the fruit base length of T2 generation yellow pear-shaped tomatoes overexpressing the CgABR gene; where #OE: tomatoes overexpressing the CmABR gene; WT: wild-type tomatoes; Bar = 1cm. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] All raw materials and equipment used in this invention are commercially available products that can be directly purchased from the market, and the primer sequences used are synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0029] Example 1:

[0030] Transcriptome data (RNA-seq) of the fruit base development process of Guanxi Honey Pomelo (long-base pomelo germplasm) and Pingshan Pomelo (short-base pomelo germplasm) were analyzed to provide the gene expression pattern of CgABR during the fruit base development process of Guanxi Honey Pomelo (long-base pomelo germplasm) and Pingshan Pomelo (short-base pomelo germplasm).

[0031] The results are as follows Figure 2 As shown, CgABR was upregulated during the development of the fruit base in both Guanxi honey pomelo and Pingshan pomelo. However, the CgABR gene expression level in Pingshan pomelo was significantly higher than that in Guanxi honey pomelo, indicating that CgABR negatively regulates the length of the pomelo fruit base, that is, the higher the expression level, the shorter the fruit base length.

[0032] Example 2

[0033] (1) Cloning of the full-length gene: Using total cDNA obtained by reverse transcription of RNA from the ovary of *Pomelo guanxiensis* 30 days after flowering as a template, PCR amplification was performed to obtain the full-length CgABR gene, the sequence of which is shown in SEQ ID NO.1. The PCR amplification primer sequences are as follows:

[0034] Forward primer: 5'-ATGATAAAGGTGGCGAATCAGC-3',

[0035] Reverse primer: 5'-TTATCCTGAAGGAGGATAATAACTAGAAG-3'.

[0036] (2) CgABR overexpression in yellow pear-shaped tomatoes

[0037] 1) Construction of CgABR overexpression vector

[0038] Using PBI121 as the expression vector, the full-length CgABR gene was fused with the vector using homologous recombination to construct an overexpression vector, and genetic transformation was mediated by Agrobacterium GV3101.

[0039] 2) CgABR overexpression in yellow pear-shaped tomatoes

[0040] Explant culture: Tomato seeds were placed in sterile Erlenmeyer flasks, first washed with sterile water, then disinfected with 75% alcohol for 40 seconds, then disinfected with 84 disinfectant for 7 minutes, washed three times with sterile water, and soaked in sterile water for 1 hour. The sterilized seeds were sown on germination medium and cultured in the dark for 3-4 days. After the seeds sprouted, they were placed in a light-controlled tissue culture incubator for 4-5 days (culture conditions: 23±2℃, 16h / d light, 8h / d darkness). When the cotyledons were fully expanded, the cotyledons were removed with a scalpel, the cotyledon petioles and tips were cut off, and the middle part was cut into 2-3 segments as explants and inoculated into the pre-culture medium.

[0041] Tomato genetic transformation: Agrobacterium GV3101 was streaked on LB medium containing 5 mg / L Kan and incubated at 28°C for 2 days. Single colonies were picked and plated on a new selection medium, and incubated at 28°C for another 2 days. Colonies were washed off with MS liquid medium and the OD600 was adjusted to 0.1. After 10–15 min of infection, the explants were dried in sterile filter paper. The dried explants were inoculated into co-culture medium and incubated in the dark at 23±2°C for 2 days. The callus obtained from co-culture was inoculated into selection medium containing HYG and incubated in the dark at 23°C for 15–30 days. The selected callus was inoculated into differentiation medium and incubated in the dark at 23°C for 30–40 days. When the differentiated seedlings grew to about 2–3 cm, they were removed from the callus and inoculated into rooting medium and incubated in the dark at 23°C for 10–15 days.

[0042] 3) Detection of transgenic plants: When the transgenic seedlings have grown 5-8 tender leaves, randomly select 2-3 leaves and extract tomato genomic DNA using the CTAB method. Perform PCR using HYG-F / HYG-R primers to screen for positive transgenic plants. A 20 μL PCR extension system contains: 10 μL 2×Taq PCR Mix (Takara), 0.5 μL each primer, 1 μL DNA, and 8 μL ddH2O. PCR reaction conditions: 94℃ pre-denaturation for 3 min; 35 cycles of 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 30 s; extension at 72℃ for 5 min. The HYG-F / HYG-R sequences are as follows:

[0043] HYG-F: 5'-CGGTGTCGTCCATCACAGTTT-3';

[0044] HYG-R:5'-GCCTGACCTATTGCATCTCCC-3':

[0045] Total RNA was extracted from leaves of plants that tested positive for PCR using the Trizol method. cDNA was synthesized using a Takara reverse transcription kit for semi-quantitative PCR detection of the target gene expression in transgenic plants. The semi-quantitative PCR reaction system consisted of: 10 μL of 2×Taq PCR Mix (Takara), 0.5 μL of each primer, 1 μL of cDNA, and 8 μL of ddH2O. PCR conditions were: 94℃ pre-denaturation for 3 min; 23 cycles of 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 30 s; and a final extension at 72℃ for 5 min.

[0046] The quantitative primers for the CgABR gene are:

[0047] qCgABRF: 5'-TTACATCAAGCGTTGGAAGTTCTC-3';

[0048] qCgABRR: 5'-CCTGTATCTTTCTCCTGGCTCT-3';

[0049] The primer sequence for the internal reference gene Actin is as follows:

[0050] ActinF: 5'-CAGCAGATGTGGATCTCAAA -3';

[0051] ActinR: 5'-CTGTGGACAATGGAAGGAC-3'.

[0052] (4) Evaluation of the fruit base length of transgenic tomatoes

[0053] After self-pollination, T0 generation positive plants produce T1 generation seeds. After sowing, when the plants have grown 5-8 tender leaves, PCR screening is performed using HYG-F / HYG-R primers to select T1 generation positive transgenic plants. After self-pollination, T1 generation positive plants produce T2 generation seeds. After sowing, when the plants have grown 5-8 tender leaves, PCR screening is performed using HYG-F / HYG-R primers to select T2 generation positive transgenic plants. After the T2 generation positive plants bear fruit, the fruit base length of the T2 generation positive plants is evaluated.

[0054] The results are as follows Figure 3 As shown, the fruit base length of T2 generation positive plants overexpressing CgABR was significantly shorter compared to that of wild-type plants.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Citrus fruits CgABR The application of the gene in negatively regulating the fruit base length of the pear-shaped tomato is characterized by: The citrus CgABR The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Citrus fruits CgABR The application of genes in the cultivation of transgenic plants is characterized by: Using a plant expression vector and an Agrobacterium-mediated method, the citrus fruit containing the above-mentioned citrus fruit was expressed. CgABR A recombinant gene expression vector was introduced into a recipient plant to obtain a transgenic plant line; the fruit stalk of the transgenic plant line was shorter than that of the recipient plant; the citrus fruit... CgABR The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plant is a yellow pear-shaped tomato.

3. The citrus fruit according to claim 2 CgABR The application of genes in the cultivation of transgenic plants is characterized by: The plant expression vectors mentioned include the binary plant binary expression vector PBI121.

4. The citrus fruit according to claim 2 CgABR The application of genes in the cultivation of transgenic plants is characterized by: The Agrobacterium mentioned includes Agrobacterium GV3101.

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