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

Abstract

The invention discloses an application of a citrus CgABR gene in regulating and controlling the length of a fruit base of a pomelo fruit. The invention proves that the CgABR gene negatively regulates the length of the fruit base of the pomelo fruit for the first time. According to the invention, the tomato is further taken as a model to research the plant, and experiments prove that compared with the fruit base length of a wild plant, the fruit base length of a T2-generation positive plant with overexpressed CgABR is obviously shortened, so that a foundation is laid for providing a new gene resource for improving the fruit shapes of citrus plants, particularly teak plants, by utilizing genetic engineering.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to the application of citrus CgABR gene in regulating the length of the fruit stalk of pomelo fruits. Background Art

[0002] Pomelo (Citrus maxima (Burm.) Merr.) is an important citrus fruit. The fruit stalk, also called the fruit neck, fruit collar or fruit handle, refers to the distance from the fruit stalk of the pomelo fruit to the pulp ( Figure 1 ). Varieties such as Shatian pomelo and Guanxi honey pomelo are long-fruit-stalk varieties. A longer fruit stalk significantly reduces the economic benefits of the fruit. The main manifestations are: (1) A longer fruit stalk will significantly reduce the edible rate and appearance quality of the fruit. (2) During the picking, storage and transportation of the fruit, due to its special structure, the fruit stalk part of the pomelo fruit is easily damaged when subjected to external forces, resulting in a significant reduction in the shelf life and commerciality of the fruit. (3) When storing and transporting pomelo fruits with a longer fruit stalk, a large amount of transportation space is occupied, increasing the transportation cost. To sum up, the length of the fruit stalk of pomelo fruits has become a bottleneck affecting the development of the pomelo industry.

[0003] At present, there is still no report on the research of genes related to regulating the length trait of the fruit stalk of pomelo fruits. Therefore, exploring and utilizing genes that regulate the length of the fruit stalk of pomelo fruits is of great significance for improving the fruit shape of pomelo fruits by means of genetic engineering. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide the application of citrus CgABR gene in regulating the length of the fruit stalk of pomelo fruits.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

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

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

[0008] A. A homologous nucleic acid molecule of the citrus CgABR gene;

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

[0010] C. A recombinant vector, expression cassette, transgenic plant tissue or recombinant bacterium containing the citrus CgABR gene or its homologous nucleic acid molecule.

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

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

[0013] 1) Application of negatively regulating the length of the fruit base of plants;

[0014] 2) Application of improving the fruit shape of plants;

[0015] 3) Application of cultivating transgenic plants.

[0016] Furthermore, the plant is any one of pomelo and tomato.

[0017] Furthermore, the pomelo includes pomelos with long fruit bases such as Guanxi pomelo and Shatian pomelo; the tomato includes yellow pear-shaped tomato (Lycopersicon esculentum).

[0018] Furthermore, the transgenic includes introducing a recombinant expression vector containing the citrus CgABR gene into a receptor plant by an Agrobacterium-mediated method using a plant expression vector to obtain a transgenic plant line; the fruit base of the transgenic plant line is shorter than that of the receptor 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] The present invention proves for the first time that the CgABR gene negatively regulates the length of the fruit base of pomelo. The present invention further uses tomato as a model research plant for experiments and proves that compared with the fruit base length of wild-type plants, the fruit base length of the T2 generation positive plants overexpressing CgABR is significantly shorter, which lays a foundation for providing new gene resources for improving the fruit shape of citrus plants, especially pomelo plants, by genetic engineering. Description of the Drawings

[0023] Figure 1 It is an example diagram of the fruit base of pomelo.

[0024] Figure 2 It is an analysis diagram of the gene expression of CgABR during the fruit base development of Guanxi pomelo (GX) and Pingshan pomelo (PS); 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;

[0025] Figure 3The figure shows the measurement results of the fruit stalk length of the T2 generation of yellow pear-shaped tomatoes overexpressing the CgABR gene; among them, #OE: tomato overexpressing the CmABR gene; WT: wild-type tomato; Bar = 1 cm. Detailed implementation mode

[0026] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation mode of the present invention is not limited thereto.

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

[0028] All kinds of raw materials and equipment used in the present invention are conventional commercially available products, which can be directly obtained through market purchase, and the primer sequences used are all synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0029] Example 1:

[0030] Analyze 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), and provide the gene expression patterns of CgABR during the fruit stalk development of Guanxi honey pomelo (long fruit stalk pomelo germplasm) and Pingshan pomelo (short fruit stalk pomelo germplasm).

[0031] The results are as Figure 2 shown. CgABR is up-regulated during the fruit stalk development of both Guanxi honey pomelo and Pingshan pomelo, but the expression level of the CgABR gene in Pingshan pomelo is significantly higher than that in Guanxi honey pomelo, indicating that CgABR negatively regulates the fruit stalk length of pomelo fruits, that is, the higher the expression level, the shorter the fruit stalk length.

[0032] Example 2

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

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

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

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

[0037] 1) Construction of the CgABR overexpression vector

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

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

[0040] Explant culture: Tomato seeds were placed in a sterile Erlenmeyer flask, first washed with sterile water, then disinfected with 75% alcohol for 40 s, then disinfected with 84 disinfectant for 7 min, washed 3 times with sterile water, and soaked in sterile water for 1 h. The sterilized seeds were sown on the germination medium and incubated in the dark for 3 - 4 d. After the seeds germinated and showed white tips, they were placed in a light tissue culture incubator and grown for 4 - 5 d (culture conditions: 23 ± 2 °C, 16 h / d light, 8 h / d dark). When the cotyledons were fully expanded, the cotyledons were excised with a scalpel, the petioles and tips of the cotyledons were removed, and the middle part was cut into 2 - 3 segments as explants and inoculated into the pre-culture medium;

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

[0042] 3) Detection of transgenic plants: When the transgenic seedlings had grown 5 - 8 young leaves, 2 - 3 of them were randomly picked, and the genomic DNA of tomatoes was extracted by the CTAB method. PCR was performed using HYG-F / HYG-R as primers to screen for positive transgenic plants. The 20 μL PCR amplification system contained: 10 μL of 2×Taq PCR Mix (Takara), 0.5 μL of each primer, 1 μL of DNA, and 2 8 μL of ddH

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

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

[0045] Total RNA of the leaves of the plants with positive PCR detection was extracted by the Trizol method, and cDNA was reverse-transcribed and synthesized using the reverse transcription kit of Takara Company for semi-quantitative PCR to detect the expression of the target gene in transgenic plants. The semi-quantitative PCR reaction system was as follows: 2×Taq PCR Mix (Takara) 10 μL, each primer 0.5 μL, cDNA 1 μL, ddH 2 O 8 μL. PCR reaction conditions: pre-denaturation at 94 °C for 3 min; 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 23 cycles; extension at 72 °C for 5 min.

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

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

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

[0049] The primer sequences for the internal reference gene Actin were:

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

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

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

[0053] The T0 generation of positive plants self-crossed to produce T1 generation seeds; after the positive T1 generation seeds were sown, when the plants grew 5 - 8 young leaves, HYG-F / HYG-R was further used as primers for PCR to screen out the positive transgenic plants of the T1 generation. The T1 generation of positive plants self-crossed to produce T2 generation seeds; after the T2 generation seeds were sown, when the plants grew 5 - 8 young leaves, HYG-F / HYG-R was further used as primers for PCR to screen out the positive transgenic plants of the T2 generation. After the T2 generation of positive plants fruited, the fruit stalk lengths of the fruits of the T2 generation of positive plants were evaluated.

[0054] The results were as Figure 3 shown. Compared with the fruit stalk lengths of the wild-type plants, the fruit stalk lengths of the T2 generation of positive plants overexpressing CgABR were significantly shorter.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A citrus CgABR gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The citrus CgABR gene-related biological material according to claim 1, characterized in that: The biomaterial is at least one of the following biomaterials: A. Citrus CgABR gene homologous nucleic acid molecule; B. the protein encoded by the citrus CgABR gene or its homologous amino acid sequence; C. A recombinant vector, expression cassette, transgenic cell line, transgenic plant tissue or recombinant bacteria containing the citrus CgABR gene or its homologous nucleic acid molecule.

3. The citrus CgABR gene-related biological material according to claim 2, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

4. The use of the citrus CgABR gene according to claim 1, characterized in that: The application is any one or more of the following applications 1), 2), and 3): 1) Application of negative regulation of plant fruit base length; 2) Application of improving plant fruit shape; 3) Application of cultivating transgenic plants.

5. The use of the citrus CgABR gene according to claim 4, characterized in that: The plant is any one of pomelo and tomato.

6. The use of the citrus CgABR gene according to claim 5, characterized in that: The pomelo includes Guanxi honey pomelo and Shatian pomelo.

7. The use of the citrus CgABR gene according to claim 5, characterized in that: The tomatoes include yellow pear-shaped tomatoes.

8. The use of the citrus CgABR gene according to any one of claims 4 to 7, characterized in that: The transgenic method comprises using a plant expression vector to introduce a recombinant expression vector containing the citrus CgABR gene into a recipient plant by an Agrobacterium-mediated method to obtain a transgenic plant strain; the fruit base of the transgenic plant strain is shorter than that of the recipient plant.

9. The use of the citrus CgABR gene according to claim 8, characterized in that: The plant expression vector comprises a binary plant binary expression vector PBI121.

10. The use of the citrus CgABR gene according to claim 8, characterized in that: The Agrobacterium includes Agrobacterium GV3101.

Images