Litchi LcCPC3 gene and its encoded protein in delaying plant flowering

Abstract

This invention discloses the application of the litchi LcCPC3 gene and its encoded protein in delaying plant flowering. The nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1; the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. This invention clarifies that the litchi LcCPC3 gene participates in regulating the flowering time of litchi and is an important regulatory gene for litchi flowering. By utilizing the litchi LcCPC3 gene, the flowering time of litchi can be regulated and flowering can be stabilized, which has important application value for achieving stable litchi yield.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology. Specifically, this invention relates to the application of the litchi LcCPC3 gene and its encoded protein in delaying flowering in plants. Background Technology

[0002] Globally, due to the extremely strict requirements of low temperatures for lychee flowering, the main economic cultivation areas for lychees are distributed within the latitude zone of 17° to 32° north and south. In my country, lychees are widely cultivated in Guangdong, Guangxi, Hainan, and southern Fujian, and are one of my country's most competitive specialty fruits in the international market.

[0003] The lychee harvest season is relatively concentrated, mainly from mid-May to early July, with a focus on mid-to-late-ripening varieties. This results in immense sales pressure and a high risk of farmers suffering losses due to low fruit prices. Therefore, extending the lychee harvest season is crucial for the healthy development of the lychee industry.

[0004] Flowering is a prerequisite for litchi fruit production. Besides being influenced by external factors, litchi flowering is also regulated by its own genes. By identifying genes that can delay litchi flowering time, clarifying their functions, and applying them in breeding, we can provide a genetic basis for staggered litchi marketing and the selection of new litchi varieties. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a new gene that regulates the flowering of litchi.

[0006] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0007] In a first aspect, the present invention provides the application of the litchi LcCPC3 gene in delaying flowering in plants, wherein the nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides the application of a protein encoded by the litchi LcCPC3 gene in delaying flowering in plants, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] In a third aspect, the present invention provides the application of a recombinant expression vector overexpressing the litchi LcCPC3 gene in delaying flowering in plants, wherein the nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1.

[0010] In a fourth aspect, the invention provides the application of engineered bacteria transformed with the recombinant expression vector overexpressing the litchi LcCPC3 gene in delaying flowering in plants.

[0011] In a fifth aspect, the present invention provides a biological agent for delaying the flowering of litchi, wherein the active ingredient is the aforementioned engineered bacteria.

[0012] In a sixth aspect, the present invention provides a method for delaying flowering of litchi, comprising the following steps: increasing the expression of the litchi LcCPC3 gene or its encoded protein in litchi plants, wherein the nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1 and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0013] In this invention, by comparing the flowering and heading days of transgenic Arabidopsis thaliana plants overexpressing the litchi LcCPC3 gene and wild-type Arabidopsis thaliana plants, it was found that the flowering time of the transgenic Arabidopsis thaliana plants was significantly later than that of the wild-type plants. This indicates that the litchi LcCPC3 gene participates in regulating the flowering time of litchi and is an important regulatory gene for litchi flowering. Utilizing the litchi LcCPC3 gene can regulate the flowering time of litchi and stabilize flowering, which has significant application value for achieving stable litchi yield.

[0014] In addition, compared with wild-type Arabidopsis plants, transgenic plants overexpressing the litchi LcCPC3 gene had significantly more rosette leaves at flowering time, and the leaves were significantly shorter and narrower. Attached Figure Description

[0015] Figure 1 The results are for the detection of Arabidopsis thaliana plants overexpressing the LcCPC3 gene in Example 2 of this invention; lanes 1-17 are Arabidopsis thaliana positive plants; "+" is positive control; "-" is negative control; "O" is water.

[0016] Figure 2 The image shows the flowering phenotype of Arabidopsis thaliana plants at 28 days in Example 3 of this invention; from left to right, they are WT, overexpressing plant OE1, and overexpressing plant OE2.

[0017] Figure 3 The results show the number of flowering days of Arabidopsis thaliana plants in Example 3 of this invention. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0020] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2013), or as recommended by the manufacturer.

[0021] In some embodiments of the present invention, the application of the litchi LcCPC3 gene in delaying flowering of plants is disclosed, wherein the nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1.

[0022] In some embodiments, the plant is lychee or Arabidopsis thaliana.

[0023] In other embodiments of the present invention, the application of the protein encoded by the litchi LcCPC3 gene in delaying flowering of plants is disclosed, the amino acid sequence of which is shown in SEQ ID NO:2.

[0024] In some embodiments, the plant is lychee or Arabidopsis thaliana.

[0025] In some embodiments of the present invention, the application of a recombinant expression vector overexpressing the litchi LcCPC3 gene in delaying flowering of plants is disclosed, wherein the nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1.

[0026] In some embodiments, the recombinant expression vector is pCAMBIA1390-LcCPC3.

[0027] In some embodiments, the plant is lychee or Arabidopsis thaliana.

[0028] In some embodiments of the present invention, the application of engineered bacteria transformed with the recombinant expression vector overexpressing the litchi LcCPC3 gene is disclosed in delaying flowering of plants, wherein the nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1.

[0029] In some embodiments, the engineered bacteria is Agrobacterium.

[0030] In some embodiments, the plant is lychee or Arabidopsis thaliana.

[0031] In other embodiments of the present invention, a biological agent for delaying litchi flowering is disclosed, the active ingredient of which is the aforementioned engineered bacteria.

[0032] In some embodiments of the present invention, a method for delaying flowering of litchi is disclosed, comprising the following steps: increasing the expression of the litchi LcCPC3 gene or its encoded protein in litchi plants, wherein the nucleotide sequence of the litchi LcCPC3 gene is shown in SEQ ID NO:1 and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0033] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1: Obtaining the LcCPC3 gene from litchi

[0035] Includes the following steps:

[0036] 1. RNA extraction and cDNA cloning

[0037] Approximately 1g of young, tender leaves from the 'Feizixiao' litchi variety were selected. After adding liquid nitrogen, the tissues and cells were lysed using a mortar and pestle, and then quickly transferred to 2.0mL centrifuge tubes. RNA was extracted from the litchi leaves using a plant total RNA extraction kit (RNAprep pure Tissue Kit, TIANGEN). The RNA concentration was detected using a spectrophotometer, and the integrity of the RNA was assessed by agarose gel electrophoresis.

[0038] Next, using the Prime Script Reverse Transcriptase kit (Takara) from Dalian Takara Bio Co., Ltd., the reverse transcription primers, dNTPs, template RNA, and RNase-free H2O were mixed (total volume 20 μL), denatured and annealed at 65°C for 5 minutes, and then placed on ice. 5 μL of 5X reverse transcriptase was then added, and reverse transcription was performed (30°C for 10 minutes, 42°C for 25 minutes, and 95°C for 5 minutes) to produce cDNA. The cDNA was stored at -20°C for later use.

[0039] 2. Amplification of the LcCPC3 gene

[0040] Using total cDNA after reverse transcription as a template, PCR amplification was performed using LcCPC3-F: ATGGATAAGCGTCGC (SEQ ID NO:3) and LcCPC3-R: AGAATTGCATCTCTT (SEQ ID NO:4) as forward and reverse primers. The PCR products were sequenced, recovered, and purified to obtain the litchi LcCPC3 gene fragment (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2).

[0041] The PCR reaction system (20 μL) consisted of: 1 μL of forward primer (10 μmol / L), 1 μL of reverse primer (10 μmol / L), 1 μL of cDNA, 10 μL of SuperMIX 2X, and 7 μL of deionized water. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 58℃ annealing for 2 minutes, 72℃ extension for 45 seconds, 34 cycles; 72℃ for 8 minutes.

[0042] SEQ ID NO:1:

[0043] ATGGATAAGCGTCGCCGGAAGCAAGCCAAGACTAGTAGTTGTGCTCTGA

[0044] AGAGGTGAGCAGTATTGAATGGGAGTTCATAAACATGTCGGAACAAGAGG

[0045] AGGATCTCATTCATAGAATGTATAAACTCGTTGGAGACAGGTGGGCACTGA

[0046] TAGCCGGACGGATTCCGGGCAGAAAAGCAGAAGAAATTGAGAGGTTTTG

[0047] GATAATGAGACATGGAGAAGTGTTTGCAAATAGAAGAAAAGAGCTCAAGA

[0048] GATGCAATTCTTGA

[0049] SEQ ID NO:2:

[0050] MDKRRRKQAKTSSCCSEEVSSIEWEFINMSEQEEDLIHRMYKLVGDRWALIA

[0051] GRIPGRKAEEIERFWIMRHGEVFANRRKELKRCNS

[0052] Example 2: Construction of recombinant expression vector for overexpressing litchi LcCPC3 and obtaining positive plants

[0053] Includes the following steps:

[0054] 1. The PCR amplification product of Example 1 was recovered and purified. The LcCPC3 gene was ligated into the vector pCAMBIA1390 plasmid (preserved in the applicant's laboratory and commercially available) using in-fusion technology. Positive clones were selected for sequencing after transformation.

[0055] 2. The correct plasmid was transformed into Agrobacterium GV3101 using the liquid nitrogen freeze-thaw method. Antibiotic selection was performed using 50 mg / L Rif and 50 mg / L Kan. The obtained single clones were identified as positive by PCR.

[0056] 3. Add Agrobacterium to 50 mL LB medium containing 50 mg / L Rif and 50 mg / L Kan resistance, and incubate overnight at 28°C in a shaker. Measure the OD value; stop shaking when the OD value reaches 0.8–1.0. Centrifuge at 28°C, 6000 rpm for 10 min.

[0057] 4. Prepare 50 mL of infection solution (containing 0.11 g MS, 2.5 g sucrose, and 10 μL 400 μl / L Silwet-77) with 50 mL of bacterial culture. Infect Arabidopsis thaliana using the Agrobacterium-mediated flower immersion method. Infect for 20-30 seconds and then cover with light for 24 hours.

[0058] 5. Transformed Arabidopsis thaliana were replanted in an incubator, and T0 generation Arabidopsis thaliana seeds were collected.

[0059] 6. Plant T0 generation Arabidopsis seeds in sterilized substrate and culture them in an incubator until the plants mature. Culture conditions include a light intensity of 120–150 μmol / (m²). 2 •s), photoperiod 16h light, 8h darkness. T1 generation plants were obtained.

[0060] 7. Collect T1 generation seeds and continue to plant T1 seeds. Detect T1 generation Arabidopsis thaliana positive plants using detection primers. Detection primer F: CGGCGACGAGCCAGGGATA (SEQ ID NO:5) and detection primer R: GCACATCGTCAACCACTACAT (SEQ ID NO:6).

[0061] The results are as follows Figure 1 As shown in the figure, the results indicate that all 17 T1 Arabidopsis thaliana plants tested were positive.

[0062] Example 3: Detection of flowering phenotype in transgenic plants overexpressing the litchi LcCPC3 gene

[0063] Transgenic Arabidopsis T2 generation plants and control wild-type Arabidopsis seeds were sown in sterilized substrate (peat:vermiculite = 1:1). After flowering and heading, the total number of days of growth of Arabidopsis plants at flowering time was counted. Six plants were investigated for each transgenic line, with three replicates for each investigation.

[0064] The flowering phenotype of Arabidopsis thaliana plants at 28 days of growth is as follows: Figure 2As shown in the figure, it is clear that the wild-type Arabidopsis thaliana WT had already flowered and produced spikes after 28 days of growth, while the transgenic Arabidopsis thaliana OE1 and OE2, which overexpress the litchi LcCPC3 gene, had not yet flowered or produced spikes at this time. The statistical results of the growth days of the Arabidopsis plants are as follows: Figure 3 As shown, the results indicated that the flowering times of transgenic Arabidopsis thaliana OE1 and OE2, which overexpress the litchi LcCPC3 gene, were 32 days and 31 days, respectively, while the flowering time of wild-type Arabidopsis thaliana WT was 28 days. The flowering time of the transgenic Arabidopsis thaliana was significantly later than that of the wild-type plant. This result suggests that the litchi LcCPC3 gene is involved in regulating the flowering time of litchi.

[0065] In addition, the number of rosette leaves at flowering time was counted. The number of leaves in the overexpressing Arabidopsis thaliana plant was 14.2, which was significantly more than the number of leaves in the wild-type Arabidopsis thaliana plant (10.3). However, the leaf length and width of the overexpressing Arabidopsis thaliana plant (1.8 and 0.6, respectively) were significantly smaller than those of the wild-type Arabidopsis thaliana plant (2.1 and 0.8, respectively).

[0066] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0067] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Lychee LcCPC3 The application of genes in delaying plant flowering is characterized by, The lychee LcCPC3 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the plant is litchi or Arabidopsis thaliana.

2. Lychee LcCPC3 The application of genes encoding proteins in delaying plant flowering, characterized by, The amino acid sequence of the encoded protein is shown in SEQ ID NO:2, and the plant is litchi or Arabidopsis thaliana.

3. Overexpression of lychee LcCPC3 The application of recombinant gene expression vectors in delaying plant flowering is characterized by, The lychee LcCPC3 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the plant is litchi or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The recombinant expression vector is pCAMBIA1390- LcCPC3 .

5. The overexpression litchi according to claim 3 or 4 LcCPC3 The application of engineered bacteria with recombinant gene expression vectors in delaying flowering in plants, wherein the plants are litchi or Arabidopsis thaliana.

6. The application according to claim 5, characterized in that, The engineered bacteria is Agrobacterium.

7. A method for delaying litchi flowering, characterized in that, Includes the following steps: Increase the amount of litchi in litchi plants LcCPC3 The expression of the gene or its encoded protein, in the litchi LcCPC3 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

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