Gene for regulating flowering time of loquat and encoded protein and application thereof

Abstract

The application discloses a gene for regulating flowering time of loquat, an encoded protein and application thereof, and belongs to the field of molecular biology. The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the corresponding encoded amino acid sequence is shown in SEQ ID No. 2. The EjEJ2 gene in the application has higher expression in flower buds and various flower organs. The EjEJ2 gene is located in the cell nucleus and the plasma membrane after transient expression in tobacco leaves. After transient transformation into loquat flower buds, it is found that overexpression of the EjEJ2 gene can advance the flowering of loquat, and transient silencing of the EjEJ2 gene can delay the flowering of loquat. The EjEJ2 gene in the application is used for regulating the flowering period of loquat, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a gene that regulates the flowering time of loquat, its encoded protein, and its applications. Background Technology

[0002] Loquat is a specialty fruit tree that sprouts in autumn, flowers in winter, bears fruit in spring, and ripens in summer. The fruit is sweet and sour, and rich in nutrients. Loquat leaves, fruit, seeds, and flowers are rich in triterpenoids, carotenoids, flavonoids, phenols, and amygdalin, among other bioactive substances, giving it high medicinal value. The flowering period is a crucial time for crop production, directly affecting the economic traits of the fruit tree. Loquat ripens relatively quickly and has a short shelf life; therefore, controlling the flowering time is an important way to extend the shelf life of loquat.

[0003] The MADS-box gene family is an important class of transcription factors regulating plant flowering. Among them, the SEPALLATA (SEP) gene is a MIKC-type MADS-box gene that regulates floral organ development. In Arabidopsis thaliana, the SEP gene has been found to participate in regulating the growth and morphogenesis of floral organs, playing a role in recognizing floral organs and regulating flowering time. Overexpression of the SEP gene promotes earlier flowering in Arabidopsis thaliana. In tomato, the SEP gene EJ2 has been found to activate the development of floral meristems and inflorescence development. However, the function of the SEP gene EjEJ2 in loquat flower development has not been reported, and its molecular mechanism for regulating flowering time remains unclear. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a gene that regulates the flowering time of loquat, its encoded protein, and its application.

[0005] The technical solution of this invention is as follows:

[0006] The amino acid sequence of loquat EjEJ2 protein is shown in SEQ ID No. 2.

[0007] The gene encoding the loquat EjEJ2 protein described above.

[0008] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0009] Vectors containing the genes described above.

[0010] Engineered bacteria containing the genes described above.

[0011] The application of the aforementioned genes, vectors, or engineered bacteria in regulating the flowering time of loquat.

[0012] Furthermore, the method of application is to overexpress the gene described in claim 2 or claim 3 in loquat, thereby promoting early flowering of loquat.

[0013] Furthermore, the method of application is to silence the gene described in claim 3 in loquat, thereby delaying the flowering time of loquat.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The EjEJ2 gene in this invention exhibits high expression in flower buds and various floral organs. It is transiently expressed in tobacco leaves, localized to the cell nucleus and plasma membrane. Transient transformation into loquat flower buds revealed that overexpression of the EjEJ2 gene advanced loquat flowering, while transient silencing of the EjEJ2 gene delayed flowering. The use of EjEJ2 in regulating loquat flowering in this invention shows great promise for future applications. Attached Figure Description

[0016] Figure 1 EjEJ2 gene clone gel electrophoresis image;

[0017] Figure 2 Tissue-specific expression analysis of EjEJ2;

[0018] Figure 3 EjEJ2 subcellular localization;

[0019] Figure 4 Continuous observation of flowering period after instantaneous injection of EjEJ2;

[0020] Figure 5 EjEJ2 instantaneous injection of relative expression level. Detailed Implementation

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial channels.

[0022] The biological materials used in this invention, 'Guozhenhao No. 3' and 'Chunhua No. 1', are both approved and commercially available loquat varieties, which can be obtained by those skilled in the art through commercial channels.

[0023] Example 1: Cloning of the EjEJ2 gene cDNA sequence

[0024] Flower buds of 'Guozhenhao No. 3' during the morphological differentiation period were collected. The outer scales of the fresh flower buds were peeled off, and 0.5g of the powder was added to liquid nitrogen and ground into powder in a mortar. Total RNA was extracted from the loquat flower buds using the CTAB method. After the RNA quality was detected by a microprotein analyzer, the powder was stored at -80℃ for later use.

[0025] Using total RNA from loquat flower buds as a template, reverse transcription of RNA was performed using SyScript III one-Tube RT SuperMix (+gDNARemover). Then, using cDNA as a template, the EjEJ2 coding region was amplified using a high-fidelity enzyme, GoldMix. Referring to the loquat genome sequence (https: / / ngdc.cncb.ac.cn / gwh / Assembly / 10399 / show), cloning primers were designed using Snapgene software: EjEJ2-F: 5'-ATGGGCAGAGGTAAAGTTGA-3'; EjEJ2-R: 5'-CTACCCAGGAATGTATCCATTAAC-3'. The reaction conditions were: 98℃, 2 min; 98℃, 10 s; 56℃, 15 s; 72℃, 15 s, for 34 cycles; 72℃, 3 min; stored at 4℃. After completion, the PCR products were collected and analyzed by agarose gel electrophoresis. The correctly amplified target band was recovered using a gel recovery and purification kit. Figure 1 The T-vector was ligated using the pClone007 Blunt Simple Vector Kit Ver.2. 10 μL of the ligation product was transformed into 50 μL of DH5α *E. coli* competent cells and incubated overnight at 37°C. The next day, single colonies were picked, and after 6 hours of shaking, bacterial culture was detected by PCR and gel electrophoresis using universal primers M13F (5'-TGTAAAACGACGGCCAGT-3') and M13R (5'-CAGGAAACAGCTATGACC-3'). Bacterial cultures with correct bands were sent for sequencing. Sequencing results were analyzed using DNAMAN software, yielding the coding region sequence of the *E. coli* EjEJ2 gene cDNA (SEQ ID No. 1). The protein sequence of the *E. coli* EjEJ2 gene cDNA was translated using the Expasy website, yielding the protein sequence of the *E. coli* EjEJ2 gene (SEQ ID No. 2).

[0026] Example 2: Tissue-specific expression analysis of the loquat EjEJ2 gene

[0027] Total RNA was extracted from nine tissue materials during the full bloom of 'Guozhenhao No. 3' and 'Chunhua No. 1' varieties, including petals, stamens, pistils, receptacles, leaves, petioles, phloem, flower buds during floret differentiation, and mature fruits. After quality testing with a microprotein analyzer, the RNA was reverse transcribed into cDNA. Real-time quantitative PCR primers were designed using Beacon Designer software: qEjEJ2-F: 5'-GTACCAAAGATGCAGTTATAC-3', qEjEJ2-R: 5'-GGAGGATCTCAACTCTTG-3'. Loquat internal control primers were qActin-F: 5'-AATGGAACTGGAATGGTCAAGGC-3', qActin-R: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3'. Real-time quantitative PCR experiments were performed using cDNA as a template with the SYBR Green I chimeric fluorescence assay, with three biological replicates for each reaction. The PCR reaction program was as follows: pre-denaturation: 95℃, 1 min; cycling reaction: 95℃, 10 s, 60℃, 20 s, for 40 cycles. The Ct values ​​were recorded, and the relative expression levels of the target gene were calculated using 2-1Tt. -ΔΔCt Calculations showed that EjEJ2 was almost not expressed in mature fruit and phloem, but its expression was relatively high in flower and flower organ-related tissues. Figure 2 This indicates that this gene may play an important role in the development of loquat flowers.

[0028] Example 3: Subcellular localization analysis of the loquat EjEJ2 gene in tobacco

[0029] EjEJ2 with stop codon removed was used to design homologous arm primers using Snapgene, GFP-EjEJ2-F: 5'-acgaacgatagccat GGTACC ATGGGCAGAGGTAAAGTTGAGC-3', GFP-EjEJ2-R:5'-gcctgccggccgcgcc GGATC C Using the plasmid correctly returned from the EjEJ2 gene cloning and sequencing as a template, PCR amplification was performed using a high-fidelity enzyme, Gold Mix. After amplification, the correct band was selected for gel extraction and recovery, and the sample was stored at -20℃.

[0030] The pC1300 vector was linearized by digestion with BamHI and KpnI rapid digestive enzymes at 37°C for 2 hours. The amplified target gene sequence was then recombined into the linearized pC1300 vector using In-Fusion enzyme (Takara), yielding pC1300-EjEJ2. This was transformed into E. coli and sent to a company for sequencing. The constructed subcellular localization vector and the empty pC1300 vector were transformed into Agrobacterium GV3101 using the freeze-thaw method. After picking and shaking the bacteria, culture PCR was performed. Cultures with correct bands were selected and added with an equal proportion of 50% glycerol, then stored at -80°C or used directly for transformation.

[0031] Fresh pC1300-EjEJ2, empty vector pC1300, and Agrobacterium nucleatum marker culture were inoculated into 10 ml of liquid culture medium (containing Kan and Rif), and shaken vigorously until the culture reached OD. 600 =0.5-0.6, centrifuge at 5000×g for 10 min to collect bacterial cells, then prepare infection solution (10mM MgCl2, 10mM MES, 20mM AS) to suspend the bacterial cells and adjust to OD. 600 =0.8-1.0, the inoculum should be prepared and used immediately. Mixing: pC1300-EjEJ2 and nuclear marker were mixed in equal proportions, incubated in the dark for 2 hours, and the bacterial solution was drawn using a disposable syringe with the needle removed. The bacterial solution was then injected into the leaf from the front side using a finger, and marked. After 12 hours of dark incubation, the leaves were placed under normal conditions for 48 hours. Tobacco leaves were then cut and the fluorescence signal was observed using a Zeiss inverted fluorescence microscope. The results showed that EjEJ2 was located in the nucleus and cell membrane (…). Figure 3 ).

[0032] Example 4: Construction of the transient overexpression vector pC1300-EjEJ2 and the gene silencing vector pTRV2-EjEJ2 of the loquat EjEJ2 gene

[0033] The pC1300 vector was linearized by digestion with BamHI and KpnI rapid digestion enzymes at 37°C for 2 hours. The pTRV2 vector was digested with BamHI and SmaI. Homologous arm primers were designed using Snapgene: PC1300-EjEJ2-F: 5'-acgaacgatagccat GGTACC ATGGGCAGAGGTAAAGTTGA-3',PC1300-EjEJ2-R:5'-gcctgccggccgcgcc GGATCC CTACCCAGGAATGTATCCATTAAC-3',pTRV2-EjEJ2-F:5'-gcctccatggg GATCC GCCGTGGCAAGCTCTATGAAT-3',pTRV2-EjEJ2-R:5'-cttcgggacatgc CCGGG Using the plasmid correctly returned from the EjEJ2 gene clone sequencing as a template, PCR amplification was performed using a high-fidelity enzyme, Gold Mix. After amplification, the correct bands were selected for gel extraction and recovery, and stored at -20℃. The cloned target gene was recombined into linearized pC1300 and pTRV2 vectors using In-Fusion enzyme (Takara), respectively, and transformed into E. coli before being sent to the company for sequencing. The constructed overexpression vector, gene silencing vector, and empty vectors pC1300, pTRV2, and pTRV1 were transformed into Agrobacterium GV3101, respectively. After picking and shaking the bacteria, PCR was performed on the bacterial culture. Bacterial cultures with correct bands were selected and mixed with an equal proportion of 50% glycerol, and stored at -80℃ or used directly for transformation.

[0034] Example 5: Transient injection of loquat flower buds with overexpression vector pC1300-EjEJ2 and gene silencing vector pTRV2-EjEJ2

[0035] Fresh Agrobacterium suspensions of pC1300-EjEJ2, pTRV2-EjEJ2, and empty vectors pC1300, pTRV2, and pTRV1 were inoculated into 30 ml of liquid culture medium (containing Kan and Rif), and shaken vigorously until the bacterial suspension reached OD. 600 =0.5-0.6, centrifuge at 5000×g for 10 min to collect bacterial cells, then prepare infection solution (10mM MgCl2, 10mM MES, 20mM AS) to suspend the bacterial cells and adjust to OD. 600 =0.8-1.0, the inoculum should be prepared and used immediately. After the suspension has been allowed to stand in the dark for 2 hours, 0.5 mL of bacterial suspension is injected from bottom to top at a depth of 0.5-1 cm from the base of the flower bud of 'Guozhenhao No. 3' using a disposable sterile syringe. At the same time, empty vector and distilled water are injected as controls.

[0036] Example 6: Phenotypic Identification of Transient Overexpression and Gene Silencing in Loquat Flower Buds

[0037] Post-treatment observation and photographic analysis of loquat flower buds treated with instantaneous injection revealed that overexpression of the EjEJ2 gene advanced the flowering period of 'Guozhenhao No. 3' loquat by one week, while silencing the EjEJ2 gene delayed flowering by two weeks. Figure 4 ).

[0038] Transiently injected flower buds were used, and RNA was extracted using the CTAB method. The RNA was then reverse transcribed into cDNA. Water treatment (CK) and cDNA containing the empty vector pC1300 and pTRV2 were used as controls. Real-time quantitative analysis revealed that the expression level of overexpressed EjEJ2 was nearly 6 times higher than that of CK and the empty vector, while gene silencing of EjEJ2 decreased the transcription level by 9 times. These results indicate that transient transformation of EjEJ2 was successful, and that overexpression of this gene promotes flowering in 'Guozhenhao 3' loquat, while silencing the gene delays flowering.

Claims

1. A loquat EjEJ2 protein, the amino acid sequence of which is shown as SEQ ID No.

2.

2. A gene encoding the loquat EjEJ2 protein of claim 1.

3. The gene of claim 2, wherein, The nucleotide sequence of the gene is shown as SEQ ID No.

1.

4. A vector containing the gene of claim 2 or 3.

5. An engineered bacterium containing the gene of claim 2 or 3.

6. Use of the gene of claim 2 or 3 or the vector of claim 4 or the engineered bacterium of claim 5 in regulating the flowering time of loquat.

7. Use according to claim 6, characterized in that, The method of the use is overexpression of the gene of claim 2 or claim 3 in loquat, thereby promoting early flowering of loquat.

8. Use according to claim 6, characterized in that, The method of the use is silencing of the gene of claim 3 in loquat, thereby delaying the flowering time of loquat.

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