Gene for regulating and controlling flowering time of loquat as well as encoding protein and application of gene

Abstract

The invention discloses a gene for regulating and controlling the flowering time of loquat as well as an encoded protein and application thereof, and belongs to the field of molecular biology, the nucleotide sequence of the gene is shown as SEQ ID No.1, and the amino acid sequence of the corresponding code is shown as SEQ ID No.2. The EjEJ2 gene disclosed by the invention has relatively high expression in flower buds and various flower organs. Transient expression is achieved in tobacco leaves and located in cell nucleuses and plasma membranes. Instantaneous transformation of the EjEJ2 gene into the loquat flower bud shows that the overexpression of the EjEJ2 gene advances the flowering of the loquat, and the instantaneous silence of the EjEJ2 gene delays the flowering of the loquat. The EjEJ2 is used for regulating and controlling the flowering phase of the loquat and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a gene for regulating the flowering time of loquat, its encoded protein, and applications thereof. Background Art

[0002] Loquat is a characteristic fruit tree. It germinates in autumn, flowers in winter, bears fruits in spring, and ripens in summer. The fruits are sweet and sour, delicious, and rich in nutrients. Loquat leaves, fruits, seeds, and flowers are rich in bioactive substances such as triterpenoids, carotenoids, flavonoids, phenols, and amygdalin, and have high medicinal value. The flowering period is a crucial period in crop production and directly affects the economic traits of fruit trees. The ripening period of loquat is relatively concentrated and the shelf life is short. Regulating 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 that regulate plant flowering. Among them, the SEPALLATA (SEP) gene is a MIKC-type MADS-box gene that regulates flower organ development. In Arabidopsis thaliana, it has been found that the SEP gene is involved in regulating the growth and morphogenesis of flower organs, plays a role in identifying flower organs and regulating the flowering time, and overexpression of the SEP gene promotes early flowering in Arabidopsis thaliana. In tomatoes, it has been studied that the SEP gene EJ2 can activate the occurrence of floral meristems and inflorescence development. However, the function of the SEP gene EjEJ2 in flower development in loquat has not been reported, and its molecular mechanism for regulating the flowering time is not clear. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a gene for regulating the flowering time of loquat, its encoded protein, and applications thereof.

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

[0006] Loquat EjEJ2 protein, whose amino acid sequence is shown in SEQ ID No.2.

[0007] A gene encoding the above-mentioned loquat EjEJ2 protein.

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

[0009] A vector containing the above-mentioned gene.

[0010] An engineered bacterium containing the above-mentioned gene.

[0011] The application of the above-mentioned gene, vector, or engineered bacterium in regulating the flowering time of loquat.

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

[0013] Furthermore, the method of the application is: silencing 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 the present invention is highly expressed in flower buds and various floral organs. Transient expression in tobacco leaves is localized to the nucleus and plasma membrane. It was found that transient transformation into loquat flower buds showed that overexpression of the EjEJ2 gene advanced the flowering of loquat, while transient silencing of the EjEJ2 gene delayed the flowering of loquat. The EjEJ2 in the present invention is used for regulating the flowering period of loquat and has good application prospects. Description of the Drawings

[0016] Figure 1 Gel electrophoresis map of EjEJ2 gene cloning;

[0017] Figure 2 Analysis of tissue-specific expression of EjEJ2;

[0018] Figure 3 Subcellular localization of EjEJ2;

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

[0020] Figure 5 Relative expression level after transient injection of EjEJ2. Detailed Embodiments

[0021] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from commercial channels unless otherwise specified.

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

[0023] Example 1 Cloning of the cDNA Sequence of the EjEJ2 Gene

[0024] Collect flower buds at the morphological differentiation stage of 'Guozhenhao No. 3', remove the outer scales from the fresh flower buds, take 0.5 g and add liquid nitrogen, and grind the flower buds into powder in a mortar. Use the CTAB method to extract the total RNA in the loquat flower buds. After detecting the quality of the RNA with a microprotein analyzer, store it at -80 °C for later use.

[0025] Using the total RNA of loquat flower buds as a template, reverse transcription of RNA was carried out using SyScript III one-Tube RT SuperMix (+gDNARemover). Then, using the cDNA as a template, the coding region of EjEJ2 was amplified using the high-fidelity enzyme gold medal Mix. 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℃ for 2 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 15 s, for 34 cycles, and 72℃ for 3 min; stored at 4℃. After completion, the PCR product was taken for agarose gel electrophoresis detection, and the correctly amplified target band was recovered using a gel recovery 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 transferred into 50 μL of DH5α Escherichia coli competent cells and cultured overnight in an inverted position in a 37℃ incubator. The next day, single colonies were picked, and after shaking the bacteria for 6 h, colony PCR and gel electrophoresis detection were carried out using the universal primers M13F: 5’-TGTAAAACGACGGCCAGT-3’, M13R: 5’-CAGGAAACAGCTATGACC-3’. The bacterial solution with the correct band was sent to the company for sequencing, and the sequencing results were compared and analyzed using DNAMAN software to obtain the coding region sequence (SEQ ID No.1) of the cDNA of the loquat EjEJ2 gene. The coding region sequence of the cDNA of the loquat EjEJ2 gene was translated into a protein sequence using the Expasy website to obtain the protein sequence (SEQ ID No.2) of the EjEJ2 gene.

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

[0027] Total RNA was extracted from nine tissue materials including petals, stamens, pistils, receptacles, leaves, petioles, phloems, flower buds at the floret differentiation stage, and fruits at the mature stage of 'Guozhenhao 3' and 'Chunhua 1'. After detecting the quality with a microprotein analyzer, it was reverse transcribed into cDNA. Real-time fluorescence quantitative PCR primers were designed using Beacon Designer software, qEjEJ2-F: 5’-GTACCAAAGATGCAGTTATAC-3’, qEjEJ2-R: 5’-GGAGGATCTCAACTCTTG-3’, and the loquat reference primer qActin-F: 5’-AATGGAACTGGAATGGTCAAGGC-3’, qActin-R: 5’-TGCCAGATCTTCTCCATGTCATCCCA-3’. Using cDNA as a template, a real-time fluorescence quantitative PCR experiment was carried out by SYBR Green I chimeric fluorescence method, and 3 biological replicates were set for each reaction. The PCR reaction program was pre-denaturation: 95°C, 1 min; cycle reaction: 95°C, 10 s, 60°C, 20 s, for 40 cycles, and the obtained Ct values were recorded. The relative expression level of the target gene was calculated using 2 -ΔΔCt -ΔΔCt method. The results showed that EjEJ2 was hardly expressed in mature fruits and phloems, but had relatively high expression levels in tissues related to flowers and floral organs ( Figure 2 ), indicating that this gene might play an important role in loquat flower development.

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

[0029] To remove the stop codon of EjEJ2, homologous arm primers were designed using Snapgene, GFP-EjEJ2-F: 5’-acgaacgatagccat GGTACC ATGGGCAGAGGTAAAGTTGAGC-3’, GFP-EjEJ2-R: 5’-gcctgccggccgcgcc GGATC C CCCAGGAATGTATCCATTAACATGC-3’. Using the plasmid with correct cloning and sequencing of the EjEJ2 gene as a template, PCR amplification was carried out using the high-fidelity enzyme GoldMix. After the amplification, the correct band was selected for gel cutting and recovery, and stored at -20°C.

[0030] The pC1300 vector was linearized by digestion with the fast-cutting enzymes BamHⅠ and KpnI at 37°C for 2 h. The amplified target gene sequence was recombined into the linearized pC1300 vector using In-Fusion enzyme (Takara) to obtain pC1300-EjEJ2, which was then transformed into Escherichia coli and sent to the company for sequencing. The constructed subcellular localization vector and the empty vector pC1300 were transformed into Agrobacterium tumefaciens GV3101 by the freeze-thaw method. After picking and culturing the bacteria, colony PCR was performed, and the bacterial solution with the correct band was mixed with an equal proportion of 50% glycerol and stored at -80°C or directly used for transformation.

[0031] The fresh bacterial solutions of pC1300-EjEJ2, the empty vector pC1300, and the nuclear marker were respectively inoculated into 10 ml of liquid medium (containing Kan and Rif), and cultured vigorously until the OD of the bacterial solution 600 = 0.5 - 0.6. The cells were collected by centrifugation at 5000×g for 10 min, and then an infection solution (10 mM MgCl 2 , 10 mM MES, 20 mM AS) was prepared to suspend the cells and adjusted to OD 600 = 0.8 - 1.0. The infection solution was prepared freshly and used immediately. Mixing: pC1300-EjEJ2 and the nuclear marker were mixed in equal proportion, and left to stand in the dark for 2 h. The syringe needle was removed, and the bacterial solution was aspirated. Pressing the finger against the front of the leaf, the bacterial solution was infiltrated from the front, and marks were made. After culturing in the dark for 12 h, it was cultured under normal conditions for 48 h. The tobacco leaves were cut, and the fluorescence signal was observed with a Zeiss inverted fluorescence microscope. The results showed that EjEJ2 was localized 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 Eriobotrya japonica EjEJ2 gene

[0033] The pC1300 vector was linearized by digestion with the fast-cutting enzymes BamHⅠ and KpnI at 37°C for 2 h. 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 GATCCGCCGTGGCAAGCTCTATGAAT-3’, pTRV2-EjEJ2-R: 5’-cttcgggacatgc CCGGG AGTGGAGTTGTTTCCTCCCAAGG-3’. Using the plasmid with the correct cloned and sequenced EjEJ2 gene as a template, PCR amplification was performed using the high-fidelity enzyme GoldMix. After the amplification, the correct band was selected for gel extraction and preservation at -20°C. The cloned target gene was recombined into the linearized pC1300 and pTRV2 vectors using In-Fusion enzyme (Takara) respectively. After transformation into Escherichia coli, it was sent to the company for sequencing. The constructed overexpression vector, gene silencing vector, and empty vectors pC1300, pTRV2, and pTRV1 were transformed into Agrobacterium tumefaciens GV3101 respectively. After picking and culturing the bacteria, colony PCR was performed, and the bacterial solution with the correct band was added with an equal proportion of 50% glycerol and stored at -80°C or directly used for transformation.

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

[0035] The fresh Agrobacterium tumefaciens bacterial solutions of pC1300-EjEJ2, pTRV2-EjEJ2, and empty vectors pC1300, pTRV2, and pTRV1 were inoculated into 30 ml of liquid medium (containing Kan, Rif), and shaken vigorously until the OD of the bacterial solution 600 = 0.5 - 0.6. The bacteria were collected by centrifugation at 5000×g for 10 min. Subsequently, an infection solution (10 mM MgCl 2 , 10 mM MES, 20 mM AS) was prepared to suspend the bacteria and adjusted to OD 600 = 0.8 - 1.0. The infection solution was prepared freshly before use. After the suspension was left standing in the dark for 2 h, 0.5 mL of the bacterial solution was injected from the bottom up at 0.5 - 1 cm from the base of the flower buds of ‘Guozhenhao No. 3’ using a disposable sterile syringe. At the same time, the empty vector and water were injected as controls.

[0036] Example 6 Phenotypic identification of transient overexpression and gene silencing in loquat flower buds

[0037] The loquat flower buds subjected to transient injection were continuously photographed and observed after treatment. By counting the flowering time, it was found that overexpression of the EjEJ2 gene advanced the flowering period of ‘Guozhenhao No. 3’ loquat by one week, and silencing of the EjEJ2 gene delayed flowering by two weeks ( Figure 4 ).

[0038] Take the flower buds for instantaneous injection, extract RNA by the CTAB method, reverse transcribe it into cDNA, use the cDNA of the water treatment (CK), empty vector pC1300 and pTRV2 as controls, and perform real-time fluorescence quantitative analysis. It is found that the expression level of overexpressed EjEJ2 is nearly 6 times higher than that of CK and the empty vector. The transcriptional level of gene-silenced EjEJ2 decreases by 9 times. The results show that the transient transformation of EjEJ2 is successful, and overexpression of this gene can promote the flowering of 'Guozhenhao No. 3' loquat, while silencing this gene delays the flowering of loquat.

Claims

1. Loquat EjEJ2 protein, whose amino acid sequence is shown in SEQ ID No.

2.

2. A gene encoding the loquat EjEJ2 protein according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. A vector containing the gene according to claim 2 or 3.

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

6. Use of the gene according to claim 2 or 3, the vector according to claim 4, or the engineered bacteria according to claim 5 in regulating the flowering time of loquat.

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

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

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