Gene EjTGA1 related to regulation and control of flowering phase and growth development of loquat as well as encoding protein and application of gene EjTGA1

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an EjTGA1 gene related to loquat flowering phase and growth development regulation, a protein coded by the EjTGA1 gene and application of the EjTGA1 gene. The full length of the cDNA sequence of the gene is as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein of the gene is as shown in SEQ ID NO.2. The EjTGA1 gene disclosed by the invention is predominantly expressed in the loquat flower. An expression vector pCambia2300-EjTGA1-eGFP is introduced into wild type arabidopsis thaliana through an agrobacterium tumefaciens-mediated inflorescence infection method, the overexpression of transgenic arabidopsis thaliana 35S:: EjTGA1 promotes the arabidopsis thaliana to bloom in advance, and the leaf area and the rosette leaf number are increased. The transgenic arabidopsis thaliana plant material obtained by using the loquat EjTGA1 gene can advance flowering and fruiting time and regulate growth and development, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology, and specifically relates to a gene EjTGA1 related to the regulation of loquat flowering period and growth and development, and a coded protein and application thereof. Background Art

[0002] Loquat belongs to the genus Eriobotrya in the Rosaceae family. It is an evergreen fruit tree native to the subtropical regions of my country. Its fruit matures in early summer, which is the off-season for fruits. The length of the shelf life affects the economic benefits of loquat. Promoting early maturity of loquat by advancing the flowering period is an important measure to extend the shelf life. Therefore, it is of great significance to study the expression characteristics of key genes regulating flowering period in regulating the development of loquat flowers, and it can accumulate gene resources for the directional genetic improvement of flowering period of loquat varieties.

[0003] The bZIP gene family is one of the important transcription factor families that regulate plant development. It contains a basic region for specific DNA binding and a leucine zipper domain for dimerization, and can bind to specific DNA sequences in the form of dimers. TGA transcription factors can bind to TGACG / as-1 and belong to group D of the bZIP family. They are involved in plant growth and development and responses to biotic or abiotic stresses. There are 10 TGA transcription factors in Arabidopsis thaliana, which are divided into 5 groups (group IV), among which members of groups I, II and III are widely involved in the disease resistance of plants. Studies have shown that the AtTGA7 gene plays an important role in the control of flowering time, and the Arabidopsis tga7 mutant will cause Arabidopsis to show delayed flowering. However, the molecular mechanism of TGA gene regulating the flowering period and growth and development of woody plants has not been studied and reported, and its related mechanism is still unclear. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a gene EjTGA1 related to the regulation of loquat flowering period and growth and development, as well as its encoded protein and application.

[0005] First, the present invention provides loquat flowering period and growth and development regulation-related protein EjTGA1, which is:

[0006] 1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

[0007] 2) A protein derived from 1) with equivalent activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2.

[0008] The present invention also provides a gene encoding the loquat EjTGA1 protein.

[0009] Preferably, the sequence of the gene is shown as SEQ ID No.1.

[0010] The invention also provides an overexpression vector, a host cell and an engineering bacterium containing the gene.

[0011] The invention also provides use of the gene in promoting flowering of plants.

[0012] In a specific embodiment of the present invention, the gene is transferred into the plant genome and overexpressed in the transgenic plant to advance the flowering and fruiting time of the plant.

[0013] The invention also provides the use of the gene in regulating plant growth and development.

[0014] In a specific embodiment of the present invention, the gene is transferred into the plant genome and overexpressed in transgenic plants to increase leaf area and rosette leaf number.

[0015] The present invention separated an EjTGA1 gene closely related to the development and regulation of loquat flowers from loquats, and found that it was localized in the cell nucleus. Real-time fluorescence quantitative PCR confirmed that the EjTGA1 gene was dominantly expressed in loquat flowers, and it can be inferred that EjTGA1 plays a key role in flower tissues. A plant overexpression vector of the EjTGA1 gene was constructed by genetic engineering means, and it was transferred into wild-type Arabidopsis for overexpression, which can advance the flowering time of Arabidopsis and promote the fruiting time; it can increase the leaf area and the number of rosette leaves, and can regulate plant growth and development. The present invention provides a good application prospect for the regulation of plant flowering period and growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The electrophoresis photographs of the coding region sequence verification of loquat EjTGA1 gene are shown. Among them, M is DL5000 DNA marker, 1 is the PCR product of EjTGA1 gene ORF; the arrow indicates the target gene band amplified by PCR.

[0017] Figure 2 The figure shows the comparison of the amino acid sequence of the protein encoded by loquat EjTGA1 with the sequences of apple, upland cotton and sweet cherry, all of which have bZIP and DOG1 domains.

[0018] Figure 3 The figure shows the subcellular localization of loquat EjTGA1 gene transiently expressed in tobacco leaves, showing that the expression product of the gene is localized in the cell nucleus. eGFP: enhanced green fluorescent protein; mCherry: nuclear localized VirD2NLS-mCherry; Bright: bright field imaging; Merged: the merged image of eGFP, VirD2NLS-mCherry and Bright.

[0019] Figure 4 The figure shows that the expression of loquat EjTGA1 gene in different loquat tissues shows significant differences.

[0020] Figure 5 The figure shows the PCR identification of transgenic Arabidopsis TGA1 positive plants. Among them, M5000 is the DNA molecular mass standard (DL5000), WT is the wild-type Arabidopsis negative control, and P is the pCambia2300-EjTGA1-eGFP plasmid.

[0021] Figure 6 The pictures show the flowering time of wild-type Arabidopsis before and after transgenic and the expression analysis of EjTGA1 gene. A shows that overexpression of EjTGA1 gene can lead to an early flowering time of transgenic Arabidopsis by about 7 days compared with non-transgenic wild-type Arabidopsis; B shows the expression level of EjTGA1 gene in transgenic Arabidopsis.

[0022] Figure 7 The figure shows the plant growth phenotype analysis of wild-type Arabidopsis before and after transgenic. A and C show that overexpression of the EjTGA1 gene can lead to an increase in the leaf area of ​​transgenic Arabidopsis compared with non-transgenic wild-type Arabidopsis; B and D show that overexpression of the EjTGA1 gene can lead to an increase in the number of rosette leaves of transgenic Arabidopsis. DETAILED DESCRIPTION

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions.

[0024] Example 1 Cloning of loquat EjTGA1 gene cDNA sequence

[0025] Different tissues of loquat were collected, sampled quickly, placed in cryopreservation tubes, quickly frozen in liquid nitrogen for 2 h, and then placed in a -80°C ultra-low temperature refrigerator for use. Total RNA from each tissue of loquat was extracted using an RNA extraction kit.

[0026] Specific primers EjTGA5'-ATGAGCTATTCAGCAACTCAATTTG-3' and EjTGA5'-TCATGTTGGTTTGCGGGGAC-3' were designed at both ends of the full-length sequence of the loquat EjTGA1 gene coding region. The reaction conditions were 95℃5min; 95℃30s, 56℃30s, 72℃20s, 35 cycles; 72℃10min. After the PCR reaction was completed, the target band was cut out after detection by 1% agarose gel electrophoresis, and the PCR product was recovered using an agarose gel DNA recovery kit ( Figure 1). After being connected to the pMD19-T vector, it was transformed into E. coli competent cells, and a single clone was picked and sequenced to verify the coding region sequence of the EjTGA1 gene.

[0027] The PCR sequencing results of the coding region sequence verification experiment were analyzed using DNAMAN software to perform sequence splicing and obtain the coding region sequence of loquat EjTGA1 gene cDNA (SEQ ID No. 1).

[0028] Oligo7 software was used to translate the coding region sequence of the cDNA of the loquat EjTGA1 gene into a protein sequence (SEQ ID No. 2). The NCBI online tool Basic Local Alignment Search Tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to search and download the homologous sequence of EjTGA1 encoding amino acids. Furthermore, the amino acid sequence of the protein encoded by the loquat EjTGA1 gene was compared with the apple MdTGA7 (XP_008338800.2), white pear PbTGA7 (XP_009367342.2) protein and apple MdTGA3-like (XP_050123759.1) for amino acid specificity analysis. It was found that these sequences all have highly conserved bZIP domains and DOG1 domains ( Figure 2 ).

[0029] Example 2 Subcellular localization analysis of loquat EjTGA1 gene

[0030] The software SnapGene was used to analyze the restriction site of the ORF sequence of the EjTGA1 gene, and the restriction site primers at both ends were designed, p2300-EjTGA1-eGFP-F: 5'-cagggtacccggggatccATGAGCTATTCAGCAACTCAATTTG-3'; p2300-EjTGA1-eGFP-R: 5'-agtgtcgactctagaggatccTGTTGGTTTGCGGGGACG-3'. The pMD19-EjTGA1 plasmid with correct sequencing was used as a template for amplification to obtain the ORF sequence of the EjTGA1 gene. The target gene and the modified vector pCAMBIA2300 plasmid were extracted respectively, and the restriction endonuclease BamHI was used for restriction digestion reaction, and the results were recovered after agarose gel electrophoresis. The target gene EjTGA1 after restriction digestion and the modified pCAMBIA2300 vector were connected using Coloning Mix ligase, and the recombinant vector was transferred into E. coli competent cells, followed by bacterial liquid PCR and restriction digestion verification and sequencing to ensure that the target gene sequence was successfully connected to the vector. The constructed vector plasmid was extracted and transferred into Agrobacterium GV3101 competent cells by freeze-thaw method.

[0031] Pick a single colony of Agrobacterium from the solid YRK medium plate and inoculate it into 10 mL of liquid medium (containing Rif+kan) and culture at 28°C and 250 rpm until OD 600 =0.5. Take 5 mL of culture medium and centrifuge for 10 min to collect the cells, then add 2 mL of permeate to resuspend the cells, then centrifuge for 10 min and add 2 mL of permeate (10 mM MgCl2, 10 mM MES-KOH, pH=5.6, 150 μM acetosyringone) to resuspend the cells. Finally, dilute to OD 600 =0.03-0.1, and then the tobacco leaves were transformed. After the transformed tobacco was cultured in the dark for 1-2 days, the eGFP fluorescence was observed ( Figure 3 ). The results showed that the expression product of this gene was localized in the cell nucleus.

[0032] Example 3 Real-time fluorescence quantitative PCR analysis of loquat EjTGA1 gene

[0033] Total RNA was extracted from different tissues (root, stem, flower, branch, fruit, leaf) of the wild loquat superior line 'Guizhou 23' ('GZ23'). After detecting its integrity and concentration, it was reverse transcribed into cDNA. Using loquat cDNA as a template, the real-time fluorescence quantitative PCR primers EjTGA1-RT-F: 5'-GAGCTGGAACTTGGCAGAGC-3' and EjTGA1-RT-R: 5'-GCCATAGTGGTTCAAGCCAC-3' were designed using SnapGene software. The loquat actin gene was used as an internal reference gene, and the primers were Ej-actin-F: 5'-AATGGAACTGGAATGGTCAAGGC-3' and Ej-actin-R: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3'. PCR was used to detect its specificity. Real-time fluorescence quantitative PCR experiments were performed only when PCR specific amplification was ensured. Three biological replicates were set for each reaction. The PCR reaction program was: 95℃ pre-denaturation for 5min; 95℃ for 15s, 56℃ for 30s, 72℃ for 30s for 41 cycles, then, the melting curve was collected: the temperature was adjusted to 60℃ for 90s, pre-dissolving; then the temperature was increased at a rate of 1.0℃ / s, and the temperature was kept for 5s for each 1℃ increase until 95℃. The results showed that among the different tissues of loquat, the expression level of EjTGA1 was dominant in flowers ( Figure 4 ), it can be inferred that EjTGA1 plays an important role in floral organs.

[0034] Example 4 Construction of the plant transgenic vector pCAMBIA2300-35S-EjTGA1-eGFP of loquat EjTGA1 gene

[0035] PCR amplification was used to introduce restriction sites at both ends of the CDS region of the loquat EjTGA1 gene. The correctly sequenced pMD19-EjTGA1 plasmid was used as a template, p2300-EjTGA1-eGFP-F: 5'-cagggtacccggggatccATGAGCTATTCAGCAACTCAATTTG-3'; p2300-EjTGA1-eGFP-R: 5'-agtgtcgactctagaggatccTGTTGGTTTGCGGGGACG-3' were used as primers, and Phanta enzyme was used for PCR amplification. PCR reaction program: 95℃5min; 95℃30s, 56℃30s, 72℃30s, 35 cycles; 72℃10min. After the PCR reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and the PCR product was recovered using an agarose gel DNA recovery kit. The recovered PCR product was connected to the pCAMBIA2300-35S-eGFP vector, transferred into Escherichia coli competent cells, single clones were picked and sequenced. According to the analysis of the sequencing results, the plasmid was extracted. The pCAMBIA2300-35S-eGFP vector was cut with BamHⅠ, detected by 1% agarose gel electrophoresis, and recovered using an agarose gel DNA recovery kit. The EjTGA1 gene was connected to pCAMBIA2300-35S-eGFP using Coloning Mix ligase, and then transferred into Escherichia coli competent cells to obtain the plant transgenic expression vector pCAMBIA2300-35S-EjTGA1-eGFP. Example 5: Transferring the transgenic expression vector pCAMBIA2300-35S-EjTGA1-eGFP into Arabidopsis thaliana

[0036] Take 3 μg of pCAMBIA2300-35S-EjTGA1-eGFP plasmid, add 100 μL of Agrobacterium competent cells, and mix well; place in an ice bath for 5 minutes, transfer to liquid nitrogen for rapid freezing for 5 minutes, and quickly place in a 37°C water bath for 5 minutes; add 700 μL of YEB liquid culture medium, and shake at 28°C and 220 rpm for 2 to 3 hours; transfer the bacterial liquid to YEB (50 mL YEB + 50 μg / mL Kan + 50 μg / mL Rif) solid selection medium, spread evenly, and invert and culture at 28°C for 48 hours.

[0037] Streak the Agrobacterium containing pCAMBIA2300-35S-EjTGA1-eGFP positive clones on 25 mL solid plate medium (containing 50 μg / mL Kan + 50 μg / mL Rif) and invert and culture at 28°C for 48 h; pick a single colony to 5 mL LB liquid medium (50 μg / mL Kan and 50 μg / mL Rif), shake and culture at 200 rpm in a 28°C shaker for 15-20 h, take 100 μL to 10 mL LB liquid medium (50 μg / mL Kan and 50 μg / mL Rif) and culture overnight, then take 2 mL of the bacterial solution to 100 mL LB liquid medium (50 μg / mL Kan and 50 μg / mL Rif) and culture until OD 600 =0.8~1.5, transfer the bacterial solution to two 50mL centrifuge tubes, centrifuge at 4℃, 5000rpm for 5min, discard the supernatant, and add 50mL of invasion and infection buffer (0.5% Silwet L-77, 5% sucrose, 1 / 2MS culture medium) to each tube.

[0038] Arabidopsis seeds were vernalized at 4°C for 48 hours, then sown in nutrient soil (perlite: vermiculite: nutrient soil = 1:4:5), and cultured at a temperature of 22°C, a humidity of 70%, and 14 h light / 10 h dark conditions; the wild-type Arabidopsis plants were watered thoroughly before transgenic; when infecting, the siliques on the Arabidopsis plants to be used were cut off, and the leaves were infiltrated with pCAMBIA2300-35S-EjTGA1-eGFP Agrobacterium infection solution for 30 seconds; covered with a black plastic bag, and the high temperature and high humidity environment inside the film was maintained. After dark culture for 24 hours, the black plastic bag was uncovered and placed for normal culture; the above method was used for infection 3 times, with an interval of 7 days.

[0039] Example 6 Screening of transgenic Arabidopsis thaliana expressing loquat EjTGA1 gene

[0040] Collect mature seeds of EjTGA1 transgenic Arabidopsis, dry them and store them. Take a part of the seeds and disinfect them, evenly sow them on a 150mm culture dish containing screening medium (1 / 2MS+1.5% sucrose+Agar+50mg / ml Kan), and place them in a dark refrigerator at 4℃ for vernalization. After 2 days, transfer the culture dish containing the seeds to a light incubator and culture them for 15 days to wait for germination. Select the seedlings that grow normally and transfer them to humus soil. After watering with rooting water, cover them with plastic wrap to keep them moist. Remove the plastic wrap after 4 days and manage water and fertilizer according to the routine. After the Arabidopsis plants were cultured for about 14 days, a certain amount of leaves from each plant were taken into a 200 μl centrifuge tube and crushed, and 8 μl of lysis solution (0.3M NaOH) was added and crushed again; lysis was performed at 95°C for 1.5 min; 170 μl of neutralization solution (0.3M HCl, 1M Tris-HCl, pH=8.0) was added to the amplification tube and mixed, and 8 μl of the lysis product was taken as a template for PCR verification. The DNA of the non-transgenic wild-type Arabidopsis was used as a control, and the EjTGA1 gene of the positive transgenic Arabidopsis plants was confirmed. Primers were designed based on the full-length loquat EjTGA1 gene sequence (p2300-EjTGA1-eGFP-F: 5'-cagggtacccggggatccATGAGCTATTCAGCAACTCAATTTG-3'; p2300-EjTGA1-eGFP-R: 5'-agtgtcgactctagaggatccTGTTGGTTTGCGGGGACG-3'), and 2×Mix taq, 1 μL DNA, were used to perform PCR identification of Arabidopsis plants, and a total of 6 positive EjTGA1 transgenic wild-type Arabidopsis plants were obtained ( Figure 5 ).

[0041] Example 7 Phenotypic Identification of Transgenic Arabidopsis thaliana Expressing the EjTGA1 Gene of Loquat

[0042] The flowering time phenotypes of these Arabidopsis were observed, counted and photographed. The phenotypic results showed that overexpression of the EjTGA1 gene can cause transgenic Arabidopsis to flower and fruit earlier than wild-type Arabidopsis ( Figure 6 ); can lead to increased leaf area and rosette leaf number in transgenic Arabidopsis ( Figure 7 ).

[0043] The cDNA of non-transgenic wild-type Arabidopsis was used as a control, and the real-time fluorescence quantitative primers EjTGA1-RT-F and EjTGA1-RT-R, as well as the Arabidopsis actin gene were used as internal reference genes. The primer pairs were: Atactin-F: 5′-CTCAAGAGGTTCTCAGCAGTA-3′; Atactin-R: 5′-TCACCTTCTTCATCCGCAGTT-3′. The expression level of the EjTGA1 gene was analyzed in the positive plants of transgenic Arabidopsis.

[0044] Among these transgenic lines, overexpressed lines 1 and 6 were selected for phenotypic identification of flowering time, and the flowering time phenotypes of these Arabidopsis were observed, counted and photographed. The phenotypic results showed that compared with wild-type Arabidopsis and non-transgenic wild-type Arabidopsis, overexpression of the EjTGA1 gene can lead to earlier flowering time in transgenic Arabidopsis. Expression analysis of the EjTGA1 gene in transgenic Arabidopsis found that compared with non-transgenic wild-type Arabidopsis, the EjTGA1 gene in transgenic Arabidopsis that bloomed early was significantly more highly expressed ( Figure 6 Overexpression of the EjTGA1 gene can lead to increased leaf area and rosette leaf number in transgenic Arabidopsis ( Figure 7 ). Therefore, transgenic Arabidopsis thaliana materials with the EjTGA1 gene can be used to modify the flowering time and growth and development of plants, thereby accelerating the flowering and fruiting of plants and regulating their growth and development.

[0045] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A loquat flowering period and growth regulation-related protein EjTGA1, which is a protein composed of the amino acids shown in SEQ ID No.

2.

2. A gene encoding the loquat flowering period regulation-related protein EjTGA1 according to claim 1.

3. The gene according to claim 2, characterized in that The sequence is shown as 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 in advancing flowering of plants.

7. The use according to claim 6, characterized in that The gene is transferred into the plant genome and overexpressed in the transgenic plant, thereby advancing the flowering and fruiting time of the plant.

8. Use of the gene according to claim 2 or 3 in regulating plant growth and development.

9. The use according to claim 8, characterized in that The gene is transferred into the plant genome and overexpressed in transgenic plants, increasing leaf area and rosette leaf number.

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