Phyllostachys edulis pebbx19 gene, and its encoded protein and application

Abstract

The application discloses a Phyllostachys edulis PeBBX19 gene, a coding protein and application thereof, and belongs to the technical field of plant genetic engineering.The nucleotide sequence of the Phyllostachys edulis PeBBX19 gene disclosed by the application is shown as SEQ ID NO.1, and the amino acid sequence of the coding protein is shown as SEQ ID NO.2.The application constructs an overexpression vector of the Phyllostachys edulis PeBBX19 gene, transforms the constructed overexpression vector of the Phyllostachys edulis PeBBX19 gene into Arabidopsis, and cultivates, screens and obtains a transgenic Arabidopsis strain with delayed flowering time.The application discloses the function of the PeBBX19 gene for the first time, the transgenic Arabidopsis plant has delayed flowering time after the PeBBX19 gene is transformed into the Arabidopsis plant, obviously shows a late flowering phenotype, and the number of rosette leaves is increased; and the expression amount of a downstream regulation gene FT is significantly reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically relates to a PeBBX19 gene of a moso bamboo and its encoded protein and application. Background Art

[0002] Flowering is a crucial stage in the transition from vegetative growth to reproductive growth in plants and is crucial for species survival. Flowering in plants is regulated by both intrinsic and extrinsic factors. Six pathways govern flowering: the photoperiod pathway, the gibberellin pathway, the vernalization pathway, the temperature pathway, the age pathway, and the autonomous flowering pathway. BBX is a class of zinc-finger transcription factors involved in various plant growth and developmental processes, including flowering and abiotic stress responses. CO was the first BBX gene identified in Arabidopsis thaliana. CO induces flowering by directly binding to the promoter of the flowering-promoting gene FT and activating its expression. Other BBX genes, such as BBX4, BBX6, BBX7, BBX19, and BBX32, also regulate flowering. bbx4 mutants flower early under both short-day and long-day conditions, while plants overexpressing BBX7 under long-day conditions exhibit reduced FT expression and delayed flowering. Plants overexpressing BBX6 flower early under both short-day and long-day conditions. BBX19 plays a negative regulatory role in the photoperiod-regulated flowering pathway. BBX19 regulates flowering time by reducing the activation of FT by CO. Although the functions of BBX19 genes have been studied in model plants, the function of the PeBBX19 gene in bamboo has not been reported, especially its role in flowering time regulation.

[0003] Moso bamboo (Phyllostachys edulis), belonging to the subfamily Bamboo of the Poaceae family, is widely distributed in my country and boasts rapid growth, strong adaptability, and high economic value. Moso bamboo has a long flowering cycle, lasting over a century. However, after flowering, the entire plant dies, causing significant losses to production. Therefore, identifying the genes and mechanisms that regulate flowering in moso bamboo is of great practical significance for cultivar improvement. Summary of the Invention

[0004] In response to the aforementioned problems in the prior art, the present invention aims to provide the PeBBX19 gene from moso bamboo. Another technical problem addressed by the present invention is to provide the protein encoded by the PeBBX19 gene from moso bamboo. A further technical problem addressed by the present invention is to provide the application of the PeBBX19 gene from moso bamboo for regulating the flowering period of plants.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The nucleotide sequence of the PeBBX19 gene of moso bamboo is shown in SEQ ID NO. 1.

[0007] The amino acid sequence of the protein encoded by the PeBBX19 gene of moso bamboo is shown in SEQ ID NO. 2.

[0008] Vectors and recombinant bacteria containing the PeBBX19 gene from Moso bamboo.

[0009] Application of the bamboo PeBBX19 gene in regulating plant flowering time.

[0010] The method of regulating the flowering time of plants is to delay the flowering time of plants, including:

[0011] 1) Construct an overexpression vector of the PeBBX19 gene from Moso bamboo;

[0012] 2) Transform the constructed overexpression vector of the bamboo PeBBX19 gene into Arabidopsis thaliana;

[0013] 3) Cultivate, screen, and obtain transgenic Arabidopsis lines with delayed flowering time.

[0014] The invention discloses an application of the PeBBX19 gene of moso bamboo in regulating the number of rosette leaves of a plant, wherein the regulation of the number of rosette leaves of a plant is to promote an increase in the number of rosette leaves of the plant.

[0015] Application of the PeBBX19 gene of moso bamboo in regulating the expression of downstream genes, wherein the downstream gene is the FT gene.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) The present invention discloses for the first time that the PeBBX19 gene was cloned from leaves of moso bamboo. The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein expressed is shown in SEQ ID NO. 2.

[0018] 2) This study constructed an overexpression vector for the PeBBX19 gene in moso bamboo, transformed it into Arabidopsis thaliana, and screened and cultivated transgenic Arabidopsis lines with delayed flowering. This study revealed for the first time the function of the PeBBX19 gene. Transformation of the gene into Arabidopsis resulted in delayed flowering, a distinct late-flowering phenotype, and an increase in the number of rosette leaves.

[0019] 3) Transgenic Arabidopsis lines overexpressing the PeBBX19 gene from moso bamboo constructed by this invention showed significantly downregulated FT expression in PeBBX19-overexpressing plants compared to control plants. The results indicate that FT is a downstream regulatory gene of PeBBX19. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the agarose gel electrophoresis diagram of the PeBBX19 gene clone of Moso bamboo (lane 1 is DNA marker, lane 2 is the target fragment);

[0021] Figure 2 Figure 1 is a vector structure diagram of the expression vector pCAMBIA1302;

[0022] Figure 3 The subcellular localization map of PeBBX19 in moso bamboo (scale bar: 20 μm);

[0023] Figure 4 PCR detection images of T3 transgenic Arabidopsis plants (Control is the control plant, #1~#5 are overexpression lines pCAMBIA1302-PeBBX19-1~5);

[0024] Figure 5 Figure 2 shows the phenotypic observation of transgenic Arabidopsis (A), the number of rosette leaves (B), and the flowering time (C) (Control is the control plant, #1 is the overexpression line pCAMBIA1302-PeBBX19-1, and #2 is the overexpression line pCAMBIA1302-PeBBX19-2);

[0025] Figure 6 Figure 2 is the quantitative analysis of the FT gene in transgenic Arabidopsis and control plants. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiments of the present invention is further described below in conjunction with the examples. Unless otherwise specified in the following examples, the technical means used are conventional means well known to those skilled in the art or are carried out according to the kit and product instructions.

[0027] The plant materials used in this application are leaves of moso bamboo, which were collected in Guangxi Zhuang Autonomous Region.

[0028] Example 1

[0029] 1. PeBBX19 gene cloning

[0030] RNA was extracted from bamboo leaves using a plant total RNA extraction kit (purchased from TIANGEN) according to the kit's instructions. cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (purchased from Vazyme). The CDS sequence of the PeBBX19 gene was obtained by blasting from the bamboo genome database (http: / / gigadb.org / dataset / view / id / 100498). Primers containing restriction enzyme sites for full-length gene cloning were designed using Primer 5 software. The primer sequences are shown below:

[0031] PeBBX19-S:

[0032] 5'-GAGAACACGGGGGACTCTAGAATGCGGACGATCTGCGAT-3',

[0033] PeBBX19-A:

[0034] 5'-GCCCTTGCTCACCATGGATCCCTTGTTGGAGTATCGTTCGAAGTT-3'.

[0035] The 50 μL PCR reaction system is: 2 μL cDNA, 25 μL 2× Phanta Max Buffer a High-fidelity enzyme (purchased from Vazyme), 1.5 μL each of PeBBX19-S and PeBBX19-A, 1 μL dNTP Mix, and 19 μL deionized water.

[0036] The PCR reaction program was as follows: 95°C for 2 minutes; 32 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 55 seconds; and 72°C for 10 minutes.

[0037] The PCR product was subjected to 1.5% agarose gel electrophoresis and the target band of the correct size was detected ( Figure 1 After gel excision, a gel recovery kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was used to recover the target fragment according to the kit instructions and sent for sequencing by Sangon Biotech (Shanghai) Co., Ltd. Sequencing revealed a 633-base CDS sequence for the PeBBX19 gene (excluding the terminator), the nucleotide sequence of which is shown in SEQ ID NO. 1; the encoded protein contains 211 amino acid residues, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0038] 2. Construction of PeBBX19 gene overexpression vector

[0039] The pCAMBIA1302 target vector (vector information is as follows) was double-digested with BamH I and Xba I. Figure 2 Double enzyme digestion was performed with the pCAMBIA1302 target vector (as shown) to obtain the linearized vector. A 50 μL double enzyme digestion reaction system consisted of: 20.5 μL pCAMBIA1302 target vector, 5 μL 10× Buffer, 2 μL each of BamH I and Xba I (purchased from Thermo Fisher Scientific), and 20.5 μL deionized water. The double enzyme digestion reaction was set at 37°C for 4 hours. The linearized vector fragment was recovered by gel excision.

[0040] The linearized vector fragment and the amplified gene fragment were ligated using homologous recombination enzyme (purchased from Vazyme) to generate the recombinant overexpression vector pCAMBIA1302-PeBBX19. The ligation reaction system (10 μL) contained 2 μL of linearized vector, 3 μL of PeBBX19 amplified fragment, 2 μL of 5× CE II Buffer, 1 μL of Exnase II, and 2 μL of deionized water. The ligation reaction was incubated at 37°C for 30 minutes.

[0041] Transform 10 μL of the ligation product into competent E. coli DH5α cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Spread the product onto LB plates containing 50 μg / mL kanamycin and incubate at 37°C overnight. Select a single colony and shake it in LB liquid medium containing 50 mg / L kanamycin. After PCR analysis and sequencing verification, the recombinant plasmid pCAMBIA1302-PeBBX19 was obtained. E. coli DH5α transformation was performed according to the manufacturer's instructions.

[0042] 3. PeBBX19 subcellular localization

[0043] The recombinant plasmid pCAMBIA1302-PeBBX19 was transformed into Agrobacterium GV3101 competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). The Agrobacterium GV3101 transformation method was carried out according to the manufacturer's instructions. Single clones were selected and shaken in LB liquid medium containing 50 μg / mL rifampicin and 50 μg / mL kanamycin. The target band was detected by PCR. 1 ml of the Agrobacterium culture containing the recombinant plasmid pCAMBIA1302-PeBBX19 was aspirated and inoculated into 50 mL of LB liquid medium containing 50 μg / mL rifampicin and 50 μg / mL kanamycin. The culture was cultured at 28°C until the OD value of the culture was 0. 600 =0.76, centrifuge at 4400rpm for 14 minutes to collect the cells. Resuspend to OD 600 = 0.6, and kept in the dark for 4 hours. According to the tobacco transient expression technique, the bacterial solution was injected into tobacco leaves for subcellular localization observation.

[0044] The results are as follows Figure 3As shown, PeBBX19 is localized in the cell nucleus.

[0045] Example 2

[0046] 1. Obtaining transgenic Arabidopsis

[0047] 1 mL of Agrobacterium containing the recombinant plasmid pCAMBIA1302-PeBBX19 was inoculated into 200 mL of LB liquid medium supplemented with 50 μg / mL rifampicin and 50 μg / mL kanamycin and cultured at 28°C at 220 rpm for 2 days. The cells were harvested by centrifugation at 4200 rpm for 15 minutes and vigorously resuspended in an equal volume of infection medium (5% sucrose (w / v, g / 100 mL), 0.02% surfactant (v / v)) to obtain an infection medium containing the recombinant plasmid pCAMBIA1302-PeBBX19. Arabidopsis inflorescences were then immersed in the infection medium containing the recombinant plasmid pCAMBIA1302-PeBBX19 for 5 minutes. A control was then infected with Agrobacterium containing the pCAMBIA1302 plasmid. After infection, the cells were protected from light for one day before normal culture. Harvested seeds were sown on MS solid medium supplemented with hygromycin and positive transgenic seedlings were selected. DNA was extracted from leaves of positive seedlings using a plant genomic DNA extraction kit (purchased from Tiangen Beijing Biochemical Technology Co., Ltd.) according to the kit instructions. Full-length primers were used to detect the PeBBX19 gene band. PCR detection was performed using the same method as in Example 1.

[0048] The results are as follows Figure 4 As shown, the target band was absent in the control plants, while all five transgenic plants showed the target band, indicating positive results. Two transgenic lines (#1 and #2) were selected and further self-pollinated until the T3 generation for quantitative analysis.

[0049] 2. Phenotypic statistics and quantitative analysis of transgenic plants

[0050] T3 transgenic plants and control plants were planted under the same conditions and photographed to record their growth process. The number of rosette leaves and flowering time of 18 plants were counted for each transgenic plant.

[0051] The results are as follows Figure 5 As shown in the figure, compared with the control plants, after overexpressing the PeBBX19 gene, the flowering time of the transgenic plants was significantly delayed and the number of rosette leaves increased, showing a late flowering phenotype.

[0052] Total RNA was extracted from transgenic seedlings and cDNA was synthesized using the same method as in Example 1. The AtActin gene was used as an internal reference gene. AtFT quantitative primers were designed using Primer 5 software, and quantitative PCR was used to detect the expression of AtFT in transgenic plants.

[0053] qAtActin-S: 5'-TTGACAATTGATGCAAACAATGACG-3'

[0054] qAtActin-A: 5'-CCATTGCTTAATTCCACGGACAAAC-3'

[0055] qAtFT-S: 5'-AGTCCTAGCAACCCTCACCTCC-3'

[0056] qAtFT-A: 5'-CCTGCCAAGCTGTCGAAACA-3'

[0057] Quantitative PCR reaction system is 10 μL: TOROGreen ® qPCR Master Mix (purchased from TOROIVD) 5 μL, cDNA 0.5 μL, forward primer 0.3 μL each, reverse primer 0.3 μL, and deionized water 3.9 μL. The quantitative PCR reaction program was set as follows: 95°C for 2 minutes, 95°C for 12 seconds, and 60°C for 25 seconds, for 39 cycles.

[0058] The results are as follows Figure 6 As shown in Figure 3, compared with the control plants, the expression of AtFT was significantly downregulated in the PeBBX19 overexpressing plants. These results indicate that FT is a downstream regulatory gene of the PeBBX19 gene.

[0059] The embodiments described above are merely illustrative of the present invention and are not restrictive. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all of these modifications, changes or equivalents will fall within the scope of protection of the present invention.

Claims

1. Application of the Moso bamboo PeBBX19 gene, whose nucleotide sequence is shown in SEQ ID NO. 1, in promoting the increase of rosette leaf number in Arabidopsis thaliana.

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