Moso bamboo PeBBX19 gene as well as encoding protein and application thereof

Abstract

The invention discloses a moso bamboo PeBBX19 gene as well as an encoding protein and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the phyllostachys pubescens PeBBX19 gene disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein of the phyllostachys pubescens PeBBX19 gene is as shown in SEQ ID NO.2. The method comprises the following steps: constructing an overexpression vector of a moso bamboo PeBBX19 gene; the constructed overexpression vector of the moso bamboo PeBBX19 gene is transformed into arabidopsis thaliana; and cultivating and screening to obtain the transgenic arabidopsis thaliana strain with delayed flowering time. The invention discloses the function of the PeBBX19 gene for the first time, the flowering time of a transgenic plant is delayed after the gene is transformed into an arabidopsis thaliana plant, an obvious late flowering phenotype is shown, and the number of rosette leaves is increased; and the FT expression quantity of the downstream regulation gene is obviously reduced.

Description

Technical Field

[0001] The 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 an important stage from vegetative growth to reproductive growth of plants, and it is of great significance for species continuation. Flowering of plants is regulated by both intrinsic and extrinsic factors. Flowering of plants is regulated by six pathways, namely 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 transcription factors with zinc finger structures, which are involved in various growth and development processes of plants, including plant flowering and abiotic stress. CO is the first BBX gene identified in Arabidopsis. CO induces flowering by directly binding to the promoter of the flowering-promoting gene FT and activating its expression. Other BBX genes can also regulate plant flowering, such as BBX4, BBX6, BBX7, BBX19, and BBX32. The bbx4 mutants both flowered early under short-day and long-day conditions, while the FT expression level in the overexpression plants of BBX7 under long-day conditions was reduced, and flowering was delayed. The overexpression plants of BBX6 flowered early under short-day and long-day conditions. BBX19 plays a negative regulatory role in the flowering pathway regulated by photoperiod. BBX19 regulates flowering time by reducing the activation of FT by CO. Although the function of BBX19 genes in model plants has been studied, the function of PeBBX19 gene in bamboo has not been reported, especially its role in flowering time regulation.

[0003] Phyllostachys edulis belongs to the subfamily Bambusoideae of the Poaceae family. It is widely distributed in my country and has the advantages of rapid growth, strong adaptability, and high economic value. The flowering cycle of bamboo is very long, lasting for hundreds of years. After flowering, the whole bamboo plant dies, causing great losses in production. Therefore, exploring the flowering regulatory genes and their regulatory mechanisms of bamboo is of great practical significance for variety improvement. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a PeBBX19 gene of moso bamboo. Another technical problem to be solved by the present invention is to provide a protein encoded by the PeBBX19 gene of moso bamboo. Another technical problem to be solved by the present invention is to provide an application of the PeBBX19 gene of moso bamboo for regulating the flowering period of plants.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is 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 bamboo is shown in SEQ ID NO. 2.

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

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

[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 in 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 plants, wherein the regulation of the number of rosette leaves of plants is to promote the increase of the number of rosette leaves of plants.

[0015] Application of the bamboo PeBBX19 gene 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 is cloned from bamboo leaves, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 2.

[0018] 2) The present invention constructs an overexpression vector of the bamboo PeBBX19 gene; transforms the constructed overexpression vector of the bamboo PeBBX19 gene into Arabidopsis; and cultivates, screens, and obtains transgenic Arabidopsis strains with delayed flowering time. The function of the PeBBX19 gene is revealed for the first time. After the gene is transformed into Arabidopsis plants, the flowering time of the transgenic plants is delayed, showing an obvious late-flowering phenotype, and the number of rosette leaves increases.

[0019] 3) The transgenic Arabidopsis strain overexpressing the PeBBX19 gene of bamboo constructed by the present invention showed that the expression of FT in the PeBBX19 overexpressing plant was significantly downregulated compared with the control plant. The results showed that the FT gene is a downstream regulatory gene of the PeBBX19 gene. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0024] Figure 5 The diagrams are the phenotypic observation of transgenic Arabidopsis thaliana (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 The figure shows the quantitative analysis of 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 embodiments. Unless otherwise specified in the following embodiments, 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 material used in this application is bamboo leaves, which were collected in Guangxi Zhuang Autonomous Region.

[0028] Example 1

[0029] 1. PeBBX19 gene cloning

[0030] RNA from bamboo leaves was extracted using a plant total RNA extraction kit (purchased from TIANGEN) according to the kit 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), and full-length gene cloning primers with restriction sites were designed using Primer 5 software. The primer sequences are as follows:

[0031] PeBBX19-S:

[0032] 5'-GAGAACACGGGGGACTCTAGAATGCGGACGATCTGCGAT-3',

[0033] PeBBX19-A:

[0034] 5'-GCCCTTGCTCACCATGGATCCCTTGTTGGAGTATCGTTCGAAGTT-3'.

[0035] 50μL PCR reaction system: 2μL cDNA, 25μL 2×Phanta Max Buffer a (Purchased from Vazyme) High-fidelity enzyme, 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: 95°C for 2 minutes; 95°C for 20 seconds, 60°C for 20 seconds, 72°C for 55 seconds, 32 cycles; 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 cutting, 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 to the company for sequencing (Shanghai Sangon Biotechnology Co., Ltd.). The PeBBX19 gene CDS sequence obtained by sequencing is 633 bases in length (excluding the terminator), and its nucleotide sequence is shown in SEQ ID NO.1; its encoded protein contains 211 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2.

[0038] 2. Construction of the overexpression vector of PeBBX19 gene

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

[0040] The linearized vector fragment and the above gene amplified fragment were connected with homologous recombinase (purchased from Vazyme) to obtain the recombinant overexpression vector pCAMBIA1302-PeBBX19. The connection reaction system was 10 μL: containing 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 connection reaction program was set to: 37°C for 30 minutes.

[0041] All 10 μL of the ligation product was transferred into E. coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Spread on LB plates containing 50 μg / mL kanamycin and culture at 37°C overnight. Select monoclonal colonies and shake them in LB liquid culture medium containing 50 mg / L kanamycin. After PCR detection and sequencing verification, the recombinant plasmid pCAMBIA1302-PeBBX19 was obtained. The E. coli DH5α transformation method was carried out according to the instructions.

[0042] 3. Subcellular localization of PeBBX19

[0043] The recombinant plasmid pCAMBIA1302-PeBBX19 was transferred into Agrobacterium GV3101 competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). The transformation method of Agrobacterium GV3101 was carried out according to the instructions. Single clone colonies were selected and shaken with LB liquid culture medium containing 50μg / mL rifampicin and 50μg / mL kanamycin, and the target band was detected by PCR. 1ml of Agrobacterium culture containing the recombinant plasmid pCAMBIA1302-PeBBX19 was aspirated and inoculated into 50mL LB liquid culture medium containing 50μg / mL rifampicin and 50μg / mL kanamycin, and cultured at 28℃ until the culture OD 600 =0.76, centrifuge at 4400rpm for 14 minutes to collect the cells. Resuspend to OD 600 =0.6, and kept in dark for 4 hours. According to the tobacco transient expression technology, 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 nucleus.

[0045] Example 2

[0046] 1. Obtaining transgenic Arabidopsis

[0047] 1mL of Agrobacterium containing the recombinant plasmid pCAMBIA1302-PeBBX19 was inoculated into 200mL of LB liquid medium containing 50μg / mL rifampicin and 50μg / mL kanamycin, and cultured at 28℃ 220rpm for 2 days. The cells were collected by centrifugation at 4200rpm for 15 minutes, and the cells were vigorously resuspended with an equal volume of infection solution (5% sucrose (w / v, g / 100mL), 0.02% surfactant (v / v)) to obtain an infection solution containing the recombinant plasmid pCAMBIA1302-PeBBX19. Then, the Arabidopsis inflorescence was soaked in the Agrobacterium infection solution containing the recombinant plasmid pCAMBIA1302-PeBBX19 for 5 minutes. The Agrobacterium containing the pCAMBIA1302 plasmid was infected as a control (Control). After infection, it was protected from light for one day and then cultured normally. The harvested seeds were sown on MS solid medium containing hygromycin to screen positive transgenic seedlings. Take the leaves of the positive seedlings, use the plant genomic DNA extraction kit (purchased from Tiangen Beijing Biochemical Technology Co., Ltd.), refer to the kit instructions, extract DNA. Use the full-length primer to detect the PeBBX19 gene band. The PCR detection method is the same as in Example 1.

[0048] The results are as follows Figure 4 As shown, there was no target band in the control group, but the target band was detected in all five transgenic plants, indicating that they were positive seedlings. Two transgenic lines (#1 and #2) were selected and continued to be self-pollinated and harvested until the T3 generation for quantitative analysis.

[0049] 2. Statistics and quantitative analysis of transgenic plant phenotypes

[0050] The T3 transgenic plants and control plants were planted in the same environment, and the growth process was recorded by taking photos. The number of rosette leaves and flowering time of 18 plants were counted for each transgenic plant system.

[0051] The results are as follows Figure 5 As shown, compared with the control group 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 in the same manner as in Example 1. 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: 10 μL: TOROGreen ® qPCR Master Mix (purchased from TOROIVD) 5 μL, cDNA 0.5 μL, forward primer and reverse primer 0.3 μL each, deionized water 3.9 μL. The quantitative PCR reaction program was set as: 95°C for 2 minutes; 95°C for 12 seconds, 60°C for 25 seconds, and 39 cycles.

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

[0059] The embodiments described above are merely illustrative rather than restrictive of the present invention. 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 will fall within the scope of protection of the present invention.

Claims

1. The PeBBX19 gene of moso bamboo, whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the bamboo PeBBX19 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A vector and recombinant bacteria containing the bamboo PeBBX19 gene according to claim 1.

4. Use of the bamboo PeBBX19 gene according to claim 1 in regulating the flowering time of plants.

5. The application according to claim 4, characterized in that: The regulating the flowering time of plants is to delay the flowering time of plants.

6. The use according to claim 5, characterized in that: include: 1) Construct an overexpression vector of the PeBBX19 gene in Moso bamboo; 2) Transform the constructed overexpression vector of the bamboo PeBBX19 gene into Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis lines with delayed flowering time.

7. Use of the bamboo PeBBX19 gene according to claim 1 in regulating the number of rosette leaves of a plant.

8. The use according to claim 7, characterized in that: The regulating the number of plant rosette leaves is to promote the increase of the number of plant rosette leaves.

9. Use of the bamboo PeBBX19 gene according to claim 1 in regulating the expression level of downstream genes.

10. The use according to claim 9, characterized in that: The downstream gene is the FT gene.

Images