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

Abstract

The invention discloses a phyllostachys edulis PeSVP 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 PeSVP gene disclosed by the invention is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein of the PeSVP gene is shown as SEQ ID NO. 2. The method comprises the following steps: constructing an overexpression vector of a phyllostachys edulis PeSVP Transforming the constructed overexpression vector of the phyllostachys edulis PeSVP gene 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 PeSVP 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. The method can be used in molecular breeding for regulating and controlling the flowering phase of plants, and has important significance for cultivating new plant germplasm with higher biological yield.

Description

Technical Field

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

[0002] The transition from vegetative growth to reproductive growth in plants is achieved under the stimulation of a variety of external and internal signals. The flowering regulation pathways of plants include vernalization pathway, photoperiod pathway, temperature pathway, gibberellin pathway, autonomous flowering and age pathway. MADS-box transcription factors are involved in the regulation of plant flowering. The Arabidopsis SVP gene encodes a MADS-box transcription factor that acts as a negative regulator of flowering. Under long-day and short-day conditions, the SVP gene can delay plant flowering, and SVP gene mutations can cause plants to flower early. SVP prevents plants from flowering by directly binding to the GArG element on the promoter of FT in the phloem and SOC1 in the shoot apical meristem and inhibiting their expression. SVP can form a complex with FLC to directly inhibit the expression of SOC1 and FT. Overexpression of the rice SVP homologous gene (OsMADS55) delays the flowering time of plants and can restore the early flowering phenotype of the Arabidopsis svp mutant. SVP can also interact with AP1 to regulate flowering time. In addition, the SVP gene is also involved in other developmental regulation, such as regulating inflorescence branching, flower development and bud dormancy. The kiwifruit SVP gene can delay spring seed bud germination. Although the SVP gene of model plants has been studied in depth, the function of the PeSVP gene of bamboo is still unclear.

[0003] Moso bamboo (Phyllostachys edulis) belongs to the subfamily Bambusoideae of the Poaceae family. It has the advantages of strong adaptability and rapid growth. Moso bamboo requires a long period of nutrient storage for flowering, sometimes up to hundreds of years. The death of the bamboo forest after flowering will cause huge losses to production. Therefore, the exploration of the flowering regulatory genes and their regulatory mechanisms of Moso 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 PeSVP gene of moso bamboo. Another technical problem to be solved by the present invention is to provide a protein encoded by the PeSVP gene of moso bamboo. Another technical problem to be solved by the present invention is to provide an application of the PeSVP 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 bamboo PeSVP gene is shown in SEQ ID NO.1.

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

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

[0009] Application of PeSVP gene in regulating 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 PeSVP gene in bamboo;

[0012] 2) Transform the constructed overexpression vector of the bamboo PeSVP 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 PeSVP 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 PeSVP gene in regulating the expression of its downstream gene, 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 PeSVP 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 encoded protein is shown in SEQ ID NO. 2.

[0018] 2) The present invention constructs an overexpression vector of the bamboo PeSVP gene; transforms the constructed overexpression vector of the bamboo PeSVP gene into Arabidopsis; and cultivates, screens, and obtains transgenic Arabidopsis strains with delayed flowering time. The function of the PeSVP gene in regulating the flowering time of plants is revealed for the first time. Overexpression of the PeSVP gene increases the number of rosette leaves in plants and delays flowering time, indicating that the PeSVP gene negatively regulates plant flowering.

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

[0020] Figure 1 Agarose gel electrophoresis of the cloning of PeSVP gene in moso bamboo (lane 1 is the target fragment, lane 2 is the DNA marker);

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

[0022] Figure 3 The subcellular localization map of PeSVP in bamboo (scale bar is 10µm);

[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-PeSVP-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 and #2 are the overexpression lines pCAMBIA1302-PeSVP-1 and pCAMBIA1302-PeSVP-2);

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

[0026] The present invention is further described below in conjunction with specific examples, but the examples do not limit the form of the present invention. Unless otherwise specified in the following examples, the technical means used can refer to the methods listed in the book "Molecular Cloning Laboratory Manual" (3rd Edition) by J. Sambrook or conventional methods in the art, or 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. PeSVP gene cloning

[0030] RNA from bamboo leaves was extracted using a plant RNA extraction kit (purchased from TIANGEN) according to the kit instructions. cDNA was synthesized according to the HiScript III 1st Strand cDNA Synthesis Kit (purchased from Vazyme). The CDS sequence of the PeSVP gene was obtained from the bamboo genome database (http: / / gigadb.org / dataset / view / id / 100498), and full-length primers with restriction sites were designed using Primer 5 software. The primer sequences are as follows:

[0031] Forward Primer:

[0032] 5'-GAGAACACGGGGGACTCTAGAATGGCGCGGGAGCGG-3',

[0033] Reverse Primer:

[0034] 5'-GCCCTTGCTCACCATGGATCCCTTCCATGCAACACAAGGCAAC-3'.

[0035] The PCR reaction system is: 0.8 μL each of forward primer and reverse primer, 2.5 μL cDNA, 25 μL 2×Phanta MaxBuffer a High-fidelity enzyme (purchased from Vazyme), 19.9 μL deionized water and 1 μL dNTP Mix.

[0036] PCR reaction program: 95°C for 2 minutes and 20 seconds; 95°C for 25 seconds, 60°C for 25 seconds, 72°C for 50 seconds, 31 cycles; 72°C for 9 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 ). According to the instructions of the gel recovery kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), the target fragment was recovered and sent to the company for sequencing (Sangon Biotechnology (Shanghai) Co., Ltd.). The PeSVP gene CDS sequence obtained by sequencing is 684 bases in length (excluding the terminator), and its nucleotide sequence is shown in SEQ ID NO.1; its encoded protein contains 228 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2.

[0038] 2. Construction of PeSVP gene overexpression vector

[0039] The pCAMBIA1302 expression vector (vector information is as follows) was double-digested with BamH I and Xba I. Figure 2 Double enzyme digestion was performed with 5 μL of 10× Buffer, 1.5 μL of BamH I and Xba I (purchased from Thermo Fisher Scientific), 19.5 μL of pCAMBIA1302 vector, and 22.5 μL of deionized water. Double enzyme digestion reaction procedure: 36°C for 3.5 hours. The linearized vector fragment was recovered by gel cutting.

[0040] The PeSVP amplified fragment and the linearized vector fragment were connected with homologous recombination enzyme (purchased from Vazyme) to obtain the recombinant overexpression vector pCAMBIA1302-PeSVP. The homologous recombination connection reaction system is: Exnase Ⅱ 1μL, PeSVP amplified fragment 2.5μL, linearized vector 1μL, 5×CE Ⅱ Buffer 2μL, deionized water 3.5μL. The reaction conditions are 40℃ for 35 minutes. According to the instructions, the connection product was transferred into Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biological Company). The transformed Escherichia coli was spread on LB solid medium containing 50μg / mL kanamycin and cultured at 37℃ for 20 hours. Single clones were selected and shaken with LB liquid medium containing 50mg / L kanamycin. After PCR detection, it was sent to the company for sequencing. If the sequencing is correct, the recombinant plasmid pCAMBIA1302-PeSVP is obtained.

[0041] 3. PeSVP subcellular localization

[0042] Refer to the instructions to transfer the recombinant plasmid pCAMBIA1302-PeSVP into Agrobacterium GV3101 competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Spread the transformed Agrobacterium on LB solid medium containing 50μg / mL rifampicin and 50μg / mL, and culture at 28℃ for 3 days. Select single clones and shake the bacteria with LB liquid medium containing rifampicin and kanamycin. The PCR detection band is correct, which is the positive bacteria. Inoculate 1ml of positive bacteria in 100mL LB liquid medium containing rifampicin and kanamycin, and shake and culture at 28℃ until the bacterial solution OD 600 =0.72, centrifuge at 4200rpm for 9 minutes to collect the cells. Resuspend to OD 600 =0.6, cultured in the dark for 5 hours. According to the tobacco transient expression technique, the bacterial solution was injected into tobacco leaves and the subcellular localization was observed. Agrobacterium containing the pCAMBIA1302 plasmid was used as a control (Control).

[0043] The results are as follows Figure 3 As shown, PeSVP is localized in the cell nucleus.

[0044] Example 2

[0045] 1. Obtaining transgenic Arabidopsis

[0046] Inoculate 500 μL of Agrobacterium containing the recombinant plasmid pCAMBIA1302-PeSVP into 150 mL of LB liquid medium containing rifampicin and kanamycin, and culture at 28°C 250 rpm for 1 day. Collect the cells by centrifugation at 4100 rpm for 30 minutes, and vigorously resuspend the cells with an equal volume of infection solution (5% sucrose (w / v, g / 100 mL), 0.025% surfactant (v / v)) to obtain the infection solution containing the recombinant plasmid pCAMBIA1302-PeSVP. Soak the Arabidopsis inflorescence with the Agrobacterium infection solution containing the recombinant plasmid pCAMBIA1302-PeSVP for 10 minutes. After infection, culture in the dark for one day and then culture normally.

[0047] After the seed pods of the plants matured, the seeds were collected and sown on MS solid medium containing hygromycin, and the transgenic seedlings were selected and transplanted to the soil for growth. The leaves of the seedlings were taken and DNA was extracted according to the instructions of the plant genomic DNA extraction kit (purchased from Tiangen Beijing Biochemical Technology Co., Ltd.). The target band was detected by PCR using the full-length primers of the gene. The PCR detection method was the same as in Example 1.

[0048] The results are as follows Figure 4 As shown in the figure, the target band was detected in only five transgenic plants, indicating that PeSVP has been successfully introduced into Arabidopsis. Two lines (#1 and #2) were selected from these five transgenic lines for further self-pollination and harvesting, and subsequent experiments were carried out in the T3 generation.

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

[0050] Transgenic plants and control plants were planted in a climate chamber at the same time, and the growth process was recorded. 25 plants were planted for each transgenic line, and the number of rosette leaves and flowering time of each line were counted.

[0051] The results are as follows Figure 5 As shown, compared with the control plants, the lines overexpressing the PeSVP gene in Arabidopsis thaliana showed a late flowering phenotype, with a significantly delayed flowering time and an increased number of rosette leaves.

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

[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: 0.35 μL each of forward primer and reverse primer, 1.3 μL cDNA, TOROGreen ® 5 μL of qPCR Master Mix (purchased from TOROIVD), 3 μL of deionized water. Quantitative PCR reaction conditions: 95°C for 1 minute and 30 seconds; 95°C for 14 seconds, 60°C for 21 seconds, 42 cycles.

[0058] The results are as follows Figure 6 As shown in Figure 2, the expression of FT in PeSVP overexpressing plants was significantly downregulated compared with that in control plants. The results indicate that PeSVP regulates flowering by regulating the expression of FT 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 PeSVP gene of moso bamboo, whose nucleotide sequence is shown in SEQ ID NO.

1.

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

2.

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

4. Use of the bamboo PeSVP 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 3, characterized in that: include: 1) Construct an overexpression vector of the PeSVP gene in bamboo; 2) Transform the constructed overexpression vector of the bamboo PeSVP gene into Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis lines with delayed flowering time.

7. Use of the PeSVP gene of moso bamboo 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 PeSVP 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