PeRAV1 gene of moso bamboo and its expressed protein and application

Abstract

The present invention discloses a PeRAV1 gene of moso bamboo, its expression protein and application, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the PeRAV1 gene of moso bamboo disclosed in the present invention is shown as SEQ ID NO.1, and the amino acid sequence of its expression protein is shown as SEQ ID NO.2. The present invention constructs an overexpression vector of the PeRAV1 gene of moso bamboo; transforms the constructed overexpression vector of the PeRAV1 gene of moso bamboo into Arabidopsis thaliana; cultivates, screens and obtains transgenic Arabidopsis thaliana with delayed flowering time and / or increased number of rosette leaves. The results of the examples of the present application show that the strains overexpressing the PeRAV1 gene exhibit obvious late flowering phenotypes, increased number of rosette leaves and delayed flowering time; and the expression levels of downstream regulatory genes FT, SOC1 and GA3OX1 are significantly downregulated.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more particularly relates to a PeRAV1 gene of a moso bamboo and its expression protein and application. Background Art

[0002] Moso bamboo (Phyllostachys edulis), belonging to the subfamily Bambusoideae of the Poaceae family, is widely distributed in my country and is an important non-timber forest product, characterized by rapid growth, strong adaptability, and high economic value. Moso bamboo has a long flowering cycle, ranging from several decades to over a century. This prolonged flowering cycle has severely hampered research on bamboo genetics and breeding. Therefore, identifying the genes and mechanisms that regulate flowering in moso bamboo is of great practical significance for bamboo variety improvement.

[0003] Flowering is an important reproductive process in plants, and successful flowering and fruiting are very important for species continuation. Currently, there are six recognized flowering regulatory pathways, the most important of which are the photoperiod pathway and the vernalization pathway. The FT gene is an important node gene, and multiple flowering regulatory pathways ultimately affect flowering by affecting the expression of the FT gene. RAV family genes contain two domains, AP2 and B3, and are involved in a variety of biological processes, including plant resistance and flowering regulation. The RAV1 gene belongs to the RAV transcription factor family. Arabidopsis RAV1 overexpression plants have a significantly delayed flowering time, which is related to the inhibition of FT. Although there have been many functional studies on the RAV1 gene in model plants, the function of the PeRAV1 gene in bamboo has not been reported, especially its role in flowering regulation. Summary of the Invention

[0004] In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide the PeRAV1 gene of moso bamboo. Another technical problem to be solved by the present invention is to provide the expression protein of the PeRAV1 gene of moso bamboo. Another technical problem to be solved by the present invention is to provide the application of the PeRAV1 gene of moso bamboo in regulating the flowering time of plants, for regulating the flowering time of plants. Another technical problem to be solved by the present invention is to provide the application of the PeRAV1 gene of moso bamboo in regulating the number of rosette leaves of plants, for regulating the number of rosette leaves of plants. Another technical problem to be solved by the present invention is to provide the application of the PeRAV1 gene of moso bamboo in regulating the expression level of downstream genes.

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

[0006] A PeRAV1 gene of moso bamboo, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0007] The amino acid sequence of the protein encoded by the PeRAV1 gene of moso bamboo is shown in SEQ ID NO.2.

[0008] Vectors and recombinant bacteria containing the PeRAV1 gene from Moso bamboo.

[0009] Application of the PeRAV1 gene from Moso bamboo 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) Construction of an overexpression vector for the PeRAV1 gene in bamboo;

[0012] 2) Transform the constructed PeRAV1 gene overexpression vector into Arabidopsis thaliana;

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

[0014] Application of the PeRAV1 gene in regulating the number of rosette leaves in moso bamboo.

[0015] The regulating the number of rosette leaves of the plant is to promote an increase in the number of rosette leaves, comprising:

[0016] 1) Construction of an overexpression vector for the PeRAV1 gene in bamboo;

[0017] 2) Transform the constructed PeRAV1 gene overexpression vector into Arabidopsis thaliana;

[0018] 3) Cultivate, screen and obtain transgenic Arabidopsis thaliana with increased number of rosette leaves.

[0019] Application of the bamboo PeRAV1 gene in regulating the expression of downstream genes of the PeRAV1 gene.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention discloses a PeRAV1 gene for bamboo, whose nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. The present invention constructs an overexpression vector for the PeRAV1 gene for bamboo; transforms the constructed overexpression vector for the PeRAV1 gene for bamboo into Arabidopsis thaliana; and cultivates, screens, and obtains transgenic Arabidopsis thaliana with delayed flowering time and / or increased number of rosette leaves. The results of the examples of the present application show that the strains overexpressing the PeRAV1 gene exhibit a significant late-flowering phenotype, with increased number of rosette leaves and delayed flowering time; and the expression levels of the downstream regulatory genes FT, SOC1, and GA3OX1 are significantly downregulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the electrophoresis diagram of PCR amplification of PeRAV1 gene in bamboo (lane 1 is DNA Marker, lanes 2-3 are PCR products of PeRAV1);

[0023] Figure 2 Figure 1 is a vector structure diagram of the expression vector pCAMBIA1302;

[0024] Figure 3 The subcellular localization map of PeRAV1 in bamboo (scale bar: 10 μm);

[0025] Figure 4 PCR detection diagram of transgenic Arabidopsis thaliana (Control is the control plant, #1 is the overexpression line pCAMBIA1302-PeRAV1-1, #3 is the overexpression line pCAMBIA1302-PeRAV1-3, #4 is the overexpression line pCAMBIA1302-PeRAV1-4, and #6 is the overexpression line pCAMBIA1302-PeRAV1-6);

[0026] Figure 5 Flowering phenotype (A), rosette leaf number (B), and flowering time (C) of transgenic Arabidopsis thaliana (Control is the control plant, #1 is the overexpression line pCAMBIA1302-PeRAV1-1, and #3 is the overexpression line pCAMBIA1302-PeRAV1-3);

[0027] Figure 6 Quantitative analysis of FT, SOC1 and GA3OX1 genes in transgenic Arabidopsis and control plants. DETAILED DESCRIPTION

[0028] To make the objects, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific examples. Unless otherwise specified in the following examples, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be performed with reference 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 kits and product instructions.

[0029] The materials used in this application are plant materials, namely bamboo leaves, which were collected in Guangxi Zhuang Autonomous Region.

[0030] Example 1

[0031] 1. PeRAV1 gene cloning

[0032] Total RNA was extracted from bamboo leaves using the RNAsimpleTotal RNA Extraction Kit (purchased from TIANGEN). The RNA was then reverse-transcribed and synthesized into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (purchased from Vazyme) and stored at -20°C until ready for use. Full-length primers with restriction enzyme sites were designed using Primer 5 software based on the bamboo genome database (http: / / gigadb.org / dataset / view / id / 100498) to amplify the PeRAV1 gene. The primer sequences are shown below:

[0033] Forward primer:

[0034] 5'-GAGAACACGGGGGACTCTAGAATGGACAGCACGAGCTGCC-3',

[0035] Reverse primer:

[0036] 5'-GCCCTTGCTCACCATGGATCCGCGCGACGCGAACTCTATGC-3'.

[0037] PCR reaction system: 1.5 μL cDNA, 12.5 μL 2× Phanta Max Buffer a (purchased from Vazyme), 1 μL dNTP Mix, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 2 μL forward primer, 2 μL reverse primer, and 30 μL deionized water. Add the sample and mix thoroughly on ice.

[0038] The PCR reaction program was as follows: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 15 seconds, annealing at 61°C for 15 seconds, extension at 72°C for 1 minute 20 seconds, 35 cycles; and 72°C for 5 minutes.

[0039] The amplified product was subjected to 1% agarose gel electrophoresis and a single PCR band was detected ( Figure 1 After gel excision, the amplified fragment was recovered using a gel recovery kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) and sent to a biotechnology company (Shanghai Sangon Biotechnology Co., Ltd.) for sequencing. Sequencing results showed that the amplified fragment of the PeRAV1 gene was 1119 bases long (excluding the terminator), with the sequence shown in SEQ ID NO. 1. The encoded protein contained 373 amino acid residues, with the sequence shown in SEQ ID NO. 2.

[0040] 2. Construction of PeRAV1 gene overexpression vector

[0041] The pCAMBIA1302 vector (vector information is as follows) was double-digested with Xba I and Bam HI. Figure 2 shown).

[0042] The double enzyme digestion reaction system is: 21 μL pCAMBIA1302 plasmid, 5 μL 10× Buffer, 2 μL Bam HI, 2 μL Xba I (purchased from Thermo Fisher Scientific), 20 μL deionized water. Mix well on ice.

[0043] The double enzyme digestion reaction procedure is: enzyme digestion at 37℃ for 4 hours, followed by gel excision and recovery.

[0044] The linearized vector fragment after double enzyme digestion and the above gene amplification fragment were ligated at 37°C for 30 minutes using homologous recombinase (purchased from Vazyme).

[0045] The ligation reaction system was: 2.5 μL PeRAV1 amplified fragment, 1.5 μL linearized vector, 2 μL 5×CEⅡBuffer, 1 μL ExnaseⅡ, and 3 μL deionized water.

[0046] Immediately after the ligation reaction, place on ice. After cooling, heat shock 10 μL of the ligation product into competent E. coli DH5α cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Single colonies were selected and shaken in LB liquid medium containing kanamycin (50 mg / L). After PCR analysis and sequencing verification, the recombinant plasmid pCAMBIA1302-PeRAV1 was obtained. The recombinant vector was transformed into Agrobacterium tumefaciens GV3101 cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.) using the freeze-thaw method. After PCR verification, the cells were preserved and used. E. coli and Agrobacterium transformation methods were carried out according to the manufacturer's instructions.

[0047] 3. PeRAV1 subcellular localization

[0048] The Agrobacterium culture containing the recombinant plasmid pCAMBIA1302-PeRAV1 was cultured in LB liquid medium (containing 50 μg / mL rifampicin and 50 μg / mL kanamycin) at 28°C and shaken until the culture OD 600 After centrifugation at 4500 rpm for 13 minutes, the cells were collected and resuspended in buffer until the 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.

[0049] The results are as follows Figure 3 As shown, consistent with typical transcription factors, PeRAV1 is localized in the nucleus.

[0050] Example 2

[0051] 1. Overexpression of the PeRAV1 gene in Arabidopsis

[0052] Agrobacterium containing the PeRAV1 overexpression vector was used to infect Arabidopsis thaliana (Col-0) using the inflorescence infection method. Agrobacterium containing an empty vector was used to infect Arabidopsis thaliana as a control (Control). The specific method is as follows:

[0053] Add 200 mL of LB liquid medium (containing 50 μg / mL rifampicin and 50 μg / mL kanamycin) and 1 mL of Agrobacterium containing the recombinant plasmid pCAMBIA1302-PeRAV1 to a 250 mL conical flask and culture at 28°C in a shaker at 220 rpm for 16 hours; collect the bacteria by centrifugation at 4500 rpm in a 50 mL centrifuge tube for 25 minutes; vigorously resuspend the bacteria with an equal volume of transformation solution (5% sucrose (w / v, g / 100 mL), 0.02% surfactant (v / v)); use scissors to cut off the opened flowers and siliques on the Arabidopsis thaliana; soak the Arabidopsis inflorescence in the above-mentioned transformation solution for 7 minutes; after one day in a culture room away from light, culture normally.

[0054] T1 seeds were sown on MS solid medium supplemented with hygromycin for screening. Leaves from positive seedlings were harvested, and DNA was extracted using a plant genomic DNA extraction kit (purchased from Tiangen Beijing Biochemical Technology Co., Ltd.). Based on the nucleotide sequence of the PeRAV1 gene, quantitative primers for the PeRAV1 gene were designed using Primer 5 software (forward primer qPeRAV1-S: CTACGACGTGGCGGTGCA, reverse primer qPeRAV1-A: GGCGACGAGGTGGGGTTT). PCR was performed using these quantitative primers to detect PeRAV1 expression.

[0055] The results are as follows Figure 4 As shown, the target fragment was absent in the control plants, while it was detected in all four PeRAV1-positive seedlings, indicating that PeRAV1 had been successfully introduced into Arabidopsis. Two transgenic Arabidopsis lines (#1 and #3) with higher expression levels (brighter bands) were selected and further self-pollinated until the T3 generation for quantitative analysis.

[0056] 2. Phenotypic statistics and quantitative analysis of transgenic plants

[0057] T3 Arabidopsis plants of lines #1 and #3 were grown in a climate chamber under long-day conditions (16 hours of daylight and 8 hours of darkness). The number of rosette leaves and flowering time of 20 plants in each line were counted.

[0058] The results are as follows Figure 5 As shown in Figure 3, overexpression of PeRAV1 in Arabidopsis thaliana resulted in a significant late-flowering phenotype, with an increase in the number of rosette leaves and a delayed flowering time.

[0059] 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. Quantitative primers for AtFT, AtSOC1, and AtGA3OX1 were designed and quantitative PCR was performed to detect their expression in transgenic plants.

[0060] qAtActin-S: 5'-TTGACAATTGATGCAAACAATGACG-3',

[0061] qAtActin-A: 5'-CCATTGCTTAATTCCACGGACAAAC-3';

[0062] qAtFT-S: 5'-AGTCCTAGCAACCCTCACCTCC-3',

[0063] qAtFT-A: 5'-CCTGCCAAGCTGTCGAAACA-3';

[0064] qAtSOC1-S: 5'-ATCGAGTCAGCACCAAACCG-3',

[0065] qAtSOC1-A: 5'-TTCCTATGCCTTCTCCCAAGAG-3';

[0066] qAtGA3OX1-S: 5'-ACAAGTGGACCCCTAAAGACGA-3',

[0067] qAtGA3OX1-A: 5'-TTGGACAGGTAGCCCGAAGA-3'.

[0068] Quantitative PCR reaction system: 10 μL qPCR Master Mix (purchased from TOROIVD), 1 μL cDNA, 0.5 μL forward primer, 0.5 μL reverse primer, 8 μL deionized water.

[0069] The quantitative PCR reaction program was as follows: 95°C for 1 minute; 95°C for 10 seconds, 60°C for 30 seconds, for 38 cycles.

[0070] The results are as follows Figure 6 As shown in the figure, compared with the control plants, the expression levels of AtFT, AtSOC1, and AtGA3OX1 were significantly downregulated in the PeRAV1 overexpressing plants. The results showed that FT, SOC1, and GA3OX1 genes are downstream regulatory genes of the PeRAV1 gene.

[0071] The above description is only illustrative of the present invention and 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 them will fall within the scope of protection of the present invention.

Claims

1. A type of bamboo PeRAV1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The bamboo of claim 1 PeRAV1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. Containing the bamboo described in claim 1 PeRAV1 Gene vectors and recombinant bacteria.

4. The bamboo of claim 1 PeRAV1 The use of a gene in regulating the flowering time of a plant, wherein the regulating the flowering time of the plant is to delay the flowering time of the plant, comprises: 1) Construction of bamboo PeRAV1 Gene overexpression vectors; 2) The bamboo PeRAV1 The gene overexpression vector was transformed into Arabidopsis thaliana; 3) Cultivate, screen, and obtain transgenic Arabidopsis lines with delayed flowering time.

5. The bamboo of claim 1 PeRAV1 Use of a gene in regulating the number of rosette leaves in a plant, wherein regulating the number of rosette leaves in a plant is to promote an increase in the number of rosette leaves, comprising: 1) Construction of bamboo PeRAV1 Gene overexpression vectors; 2) The bamboo PeRAV1 The gene overexpression vector was transformed into Arabidopsis thaliana; 3) Cultivate, screen, and obtain transgenic Arabidopsis thaliana with increased rosette leaf numbers.

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