Moso bamboo PeRAV1 gene as well as expression protein and application thereof

Abstract

The invention discloses a phyllostachys edulis PeRAV1 gene as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the phyllostachys pubescens PeRAV1 gene disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the phyllostachys pubescens PeRAV1 gene is as shown in SEQ ID NO.2. The method comprises the following steps: constructing an overexpression vector of a moso bamboo PeRAV1 gene; transforming the constructed overexpression vector of the phyllostachys edulis PeRAV1 gene into arabidopsis thaliana; and cultivating and screening to obtain transgenic arabidopsis thaliana with delayed flowering time and / or increased rosette leaf number. Results of the embodiment of the invention show that a strain with overexpressed PeRAV1 gene shows an obvious late flowering phenotype, the number of rosette leaves is increased, and the flowering time is delayed; and the expression quantities of downstream regulation genes FT, SOC1 and GA3OX1 are obviously reduced.

Description

Technical Field

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

[0002] Moso bamboo (Phyllostachys edulis) belongs to the subfamily Bambusoideae of the Poaceae family. It is widely distributed in my country and is an important non-timber forest product with the advantages of rapid growth, strong adaptability and high economic value. The flowering cycle of moso bamboo is very long, ranging from several decades to more than 100 years. The long flowering cycle seriously hinders the research on genetic breeding of bamboo plants. Therefore, exploring the flowering regulatory genes and their regulatory mechanisms of moso bamboo has important practical significance for variety improvement.

[0003] Flowering is an important reproductive process of plants, and smooth flowering and fruiting are very important for species continuation. There are currently six recognized flowering regulation 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 regulation 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 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 period 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 amount of downstream genes.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is 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 bamboo.

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

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

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

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

[0015] The method of regulating the number of rosette leaves of a plant is to promote an increase in the number of rosette leaves, and comprises:

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

[0017] 2) Transform the constructed bamboo 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 nucleotide sequence of the bamboo PeRAV1 gene disclosed in the present invention is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. The present invention constructs an overexpression vector of the bamboo PeRAV1 gene; transforms the constructed overexpression vector of the bamboo PeRAV1 gene 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 show 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. 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 is the vector structure diagram of the expression vector pCAMBIA1302;

[0024] Figure 3 This is the subcellular localization map of PeRAV1 in bamboo (scale bar is 10 μm);

[0025] Figure 4 It is a PCR detection diagram of transgenic Arabidopsis (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 The flowering phenotype diagram (A), rosette leaf number diagram (B) and flowering time diagram (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] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be carried out with reference to the methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0029] The plant materials used in this application are 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 RNAsimpleTotal RNA extraction kit (purchased from TIANGEN), and then reverse transcribed and synthesized into cDNA using HiScript III 1st Strand cDNA Synthesis Kit (purchased from Vazyme) and stored at -20°C for later use. Full-length primers with restriction sites were designed using Primer 5 software to amplify the PeRAV1 gene according to the bamboo genome database http: / / gigadb.org / dataset / view / id / 100498. The primer sequences are as follows:

[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, 30 μL deionized water. Add the sample on ice and mix well.

[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 and 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 cutting, the amplified fragment was recovered using a gel recovery kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), and the fragment was sent to a biological company (Shanghai Biotechnology Co., Ltd.) for sequencing. The sequencing results showed that the full length of the amplified fragment of the PeRAV1 gene was 1119 bases (excluding the terminator), the sequence was shown in SEQ ID NO.1, and the encoded protein contained 373 amino acid residues, the sequence was 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), and 20 μL deionized water. Mix well on ice.

[0043] The double enzyme digestion reaction procedure is: digest at 37℃ for 4 hours and then cut the gel for recovery.

[0044] The linearized vector fragment after double restriction digestion and the above gene amplification fragment were connected 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] After the ligation reaction is completed, it is immediately placed on ice. After cooling, 10 μL of the ligation product is transferred into Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biological Company) by heat shock method. Monoclonal colonies are selected and shaken with LB liquid medium containing kanamycin (50 mg / L). After PCR detection and sequencing verification, the recombinant plasmid pCAMBIA1302-PeRAV1 is obtained. The recombinant vector is transferred into Agrobacterium GV3101 cells (purchased from Shanghai Weidi Biological Company) by freeze-thaw method. After PCR detection is correct, the bacteria are preserved and used. The transformation methods of Escherichia coli and Agrobacterium are carried out according to the instructions.

[0047] 3. Subcellular localization of PeRAV1

[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 The cells were collected by centrifugation at 4500 rpm for 13 minutes and resuspended in buffer until the OD 600 =0.6, and kept in dark for 4 hours. According to tobacco transient expression technology, 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 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 LB liquid culture medium (containing 50 μg / mL rifampicin and 50 μg / mL kanamycin) and 1 mL Agrobacterium containing the recombinant plasmid pCAMBIA1302-PeRAV1 into 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; soak the inflorescence of Arabidopsis in the above-mentioned transformation solution for 7 minutes; after one day in a culture room away from light, culture normally.

[0054] The T1 generation seeds were sown on MS solid medium containing hygromycin for screening. The leaves of the positive seedlings were taken and DNA was extracted using a plant genomic DNA extraction kit (purchased from Tiangen Beijing Biochemical Technology Co., Ltd.). According to the nucleotide sequence of the PeRAV1 gene, the quantitative primers of the PeRAV1 gene were designed using Primer 5 software (forward primer qPeRAV1-S: CTACGACGTGGCGGTGCA, reverse primer qPeRAV1-A: GGCGACGAGGTGGGGTTT). The expression of PeRAV1 was detected by PCR using gene quantitative primers.

[0055] The results are as follows Figure 4 As shown in the figure, there was no target fragment in the control group plants, while the target fragment was detected in all four PeRAV1 positive seedlings, indicating that PeRAV1 had been successfully transferred into Arabidopsis. Two Arabidopsis transgenic lines (#1 and #3) with higher expression levels (brighter bands) were selected and continued to self-pollinate until T 3 Quantitative detection was performed.

[0056] 2. Statistics and quantitative analysis of transgenic plant phenotypes

[0057] The T of #1 and #3 strains 3 Arabidopsis plants were grown in a climate chamber under long-day conditions (16 h day, 8 h night). The number of rosette leaves and flowering time of 20 plants in each line were counted.

[0058] The results are as follows Figure 5As shown, after overexpressing PeRAV1 in Arabidopsis, the plants showed an obvious 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 in the same manner 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 used 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: 95°C for 1 min; 95°C for 10 s, 60°C for 30 s, and 38 cycles.

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

[0071] The above description is only 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. A PeRAV1 gene of moso bamboo, the nucleotide sequence of which is shown in SEQ ID NO.

1.

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

2.

3. A vector and recombinant bacteria containing the PeRAV1 gene of bamboo according to claim 1.

4. Use of the bamboo PeRAV1 gene according to claim 1 in regulating the flowering time of plants.

5. The use 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) Construction of an overexpression vector for the PeRAV1 gene in bamboo; 2) Transform the constructed bamboo PeRAV1 gene overexpression vector into Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis strains with delayed flowering time.

7. Use of the PeRAV1 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 rosette leaves of the plant is to promote the increase of the number of rosette leaves.

9. The use according to claim 8, characterized in that: include: 1) Construction of an overexpression vector for the PeRAV1 gene in bamboo; 2) Transform the constructed overexpression vector of PeRAV1 gene of bamboo into Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis thaliana with increased number of rosette leaves.

10. Use of the bamboo PeRAV1 gene according to claim 1 in regulating the expression level of downstream genes of the PeRAV1 gene.

Images