Moso bamboo PeMT2 gene as well as encoding protein and application thereof

Abstract

The invention discloses a moso bamboo PeMT2 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 edulis PeMT2 gene disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the encoding protein of the phyllostachys edulis PeMT2 gene is as shown in SEQ ID NO.2. The preparation method comprises the following steps: cloning a phyllostachys pubescens PeMT2 gene to a pCAMBIA1302 plasmid, so as to obtain a recombinant vector pCAMBIA1302-PeMT2; transforming the recombinant vector into arabidopsis thaliana, and culturing and screening to obtain a transgenic arabidopsis thaliana strain. The transgenic arabidopsis strain shows an obvious late flowering phenotype, the number of rosette leaves is increased, and the flowering time is delayed. The invention discloses the function of the PeMT2 gene for the first time, and also discloses the application of the PeMT2 gene in regulating the flowering time of plants and the quantity of rosette leaves.

Description

TECHNICAL FIELD

[0001] The present application relates to plant genetic engineering technology, in particular to a Phyllostachys edulis PeMT2 gene, a protein encoded by the gene and application thereof. BACKGROUND

[0002] DNA methylation has been widely studied in organisms since its discovery, and its functions are related to gene regulation, reproduction and development, diseases and aging, etc. DNA methylation is a defense mechanism that can resist changes that damage DNA structure. DNA methylation helps transcriptional silence, making DNA unable to be transcribed and activated, thus playing a protective role in a certain sense. DNA methylation is achieved by the activity of DNA methyltransferase. DNMT transfers a methyl group from S-adenosyl methionine to cytosine through an enzyme. MT1 (MTHFD1) encodes a methylenetetrahydrofolate dehydrogenase involved in one-carbon metabolism and DNA methylation control. In mthfd1-1 mutants, folate metabolism is impaired, leading to the accumulation of homocysteine, a hallmark of impaired methionine cycle, resulting in loss of global CHG and CHH methylation and reduction of H3K9me. DNA methylation is a relatively stable epigenetic marker. However, it is still dynamically regulated. In plants, the maintenance of DNA methylation depends on the maintenance of DNA methyltransferase during DNA replication. MT1 genes have been reported to regulate DNA methylation, but research on their role in plant flowering is still rare.

[0003] Phyllostachys edulis belongs to Bambusoideae of Poaceae and is a plant with rapid growth and a particularly long vegetative growth period. In order to flower, Phyllostachys edulis needs to undergo a long period of vegetative growth, sometimes even taking hundreds of years. The flowering mechanism of Phyllostachys edulis has not been elucidated yet. It is currently believed that age and environmental conditions may affect flowering, and it is not clear whether the methylation process affects flowering. SUMMARY

[0004] The key technical problem to be solved by the present application is to provide a Phyllostachys edulis PeMT2 gene and application thereof. The Phyllostachys edulis PeMT2 gene is cloned into a pCAMBIA1302 plasmid to obtain a recombinant vector pCAMBIA1302-PeMT2, the recombinant vector is transformed into Agrobacterium and then into Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana. Finally, the overexpression plant has increased number of rosette leaves and delayed flowering time.

[0005] To solve the above technical problems, the present application adopts the following technical solutions:

[0006] A protein encoded by a Phyllostachys edulis PeMT2 gene, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0007] An isolated polynucleotide encoding the expressed protein of the bamboo PeMT2 gene.

[0008] In some embodiments, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO. 1.

[0009] A biological material containing the polynucleotide expression cassette, recombinant vector, or recombinant cell.

[0010] The application of the biomaterial in the cultivation of transgenic plants or molecular breeding, wherein the plant is moso bamboo or Arabidopsis thaliana.

[0011] A method for delaying flowering and / or prolonging the vegetative growth period of a plant, comprising expressing the expression protein of the PeMT2 gene of moso bamboo, wherein the plant is moso bamboo or Arabidopsis thaliana.

[0012] In some embodiments, the method includes the following steps:

[0013] S1: Construct the expression vector for the PeMT2 gene of moso bamboo;

[0014] S2: Transform the expression vector of the PeMT2 gene from moso bamboo into plants or plant tissues;

[0015] S3: Cultivate and obtain transgenic plant plants, wherein the transgenic plant plants have a phenotype of delayed flowering time and / or prolonged vegetative growth period.

[0016] In some embodiments, the method for constructing the expression vector of the moso bamboo PeMT2 gene is as follows: the expression cassette of the moso bamboo PeMT2 gene is inserted into the vector backbone, the vector backbone containing plant selective marker genes and / or terminator sequences.

[0017] In some embodiments, the expression cassette of the moso bamboo PeMT2 gene is linked to a plant functional promoter.

[0018] In some embodiments, the plant functional promoter is the cauliflower mosaic virus 35S promoter.

[0019] In some embodiments, the plant is Arabidopsis thaliana, and the phenotype of increased vegetative growth is specifically manifested by an increase in the number of rosette leaves.

[0020] The beneficial effects of this invention are:

[0021] 1) This invention discloses for the first time the PeMT2 gene cloned from moso 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.

[0022] 2) This invention detected the expression of the PeMT2 gene in different tissues of moso bamboo in real time and found that it was expressed in multiple tissues.

[0023] 3) This invention reveals the function of the PeMT2 gene in moso bamboo for the first time. The PeMT2 gene in moso bamboo was inserted into the pCAMBIA1302 plasmid to obtain the recombinant vector pCAMBIA1302-PeMT2; the recombinant vector was transformed into tobacco leaves, and it was found that the protein it encodes is located in the nucleus and cytoplasm; the recombinant vector was transformed into Arabidopsis thaliana, and the resulting transgenic Arabidopsis thaliana lines had delayed flowering time, increased rosette leaves, and showed a clear late-flowering phenotype. Attached Figure Description

[0024] Figure 1 Agarose gel electrophoresis image of the PeMT2 gene clone from moso bamboo (lane 1 is the 2000 DNA Marker, lane 2 is the target gene fragment).

[0025] Figure 2 Quantitative analysis of PeMT2 gene in different tissues of moso bamboo (R for root, S for stem, L for leaf, B for shoot).

[0026] Figure 3 Subcellular localization map of PeMT2 in moso bamboo (scale bar is 20µm).

[0027] Figure 4 The image shows the PCR detection results of positive plants of Arabidopsis thaliana after transformation with PeMT2 from moso bamboo (WT is the wild-type plant, Control is the pCAMBIA1302-PeMT2 recombinant plasmid, and #1~#4 are the overexpression lines pCAMBIA1302-PeMT2-1~4).

[0028] Figure 5 The images show the phenotypic observation of transgenic Arabidopsis thaliana (A), the number of rosette leaves (B), and the flowering time (C). (Control plants are control plants, #2 is the overexpression line pCAMBIA1302-PeMT2-2, and #3 is the overexpression line pCAMBIA1302-PeMT2-3. The scale bar is 1.5 cm.) Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention are further described below in conjunction with the examples. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art or performed in accordance with the kit and product instructions.

[0030] The plant material used in this application is bamboo leaves, which were collected on the campus of Nanjing Forestry University.

[0031] Example 1

[0032] 1. PeMT2 gene cloning

[0033] RNA was extracted from bamboo leaves and cDNA was obtained by reverse transcription. RNA extraction was performed using a plant RNA extraction kit (TaKaRa) according to the kit instructions. cDNA synthesis was performed using a reverse transcription kit (TaKaRa). The PeMT2 gene CDS sequence was obtained from the bamboo genome database (http: / / gigadb.org / dataset / view / id / 100498) using BLAST. Full-length primers (containing restriction enzyme sites) were designed using Primer 5 software to clone the gene. The primer sequences are shown below:

[0034] MT2-GS:

[0035] 5'-GAGAACACGGGGGACTCTAGAATGCCCCAATGGGCCAGA-3',

[0036] MT2-GA:

[0037] 5'-GCCCTTGCTCACCATGGATCCAGCGGATATAGCTATGTTCTCCTG-3'.

[0038] The 50μL PCR reaction system consisted of: 2.5μL cDNA, 25μL 2×MegaPfu Premix (purchased from TOLOBIO) high-fidelity enzyme, 3μL each of MT2-GS and MT2-GS, and 16.5μL deionized water.

[0039] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 minutes; 95℃ for 15 seconds, 56℃ for 15 seconds, 72℃ for 15 seconds, 35 cycles; 72℃ for complete extension for 5 minutes.

[0040] Agarose gel electrophoresis was used to detect PCR results. Figure 1 The target band of the correct size was cut out, and the fragment was recovered using a gel extraction kit (purchased from Omega) and sent to the company for sequencing (Sangon Biotech (Shanghai) Co., Ltd.). The PeMT2 gene is 504 bases long (with the terminator removed), and its nucleotide sequence is shown in SEQ ID NO.1; its encoded protein contains 168 amino acids, and its amino acid sequence is shown in SEQ ID NO.2.

[0041] 2. Tissue expression results of the PeMT2 gene

[0042] RNA was extracted from the roots, stems, leaves, and shoots of moso bamboo, and cDNA was synthesized, following the same method as in Example 1. Quantitative primers for PeMT2 were designed based on the full-length PeMT2 gene sequence. Moso bamboo PeNTB was used as an internal reference gene. Real-time quantitative PCR was used to detect the expression of PeMT2 in the roots, stems, leaves, and shoots of moso bamboo.

[0043] qPeNTB-S: 5'-TCTTGTTTGACACCGAAGAGGAG-3',

[0044] qPeNTB-A: 5'-AATAGCTGTCCCTGGAGGAGTTT-3',

[0045] qPeMT2-S: 5'-CCATCGCTGCCGACATCC-3',

[0046] qPeMT2-A: 5'-GAGTCCTTCCTGCACCCCAC-3'.

[0047] The quantitative PCR reaction system consisted of: 25 μL SYBR Green Master Mix (purchased from Yisheng), 3 μL cDNA, 1 μL forward primer, 1 μL reverse primer, and 20 μL deionized water.

[0048] The quantitative PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 minutes; 95℃ for 10 seconds, 60℃ for 20 seconds, 72℃ for 20 seconds, for 40 cycles.

[0049] The results showed that ( Figure 2 The PeMT2 gene showed little difference in expression among the roots, stems, leaves, and shoots of moso bamboo, with relatively higher expression levels in the roots and shoots, suggesting that the gene may play a role in these tissues.

[0050] 3. Constructing an overexpression vector for the PeMT2 gene

[0051] The pCAMBIA1302 vector was double-digested with restriction endonucleases BamHI and XbaI. The double digestion reaction system consisted of 1 μL each of BamHI and XbaI (purchased from Thermo Fisher Scientific), 2.5 μL of 10× Buffer, 10.5 μL of pCAMBIA1302 vector, and 10 μL of deionized water. After incubation at 37°C for 4 hours, the linearized vector fragment was recovered by gel extraction.

[0052] The correctly submitted gene fragment and the recovered linearized vector fragment were ligated using homologous recombinase (purchased from TOLOBIO) to obtain the recombinant overexpression vector pCAMBIA1302-PeMT2. The total ligation volume was 20 μL: 2×Ezmax @10 μL of Universal CloneMix, 1.5 μL of linearized vector, 4.5 μL of PeMT2 amplification fragment, and 4 μL of deionized water were used. The reaction was carried out at 37℃ for 15 minutes to transform *E. coli* DH5α competent cells (purchased from TOLOBIO). Single clones were picked from LB agar plates containing 50 μg / mL kanamycin, and subjected to PCR detection via shaking. After confirming the correct band size, the cells were sent for sequencing. After successful sequencing, the recombinant plasmid was named pCAMBIA1302-PeMT2.

[0053] 4. PeMT2 subcellular localization

[0054] Following the instructions for use of Agrobacterium GV3101 competent cells (purchased from TOLOBIO), the recombinant plasmid pCAMBIA1302-PeMT2 was transformed into Agrobacterium. Single clones were selected from LB agar plates containing 50 μg / mL rifampicin and 50 μg / mL kanamycin, and cultured by shaking PCR. After confirming the correct band size, the cells were preserved. 2 ml of the preserved bacterial culture was added to 8 ml of LB liquid medium and incubated at 28°C and 220 rpm until the bacterial culture was complete. Centrifuge at 4800 rpm for 10 minutes. Following the transient expression technique for tobacco, resuspend the precipitate in the inoculum solution. After being left to stand in the dark for 6 hours, tobacco leaves were injected to observe subcellular localization.

[0055] The results showed that PeMT2 was located in the cytoplasm and nucleus. Figure 3 ).

[0056] Example 2

[0057] 1. Obtaining PeMT2 gene overexpression in Arabidopsis thaliana

[0058] Inoculate 1 mL of the preserved Agrobacterium into 150 mL of LB broth containing the appropriate antibiotic and incubate at 28°C and 220 rpm for 20 hours. Collect the bacterial cells by centrifugation at 6000 rpm for 5 minutes, resuspend the cells in 5% sucrose, and adjust... Add 0.025% surfactant and mix well to obtain the infection solution. Soak Arabidopsis inflorescences in the infection solution for 8 minutes. Control plants were infected with Agrobacterium containing the pCAMBIA1302 plasmid. Cover with plastic wrap to maintain moisture and incubate in the dark for one day before transferring to normal light and dark conditions. Harvest seeds after the seed pods of the infected plants turn yellow. Sow seeds on MS solid medium containing hygromycin and incubate for 10 days. Transplant suspected positive transgenic seedlings into soil. Take seedling leaves and extract DNA using a plant DNA extraction kit (purchased from TaKaRa) according to the kit instructions. Detect the PeMT2 gene band using MT2-GS and MT2-GS primers. PCR detection method is the same as in Example 1.

[0059] The results showed that the target band was not present in the wild-type plants, but it was detected in all four transgenic plants, and was consistent with the PeMT2 gene band in the pCAMBIA1302-PeMT2 plasmid. Figure 4 This indicates that all four transgenic plants were positive seedlings. Two transgenic lines (#2 and #3) were selected for self-pollination and seed harvesting, and the phenotype of the next generation was observed.

[0060] 2. Phenotypic statistics of transgenic plants

[0061] Control plants and transgenic plants were planted in the same climate chamber, and the number of rosette leaves and flowering time of each transgenic plant were systematically counted for 16 plants.

[0062] Compared to the control group, transgenic plants overexpressing the PeMT2 gene showed a significant increase in the number of rosette leaves and a marked delay in flowering, exhibiting a late-flowering phenotype. These results indicate that overexpression of the PeMT2 gene in bamboo can significantly increase the number of rosette leaves and delay flowering. Figure 5 ).

[0063] The embodiments described above are merely illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A protein encoded by the PeMT2 gene of moso bamboo, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. An isolated polynucleotide, characterized in that, The polynucleotide encodes the expression protein of the bamboo PeMT2 gene as described in claim 1.

3. The polynucleotide according to claim 2, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

1.

4. A biomaterial, characterized in that, The biomaterial is an expression cassette, recombinant vector, or recombinant cell containing the polynucleotides described in claim 2 or 3.

5. The application of the biomaterial according to claim 4 in the cultivation of transgenic plants or molecular breeding, characterized in that, The plant in question is either moso bamboo or Arabidopsis thaliana.

6. A method for delaying flowering and / or prolonging the vegetative growth period of plants, characterized in that, This includes expressing the PeMT2 gene of bamboo as described in claim 1 in a plant, wherein the plant is bamboo or Arabidopsis thaliana.

7. The method according to claim 6, characterized in that, Includes the following steps: S1: Construct the expression vector of the bamboo PeMT2 gene as described in claim 2 or 3; S2: Transform the expression vector of the PeMT2 gene from moso bamboo into plants or plant tissues; S3: Cultivate and obtain transgenic plant plants, wherein the transgenic plant plants have a phenotype of delayed flowering time and / or prolonged vegetative growth period.

8. The method according to claim 7, characterized in that, The method for constructing the expression vector of the moso bamboo PeMT2 gene is as follows: the expression cassette of the moso bamboo PeMT2 gene is inserted into the vector backbone, and the vector backbone contains plant selective marker genes and / or terminator sequences.

9. The method according to claim 8, characterized in that, In the expression cassette of the moso bamboo PeMT2 gene, the moso bamboo PeMT2 gene is linked to a plant functional promoter.

10. The method according to claim 9, characterized in that, The plant functional promoter is the cauliflower mosaic virus 35S promoter.

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