Petunia anther dominant expression gene PhMET1 as well as expression protein and application thereof

Abstract

The invention discloses a petunia anther dominant expression gene PhMET1 as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the petunia anther dominant expression gene PhMET1 disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the petunia anther dominant expression gene PhMET1 is as shown in SEQ ID NO.2. The method comprises the following steps: constructing a silent expression vector TRV2-PhMET1 of a petunia anther dominant expression gene PhMET1; the gene is transformed into petunia to construct a transgenic petunia strain expressed by a silent PhMET1 gene. Results of the embodiment show that by silencing the PhMET1 gene, a transgenic petunia plant with shortened flower filament length and / or reduced flower anther and / or reduced pollen grain viability and / or shrunk pollen grains is obtained, a new way is provided for improving the flower type of petunia, and a means is provided for controlling pollen spreading and cross breeding of petunia.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically relates to a petunia anther dominantly expressed gene PhMET1, an expressed protein and an application thereof. Background Art

[0002] Petunia hybrida, a member of the genus Petunia in the Solanaceae family, boasts large, numerous flowers that bloom profusely, long-lasting, and richly colored. It makes an excellent flower for flower beds and pots, can be grown in clusters naturally, or as a cut flower. In suitable climates or greenhouse cultivation, it can bloom year-round. It can be widely used in flowerbed arrangements, planter configurations, scenic spots, window sill embellishments, and home decoration, possessing great application value and research potential.

[0003] The maintenance and establishment of DNA methylation is performed by methyltransferases. MET1 is a type of methyltransferase responsible for maintaining methylation at CpG and CHG sites in plants. DNA methylation plays an important regulatory role in plant development, such as leaf growth, fruit ripening, seed development, and vernalization. Furthermore, DNA methylation is involved in anther development, such as tapetum differentiation and programmed cell death (PD). In Brassica napus and tobacco, DNA methylation promotes PD. During active tapetum cell development, DNA methylation levels are low. As the tapetum enters a degradation phase, DNA methylation levels begin to rise, reaching their highest levels during the tapetum PCD phase. Studies have found that highly modified DNA methylation is a hallmark of anther development and plays a key role in anther development. Therefore, studying the function of the methyltransferase MET1 in regulating anther development in petunia may provide useful molecular tools for genetic engineering applications in petunia. However, research on genes related to anther dominant expression in petunia is relatively limited, and there are no specific reports on the application of PhMET1, a gene related to anther dominant expression in petunia. Summary of the Invention

[0004] In response to the aforementioned problems in the prior art, the present invention aims to provide a gene, PhMET1, that is dominantly expressed in petunia anthers. Another technical problem addressed by the present invention is to provide an expression protein of the gene, PhMET1, that is dominantly expressed in petunia anthers. A further technical problem addressed by the present invention is to provide a use of the gene, PhMET1, that is dominantly expressed in petunia anthers, for regulating plant filament length, anther size, and pollen grain viability, thereby accelerating the breeding of superior varieties.

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

[0006] A petunia anther-dominantly expressed gene PhMET1 has a nucleotide sequence shown in SEQ ID NO.1.

[0007] The amino acid sequence of the expressed protein of the petunia anther dominantly expressed gene PhMET1 is shown in SEQ ID NO.2.

[0008] TRV2-PhMET1, a silent expression vector containing the petunia anther-dominantly expressed gene PhMET1.

[0009] The use of the silencing expression vector TRV2-PhMET1 in shortening the length of flower filaments includes:

[0010] 1) Construction of a silencing expression vector for the petunia anther-dominantly expressed gene PhMET1;

[0011] 2) Transforming the constructed silencing expression vector of the petunia anther-dominantly expressed gene PhMET1 into petunia;

[0012] 3) Cultivate, screen and obtain transgenic petunia plants with shortened flower filaments.

[0013] Application of the silencing expression vector TRV2-PhMET1 in reducing the size of flower anthers.

[0014] Application of the silencing expression vector TRV2-PhMET1 in inhibiting pollen grain viability.

[0015] Application of the silencing expression vector TRV2-PhMET1 in causing pollen grain shrinkage.

[0016] Application of the petunia anther-dominantly expressed gene PhMET1 in regulating flower filament length and / or regulating flower anther size and / or regulating pollen grain vitality.

[0017] Application of the petunia anther-dominantly expressed gene PhMET1 in regulating pollen grain morphology.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) This paper identifies the anther-dominantly expressed gene PhMET1 in petunia for the first time, clarifies its nucleotide sequence (SEQ ID NO.1) and the amino acid sequence of the expressed protein (SEQ ID NO.2), filling the gap in the research of genes related to anther development in petunia.

[0020] 2) The present invention constructed a silencing expression vector TRV2-PhMET1 containing the PhMET1 gene and transformed it into petunia. The results of the examples show that by silencing the PhMET1 gene, the length of the filaments of petunia flowers can be significantly shortened, providing a new approach to improving the flower shape of petunias; the volume of the anthers of the flowers is significantly reduced, which helps to regulate the development of the reproductive organs of petunias and may affect their pollination and reproductive characteristics; silencing the PhMET1 gene can lead to a significant decrease in pollen grain vitality, and most pollen grains cannot be dyed with acetyl carmine, which provides a means to control pollen dispersal and hybrid breeding in petunias; the pollen grains are shrunken in morphology, which is significantly different from the plump and round pollen grains of the wild type, providing a model for studying the mechanism of pollen grain development and regulating pollen morphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the expression map of TRV2-PhMET1 vector;

[0022] Figure 2 This is a comparison of gene expression levels between the wild type and VIGS silenced lines of petunia (EV is the wild type, and pTRV2-PhMET1:VIGS is the silenced plant);

[0023] Figure 3 A comparison of flowers of the wild-type and VIGS-silenced petunia lines (EV is the wild-type, and pTRV2-PhMET1:VIGS is the silenced plant);

[0024] Figure 4 Comparison of anthers and filaments of wild-type and VIGS-silenced petunia lines (EV is the wild-type, and pTRV2-PhMET1:VIGS is the silenced plant);

[0025] Figure 5 This is a comparison of acetylcarmine staining of the wild type and VIGS silenced petunia lines (EV is the wild type, and pTRV2-PhMET1:VIGS is the silenced plant). DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions 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.

[0027] The plant material used in this application is Petunia hybrida "W115", which was planted in the Jiangsu Provincial Key Laboratory of the College of Landscape Architecture, Nanjing Forestry University. The anthers of flowers in the an2 developmental stage of Petunia hybrida were selected, placed in a sterilized centrifuge tube, and immediately placed in liquid nitrogen for quick freezing, and then stored in a -80°C refrigerator.

[0028] Example 1

[0029] 1. Total RNA extraction from plant tissues and cDNA synthesis

[0030] In this experiment, TIANGEN Plant RNA Extraction Kit (DP432) was used to extract anther RNA from petunia an2 developmental stage flowers. TaKaRa PrimeScript TM The extracted RNA was reverse transcribed into cDNA using the RT Master Mix (Perfect Real Time) reverse transcription kit according to the instructions.

[0031] 2. Cloning of target genes

[0032] Nucleotide sequences of candidate genes were extracted using TBtools software, combining the complete petunia genome file and annotation files. After checking for sequence integrity, the distribution of restriction sites within the candidate gene coding sequence was analyzed using BioXM software (version 2.6). Suitable restriction sites were selected based on the restriction site information on the TRV2 vector. The identified restriction sites, the candidate gene CDs nucleotide sequence, and the vector sequence were input into CE design software (version 1.04). The software's double-enzyme linearized primer design function was used to design primers for the super-expression vector amplification of the candidate gene. Primer sequence information was provided to Nanjing Beluga Biotechnology Co., Ltd. for primer synthesis, and the resulting primers were stored in a refrigerator at 4°C until needed. PCR amplification of the target gene was performed using cDNA from petunia flowers at the an2 stage (PhMET1 is specifically expressed in anthers and has a higher expression level during the an2 stage) as a template. The total reaction volume was 20 μL: 1 μL Forward Primer, 1 μL Reverse Primer, 1 μL cDNA, 10 μL Primer STAR Max (high-fidelity enzyme), and 7 μL ultrapure water. The amplification program was 40 cycles of pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s.

[0033] The PCR amplification products were subjected to agarose gel electrophoresis, and the gel with clear brightness, a single band, and a length consistent with the predicted length of the target gene was excised and recovered using a gel recovery kit from Hunan Aikerui Biotechnology Co., Ltd. The full-length coding region sequence of the PhMET1 gene was ultimately obtained, 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.

[0034] The amplification primer sequences are as follows:

[0035] PhMET1-F:

[0036] 5'-agaaggcctccatggggatccCAACTTGCTGGTCTTGATGATACC-3',

[0037] PhMET1-R:

[0038] 5'-tgtcttcgggacatgcccgggGATCTTGATGTAAAATTTGCTGGATG-3'.

[0039] Example 2

[0040] 1. VIGS gene silencing system

[0041] The full-length coding region of the PhMET1 gene was cloned into the TRV2 vector ( Figure 1 ) and named the vector TRV2-PhMET1; the TRV2-PhMET1 fusion expression vector containing the target gene was transformed into Agrobacterium tumefaciens competent cells GV3101.

[0042] 2. Construction and screening of positive transgenic petunia

[0043] Prepare an infection buffer solution containing 10 mmol / L MgCl2, 10 mmol / L MES, and 150 μmol / L AS at a final concentration; take out the bacterial solution of the TRV2 empty expression vector, the TRV1 auxiliary expression vector, and the TRV2-PhMET1 fusion expression vector that has been transformed into Agrobacterium from -80°C the night before, thaw at room temperature until it is mixed with ice and water, and then insert it into ice; activate the above three bacterial solutions by adding 1 mL of bacterial solution to every 250 mL LB liquid culture medium (containing 10 μg / mL of Kana), and culture at 28°C, 200 rpm, and in the dark; when the bacterial solution OD 600 When the OD value reaches 0.8-1, centrifuge (4°C, 5000 rpm, 10 min), discard the supernatant, and collect the bacteria; resuspend the bacteria in the infection buffer prepared on the same day (resuspend to an OD value of 2.0); mix ① the TRV1 auxiliary expression vector bacterial solution and the TRV2-PhMET1 fusion expression vector bacterial solution in a volume ratio of 1:1, shake well and activate for more than 2 hours (until its OD value is 1.0); ② the TRV1 auxiliary expression vector bacterial solution and the TRV2 empty expression vector bacterial solution in a volume ratio of 1:1, shake well and activate for more than 2 hours (until its OD value is 1.0).

[0044] Use a 1mL disposable syringe to inject the mixed bacterial solution of ① and ② into the back of the petunia leaves respectively. Avoid blistering the leaves during injection. Write the corresponding gene marker on each petunia pot. After watering once, grow normally until the phenotype appears (climate incubator at 20 degrees Celsius, dark incubation for 24 hours followed by normal incubation).

[0045] A real-time fluorescence quantitative experiment was performed on the injected plants. Based on the full-length cDNA sequence of PhMET1, fluorescence quantitative primers (F: 5′-AATCCGGGGGAACTCGA-3′; R: 5′-CAAAGAGCCCCTTCCACTG-3′) were designed in the non-conserved region. The cDNA of anthers of petunia an2 developmental stage flowers was used as a template, and the petunia β-actin gene was selected as an internal reference gene. Premix Ex Taq TM The instructions were followed and different components were prepared according to the reaction system ratio. The total volume of the reaction system was 10 μL, including 0.4 μL forward and reverse primers, 5 μL SYBR, 1 μL cDNA, 0.2 μL calibration solution, and 3 μL ultrapure water. The amplification procedure was 95°C pre-denaturation for 3 min, 95°C denaturation for 45 s, 60°C annealing for 30 s, and 95°C extension for 15 s, for 40 cycles. Three biological replicates and three technical replicates were performed for each sample. After ensuring the reliability of the experimental data, 2 -ΔΔCT The expression differences of target genes were calculated by SPSS 20.0 software, and the obtained data were analyzed for significance.

[0046] The results are as follows Figure 2 As shown, the expression levels of the PhMET1 gene were compared between injected and wild-type plants. Plants with lower PhMET1 expression levels after injection than in wild-type plants were selected and defined as positive transgenic lines, resulting in positive transgenic lines 1, 2, and 8. PhMET1 expression levels in VIGS-silenced transgenic plants were significantly lower than those in wild-type plants. Expression levels also varied between different positive transgenic plants, with higher expression levels in the L1 line and lower expression levels in the L8 line. This indicates that positive plants transformed with VIGS-silenced PhMET1 exhibited variable gene expression. However, compared with wild-type plants, PhMET1 expression levels were significantly lower, confirming that PhMET1 regulates anther development in petunia. The TRV2-PhMET1-L8 line, which exhibited the lowest PhMET1 expression, was selected for subsequent functional validation.

[0047] 3. Verification of transgenic positive plants

[0048] 1) Observation of flower and anther morphology

[0049] A digital camera was used to record the size and shape of the top flowers and anthers, and the stamen height (the distance from the top of the stigma to the bottom of the ovary) of the control group and the VIGS silenced positive transgenic petunia plants.

[0050] The results are as follows Figure 3 As shown, the flower size and shape of transgenic plants were not significantly different from those of wild-type controls.

[0051] The results are as follows Figure 4 As shown, the filament length of the flowers of the transgenic plants was significantly shorter than that of the wild-type controls, and the anther size was also smaller than that of the controls.

[0052] 2) Pollen viability determination

[0053] Pollen grains were collected from the top anther of the flower for pollen viability test. Pollen viability was detected by 1% acetyl carmine pollen staining method. Pollen grains that were plump and stained red were considered viable.

[0054] The results are as follows Figure 5 As shown, after treatment with 1% acetyl carmine, most wild-type pollen grains were plump and round, evenly stained, plump in shape, and dark red; while most pollen grains collected from positive transgenic plants could not be stained and had a shrunken shape.

[0055] 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 petunia anther-dominantly expressed gene PhMET1, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein of the petunia anther-dominantly expressed gene PhMET1 according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A silent expression vector TRV2-PhMET1 containing the petunia anther-dominantly expressed gene PhMET1 according to claim 1.

4. Use of the silencing expression vector TRV2-PhMET1 according to claim 3 in shortening the length of flower filaments.

5. The use according to claim 4, characterized in that include: 1) Construction of a silencing expression vector for the petunia anther-dominantly expressed gene PhMET1; 2) Transforming the constructed silencing expression vector of the petunia anther-dominantly expressed gene PhMET1 into petunia; 3) Cultivate, screen and obtain transgenic petunia plants with shortened flower filaments.

6. Use of the silencing expression vector TRV2-PhMET1 according to claim 3 in reducing the size of flower anthers.

7. Use of the silencing expression vector TRV2-PhMET1 according to claim 3 in inhibiting pollen grain viability.

8. Use of the silencing expression vector TRV2-PhMET1 according to claim 3 in causing pollen grains to shrink.

9. Use of the petunia anther-dominantly expressed gene PhMET1 according to claim 1 in regulating flower filament length and / or flower anther size and / or pollen grain activity.

10. Use of the petunia anther-dominantly expressed gene PhMET1 according to claim 1 in regulating pollen grain morphology.

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