PhERF48, a dominant expression gene of petunia anthers, its expressed protein, and its applications.
By constructing and transforming the silent expression vector of the dominant expression gene PhERF48 of petunia anther, the development of petunia anther was regulated, and the problems of obstructed pollen formation and male infertility were solved, and the increase in pollen volume and normalization of anther development were achieved.
Patent Information
- Application Number
- CN202410243337.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-04
- Publication Date
- 2025-03-21
- Estimated Expiration
- 2044-03-04
AI Technical Summary
The prior art is difficult to effectively regulate the development degree of petunia anthers, resulting in problems such as hindering pollen formation, reducing pollen volume and male infertility.
By constructing a silent expression vector of the dominant expression gene PhERF48 of petunia anther, it was transformed into petunia leaves, and transgenic petunia plants with shorter flower filaments and poor anther development were obtained, and this gene was used to regulate the development of anthers.
The regulation of petunia anther development has been achieved, resulting in normal pollen formation, increasing the amount of pollen, and the problem of male infertility has been solved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a petunia anther dominant expression gene PhERF48 and its expression protein and application. Background Art
[0002] Petunia hybrida is a plant of the genus Petunia in the family Solanaceae Juss. It has large and numerous flowers, blooms profusely, has a long flowering period, and is rich in color. It is an excellent flower for flower beds and pots. It can also be planted in natural clusters or used as cut flowers. It can bloom all year round in suitable climates or in greenhouses. It can be widely used in flower bed layout, flower trough configuration, scenic spot display, window sill embellishment, and home decoration, and has great application value and research potential.
[0003] EFR is a type of transcription factor unique to plants. In 1994, the first AP2 transcription factor gene was identified by Jofuku et al. from the model plant Arabidopsis thaliana. Subsequently, Ohme-takagi and Shinshi et al. identified an ethylene response element binding protein containing a conserved domain from tobacco (Nicotiana tabacum). EFR transcription factors are involved in the fruit development process and play an important role in regulating the color, flavor and ripening of fruits. Based on their functional identification, transgenic technology has achieved remarkable results in the study of fruit ripening regulation in many plants. The function and expression regulation pattern of ERF genes in plants are variable. Studying the function of ERF genes in the regulation of petunia anther development can provide useful molecular tools for the application of genetic engineering to improve petunia. 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 petunia anther dominantly expressed gene PhERF48. Another technical problem to be solved by the present invention is to provide an expression protein of the petunia anther dominantly expressed gene PhERF48. Another technical problem to be solved by the present invention is to provide an application of the petunia anther dominantly expressed gene PhERF48, which is used to regulate the degree of anther development and improve the cultivation and application of petunia.
[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0006] A petunia anther dominantly expressed gene PhERF48, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0007] The amino acid sequence of the expressed protein of the petunia anther dominantly expressed gene PhERF48 is shown in SEQ ID NO.2.
[0008] A vector, recombinant bacteria or host cell containing the petunia anther dominant expression gene PhERF48.
[0009] Application of petunia anther dominant expression gene PhERF48 in promoting the growth of petunia flower filament length, including:
[0010] 1) Construction of a silencing expression vector for the petunia anther dominantly expressed gene PhERF48;
[0011] 2) Transforming the constructed silencing expression vector of the petunia anther dominantly expressed gene PhERF48 into petunia leaves;
[0012] 3) Cultivate, screen and obtain transgenic petunia plants with shortened flower filaments.
[0013] The silencing expression vector is a plant expression vector TRV2-PhERF48.
[0014] The application of the silencing expression vector TRV2-PhERF48 of the dominantly expressed gene PhERF48 in petunia anthers to cause abnormal anther development and / or anther shrinkage and / or reduced pollen vitality and / or twisted and collapsed pollen grains and / or missing and shrunk germination pores in petunia flowers.
[0015] Application of petunia anther dominantly expressed gene PhERF48 in enlarging petunia anthers.
[0016] Application of petunia anther dominantly expressed gene PhERF48 in improving petunia pollen vigor.
[0017] Application of petunia anther dominantly expressed gene PhERF48 in making petunia pollen grains plump and forming obvious germination pores.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] 1) The petunia anther dominantly expressed gene PhERF48 disclosed in the present invention has a nucleotide sequence as shown in SEQ ID NO.1, and an amino acid sequence of its encoded protein as shown in SEQ ID NO.2. The present invention constructs a silencing expression vector of the petunia anther dominantly expressed gene PhERF48; transforms the constructed silencing expression vector of the petunia anther dominantly expressed gene PhERF48 into petunia leaves; cultivates and screens to obtain transgenic petunia plants with poor anther development, blocked pollen formation, and a sharp reduction in pollen quantity, resulting in male sterility.
[0020] 2) In the VIGS silenced positive transgenic plants constructed by the present invention, the absence of the petunia anther dominantly expressed gene PhERF48 resulted in poor anther development in petunia flowers.
[0021] 3) In the VIGS silenced positive transgenic plants constructed by the present invention, the deletion of the petunia anther dominantly expressed gene PhERF48 resulted in smaller anthers.
[0022] 4) In the VIGS silenced positive transgenic plants constructed by the present invention, the absence of the petunia anther dominantly expressed gene PhERF48 resulted in reduced pollen vigor.
[0023] 5) In the VIGS silenced positive transgenic plants constructed by the present invention, the absence of the petunia anther dominantly expressed gene PhERF48 resulted in the distortion and collapse of pollen grains and / or the absence and shrinkage of the germination pores.
[0024] 6) The petunia anther dominantly expressed gene PhERF48 disclosed in the present invention enables the anthers of petunia flowers to develop normally.
[0025] 7) The petunia anther dominantly expressed gene PhERF48 disclosed in the present invention makes petunia anthers larger.
[0026] 8) The petunia anther dominantly expressed gene PhERF48 disclosed in the present invention increases the pollen vigor of petunia.
[0027] 9) The petunia anther dominantly expressed gene PhERF48 disclosed in the present invention makes petunia pollen grains plump and forms obvious germination pores. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 is the expression profile of the TRV2-PhERF48 vector;
[0029] Figure 2 This is a comparison of gene expression between the wild type and VIGS silenced strains of petunia (EV is the wild type, pTRV2-PhERF48:VIGS is the silenced plant);
[0030] Figure 3 This is a comparison of flowers of the wild type and VIGS silenced strains of petunia (EV is the wild type, pTRV2-PhERF48:VIGS is the silenced plant);
[0031] Figure 4 This is a comparison of anthers and filaments of the wild type and VIGS silenced strains of petunia (EV is the wild type, and pTRV2-PhERF48:VIGS is the silenced plant);
[0032] Figure 5 This is a comparison of acetyl carmine staining of the wild type and VIGS silenced strains of petunia (EV is the wild type, and pTRV2-PhERF48:VIGS is the silenced plant);
[0033] Figure 6This is a comparison of the scanning electron microscopy results of the wild type and VIGS silenced strains of petunia (EV is the wild type, pTRV2-PhERF48:VIGS is the silenced plant). DETAILED DESCRIPTION
[0034] 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.
[0035] The plant material used in this application is Petunia "W115", which was planted in the Jiangsu Provincial Key Laboratory of the School of Landscape Architecture of Nanjing Forestry University. The anthers of flowers in the an2 developmental period of Petunia 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.
[0036] Example 1
[0037] 1. Total RNA extraction from plant tissues and cDNA synthesis
[0038] 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.
[0039] 2. PhERF48 gene cloning experiment
[0040] Combined with the whole genome file and annotation file of petunia, TBtools software was used to extract the nucleotide sequence of the candidate gene. After checking the integrity of the sequence, BioXM software (version 2.6) was used to analyze the distribution of restriction sites on the coding nucleotide sequence of the candidate gene. Combined with the restriction sites on the TRV2 vector, suitable restriction sites were screened. The selected restriction sites, CDs nucleotide sequences of candidate genes, vector sequences and other information were input into CE design software (version 1.04). The double restriction linearization primer design function in the software was used to design the super table vector amplification primers of the candidate gene. The primer sequence information was handed over to Shanghai Jierui Bioengineering Co., Ltd. for primer synthesis, and the obtained primers were placed in a 4℃ refrigerator for standby use. The cDNA of the petunia flower an2 period was used as a template (the PhERF48 gene is specifically expressed in anthers, and the expression level is the highest in the an2 period) for PCR amplification of the target gene. The total volume of the reaction system was 20 μL, including 1 μL of Forward Primer, 1 μL of Reverse Primer, 1 μL of cDNA, 10 μL of Primer STAR Max (high-fidelity enzyme), and 7 μL of ultrapure water. The amplification program was 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing for 30 s, and 72°C extension for 45 s, for 40 cycles.
[0041] The PCR amplification product was subjected to agarose gel electrophoresis, and the gel with clear brightness, single band and length consistent with the predicted length of the target gene was cut out and recovered using the gel recovery kit of Hunan Aikerui Biotechnology Co., Ltd. Finally, the full-length coding region sequence of the PhERF48 gene was obtained, and its 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.
[0042] Amplification primer sequences:
[0043] F:agaaggcctccatggggatccTGTGTAGAGTCTAGTGGATTGCAGAA
[0044] R:tgtcttcgggacatgcccgggATTTTGCCAATCATCCTTTGGA
[0045] Example 2
[0046] 1. VIGS gene silencing system
[0047] The full-length coding sequence of PhERF48 gene was cloned into TRV2 vector ( Figure 1) and named the vector TRV2-PhERF48; the TRV2-PhERF48 fusion expression vector containing the target gene was transferred into Agrobacterium tumefaciens competent cells GV3101.
[0048] 2. Construction and screening of positive transgenic petunia
[0049] Prepare an infection buffer containing 10 mmol / L MgCl2, 10 mmol / L MES, and 150 μmol / L AS at a final concentration; take out the bacterial solution of TRV2 empty expression vector, TRV1 auxiliary expression vector, and TRV2-PhERF48 fusion expression vector that has been transformed into Agrobacterium from -80°C one night in advance, thaw it at room temperature until it is mixed with ice and water, and then insert it into ice; add 100 μmol / L MgCl2, 10 mmol / L MES, and 150 μmol / L AS to each 250 mL LB liquid medium (containing Kana The above three bacterial solutions were activated at a ratio of 10 μg / mL) to 1 mL of bacterial solution, and cultured at 28°C, 200 rpm and in the dark; when the OD600 value of the bacterial solution reached 0.8-1.0, centrifugation was performed (4°C, 5000 rpm, 10 min), the supernatant was discarded, and the bacteria were collected; the bacteria were resuspended in the infection buffer prepared on the same day (resuspended to an OD value of 2.0); ① TRV1 auxiliary expression vector bacterial solution and TRV2-PhERF48 fusion expression vector bacterial solution were mixed in a volume ratio of 1:1, and activated for more than 2 hours (until its OD value was 1.0) after sufficient shaking and mixing; ② TRV1 auxiliary expression vector bacterial solution and TRV2 empty expression vector bacterial solution were mixed in a volume ratio of 1:1, and activated for more than 2 hours (until its OD value was 1.0) after sufficient shaking and mixing.
[0050] Use a 1mL disposable syringe to inject the mixed bacterial solution of ① and ② into the back of the petunia leaves respectively. Avoid making blisters on 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 culture for 24 hours and then normal culture).
[0051] A real-time fluorescence quantitative experiment was performed on the injected plants. Based on the full-length cDNA sequence of PhERF48, fluorescence quantitative primers (F: 5′-GGTCCCTCAAGAGTCTTCCAAG-3′; R: 5′-CTCTGGGTTCTGCTGGTCGAT-3′) were designed in the non-conserved region. The cDNA of anthers of flowers in the an2 developmental period of petunia was used as a template, and the petunia β-actin gene was selected as an internal reference gene. Premix Ex Taq TMInstructions, prepare different components according to the reaction system ratio. The total volume of the reaction system is 10μL, forward and reverse primers 0.4μL, SYBR 5μL, cDNA 1μL, calibration solution 0.2μL, ultrapure water 3μL. The amplification program is 95℃ pre-denaturation for 3min, 95℃ denaturation for 45s, 60℃ annealing for 30s, 95℃ extension for 15s, 40 cycles. Each sample is set up with 3 biological replicates and 3 technical replicates. Ensure that the experimental data is reliable before using 2 -ΔΔCT The expression difference of target genes was calculated by the SPSS 20.0 software.
[0052] The results are as follows Figure 2 As shown, the expression level of PhERF48 gene between the injected plants and the wild-type plants was obtained, and the plants with lower expression level of PhERF48 gene after injection than that of the wild-type plants were selected and defined as positive transgenic lines, thereby obtaining positive transgenic lines 2 / 4 / 6. The expression level of PhERF48 gene in VIGS-silenced positive transgenic plants was significantly lower than that of the wild type; at the same time, the expression levels were also different between different positive transgenic plants, with higher expression levels of PhERF48 gene in L4 plants and lower expression levels in L2 plants, indicating that there were also differences in gene expression strength in the positive plants obtained by genetic transformation of petunia by VIGS-silenced PhERF48 gene; however, compared with wild-type petunia plants, the expression level of PhERF48 gene was significantly reduced, and this result also verified that the PhERF48 gene regulated the anther development of petunia. The TRV2-PhERF48-L2 plant with the lowest expression level of PhERF48 gene was selected for subsequent functional verification.
[0053] 3. Transgenic positive plant verification
[0054] (1) Observation of flower and anther morphology
[0055] 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.
[0056] The results are as follows Figure 3 As shown, there was no significant difference in flower size and shape between the transgenic plants and the wild-type controls.
[0057] 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.
[0058] (2) Pollen viability determination
[0059] Pollen grains were collected from the top anther of the flower for pollen viability test, and the pollen viability was detected by 1% acetyl carmine pollen staining method. Pollen grains that were plump and stained red were considered viable.
[0060] The results are as follows Figure 5 As shown, after treatment with 1% acetyl carmine, most wild-type pollen grains were full and round, evenly stained in dark red, while most of the pollen grains collected from positive transgenic plants could not be stained.
[0061] (3) Pollen grain scanning electron microscopy
[0062] The anthers at the top of the flowers of the wild-type and VIGS-silenced positive transgenic plants were collected and fixed overnight at 4°C with 1% osmium tetroxide buffer and glutaraldehyde buffer. These anthers were then dehydrated in an ethanol series and dried using a critical point dryer and then coated with gold in a sputter coater (Bray et al., 1993). The pollen was then observed using a scanning electron microscope (model jsm-6390lv, JEOL Co., Japan) at accelerating voltages of 500x, 2000x, and 8000x.
[0063] The results are as follows Figure 6 As shown, scanning electron microscopy results showed that the wild-type pollen grains were plump and spherical, with thick reticular outer walls carved with obvious apical processes and branches, and large and numerous germination pores; in contrast, most of the pollen grains of the VIGS-silenced strain were obviously twisted and collapsed, and the germination pores were missing or shrunk.
[0064] 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. Silencing the Genes Predominantly Expressed in Petunia Anthers PhERF48 Application of the method in shortening the length of filaments of petunia flowers, wherein the anthers of petunia preferentially express genes PhERF48 The nucleotide sequence is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that: include: 1) Construction of petunia anther dominant expression gene PhERF48 Silencing expression vector; 2) The constructed petunia anther dominant expression gene PhERF48 The silencing expression vector was transformed into petunia leaves; 3) Cultivate, screen and obtain transgenic petunia plants with shortened flower filaments.
3. The use according to claim 2, characterized in that: The silent expression vector is a plant expression vector TRV2- PhERF48 .
4. Genes Predominantly Expressed in Petunia Anthers PhERF48 The silent expression vector TRV2- PhERF48 Application of the method for reducing the size of petunia anthers and / or reducing pollen vitality, wherein the petunia anthers preferentially express genes PhERF48 The nucleotide sequence is shown in SEQ ID NO.
1.
5. Genes Predominantly Expressed in Petunia Anthers PhERF48 The silent expression vector TRV2- PhERF48 Application of the method in causing petunia pollen grains to twist and collapse and / or germination pores to be missing or shrunk, wherein the petunia anthers preferentially express genes PhERF48 The nucleotide sequence is shown in SEQ ID NO.
1.
6. Silencing the Genes Predominantly Expressed in Petunia Anthers PhERF48 Application of the method in making petunia anthers smaller, wherein the petunia anthers preferentially express genes PhERF48 The nucleotide sequence is shown in SEQ ID NO.
1.
7. Silencing the Genes Predominantly Expressed in Petunia Anthers PhERF48 Application of the method in reducing the vigor of petunia pollen, wherein the petunia anthers preferentially express genes PhERF48 The nucleotide sequence is shown in SEQ ID NO.
1.
8. Silencing the Genes Predominantly Expressed in Petunia Anthers PhERF48 Application of the method in causing petunia pollen grains to twist and collapse, wherein the petunia anthers preferentially express genes PhERF48 The nucleotide sequence is shown in SEQ ID NO.
1.
9. Silencing the Genes Predominantly Expressed in Petunia Anthers PhERF48 Application of the method in causing the germination pores of petunia pollen grains to be missing or contracted, wherein the anther of petunia preferentially expresses a gene PhERF48 The nucleotide sequence is shown in SEQ ID NO. 1.