Application of PsPILS1b gene in regulating flowering time of plants

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of a PsPILS1b gene in regulating flowering of plants. The PsPILS1b gene is first excavated in peony flowers, a coding sequence of the PsPILS1b gene is shown as SEQ ID NO:1, and experiments prove that the PsPILS1b gene is involved in auxin transport in petals, promotes and maintains blooming of flowers. Meanwhile, the PsPILS1b gene is overexpressed in Arabidopsis, and a transgenic group is obviously delayed in petal abscission position, and the flowering period of the Arabidopsis is prolonged, further verifying that the PsPILS1b gene is involved in the peony petal senescence process, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of the PsPILS1b gene in regulating plant flowering period. Background Technology

[0002] Peonies have high ornamental, medicinal, and oil-producing value. They are commonly used in courtyards and potted plants, possessing significant ornamental and economic value. However, the natural flowering period of peonies is relatively short, with each flower lasting only 3-5 days, severely limiting their direct and indirect economic benefits.

[0003] Peony is a perennial woody plant, and conventional hybridization breeding is very inefficient. The genetic basis and molecular regulatory mechanisms of peony flowering are poorly understood, making it difficult to address the short flowering period through breeding and cultivation techniques. Therefore, delaying petal senescence and extending the flowering period have become key to overcoming the limitations on the economic benefits of peonies. Identifying genes related to petal senescence and revealing the regulatory mechanisms of petal senescence is of great guiding significance for accelerating the breeding of new peony varieties with longer flowering periods. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the purpose of this invention is to provide the application of the PsPILS1b gene in regulating plant flowering time.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the application of PsPILS1b protein or related biological materials in regulating plant flowering time, wherein the PsPILS1b protein is any one of the following: (A1) A protein with the amino acid sequence SEQ ID NO:2; The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0007] A second aspect of the present invention provides a method for delaying the flowering period of plants, the method comprising: using genetic engineering techniques to upregulate or enhance the expression of the PsPILS1b gene in plants; the sequence of the PsPILS1b gene is shown in SEQ ID NO:1.

[0008] A third aspect of the invention provides the application of the method described in the second aspect in plant breeding or quality improvement.

[0009] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention is the first to discover the PsPILS1b gene in peony flowers. Experiments have shown that PsPILS1b participates in auxin transport in petals, promoting and maintaining flower blooming. Silencing PsPILS1b affects auxin transport in petals. Even with continuous IAA supplementation, the lack of related transport proteins affects the dynamic transport of IAA in petals, accelerating petal senescence. Simultaneously, overexpression of the PsPILS1b gene in Arabidopsis thaliana significantly delayed petal abscission and prolonged the flowering period, further verifying that the PsPILS1b gene participates in the senescence process of peony petals and has promising application prospects. Attached Figure Description

[0010] Figure 1 This invention provides a heatmap analysis of the expression of the peony opening process and PsPILS family genes based on the five stages of peony opening, using petal transcriptome sequencing.

[0011] Figure 2 This is a tissue-specific expression analysis of the PsPILS1b gene in this embodiment of the invention.

[0012] Figure 3 This is a schematic diagram of the ectopic expression of the PsPILS1b promoter in Arabidopsis inflorescence in an embodiment of the present invention.

[0013] Figure 4 This is a schematic diagram of the opening process of peony cut flowers in an embodiment of the present invention.

[0014] Figure 5 The results of the expression analysis of PsPILS1b in response to auxin in the embodiments of the present invention are shown.

[0015] Figure 6 This is the tobacco subcellular localization of PsPILS1b in an embodiment of the present invention.

[0016] Figure 7 This is a schematic diagram of peony shedding caused by PsPILS1b gene silencing in an embodiment of the present invention.

[0017] Figure 8 In this embodiment of the invention, qRT-PCR was used to determine the silencing status and shedding time of the target gene in the sample.

[0018] Figure 9 This is a schematic diagram showing the changes in senescence indicators of peony petals after PsPILS1b gene silencing in an embodiment of the present invention. A represents proline, B represents superoxide dismutase, and C represents malondialdehyde.

[0019] Figure 10 This is a statistical analysis of the floral organ abscission phenotype and abscission location in the Arabidopsis thaliana control group and the 35S:PsPILS1b group in this embodiment of the invention.

[0020] Figure 11 The results of petal tearing force determination in the Arabidopsis thaliana control group and the 35S:PsPILS1b group in this embodiment of the invention are shown.

[0021] Figure 12 The relative expression levels of the AtBOP gene (A) and the AtPG gene (B) in the flower organs of Arabidopsis thaliana in this embodiment of the invention are shown. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0024] In a typical embodiment of the present invention, the application of PsPILS1b protein or related biological materials in regulating plant flowering period is provided, wherein the PsPILS1b protein is any one of the following: (A1) A protein with the amino acid sequence SEQ ID NO:2; The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0025] In some embodiments, the relevant biological material is a nucleic acid molecule capable of expressing the PsPILS1b protein, or an expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line containing the nucleic acid molecule.

[0026] In some implementations, the regulation of plant flowering time is achieved by regulating the transport capacity of auxin or petal shedding.

[0027] In some implementations, the regulation of plant flowering period is manifested as: reducing the expression level and / or activity of the PsPILS1b protein, thereby decreasing the transport capacity of the plant auxin or promoting petal abscission, and shortening the plant flowering period; or increasing the expression level and / or activity of the PsPILS1b protein, thereby increasing the transport capacity of the plant auxin or delaying petal abscission, and prolonging the plant flowering period.

[0028] In some implementations, the auxin is IAA.

[0029] In some embodiments, increasing the expression level and / or activity of the PsPILS1b protein increases the tearing force of plant petals, delays petal shedding, and prolongs the flowering period of the plant.

[0030] In some embodiments, the nucleic acid molecule capable of expressing the PsPILS1b protein is any one of the following: (B1) The DNA molecule shown in SEQ ID NO:1; DNA molecules that have 99%, 95%, 90%, 85% or more homology to the DNA sequences defined in (B2) and (B1) and encode the PsPILS1b protein.

[0031] In some implementations, the plant includes, but is not limited to, peony or Arabidopsis thaliana.

[0032] In another typical embodiment of the present invention, a method for delaying the flowering period of plants is provided, the method comprising: using genetic engineering techniques to upregulate or enhance the expression of the PsPILS1b gene in plants; the sequence of the PsPILS1b gene is shown in SEQ ID NO:1.

[0033] In some embodiments, the upregulation or enhancement of the expression of the PsPILS1b gene in plants is achieved by overexpressing the PsPILS1b gene in plants.

[0034] In some implementations, the enhancement may be selected from the following (1) to (6), or an optional combination thereof: (1) Enhanced by introducing a plasmid containing the PsPILS1b gene; (2) Enhanced by increasing the copy number of the PsPILS1b gene on the chromosome; (3) Enhanced by altering the promoter sequence of the PsPILS1b gene on the chromosome; (4) Enhanced by operatively linking a strong promoter to the PsPILS1b gene; (5) Enhancement through the introduction of enhancers; (6) Enhanced by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

[0035] Expression vectors carrying the target gene can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation.

[0036] In another typical embodiment of the present invention, the application of the above method in plant breeding or quality improvement is provided.

[0037] In some embodiments, the breeding method includes transgenic or asexual reproduction.

[0038] In some embodiments, the plant includes, but is not limited to, peony or Arabidopsis thaliana.

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0040] The main sources of experimental materials used in the following examples are as follows: The experimental plant material consisted of robust peony plants of the variety 'Snow Reflecting Peach Blossom'. Paeonia suffruticosa 'Xueyingtaohua'); the Arabidopsis thaliana variety is Columbia type ( Arabidopsis thaliana The tobacco variety is Benedictine (Tobacco Benedict). Nicotiana benthamiana The subcellular localization vector pSoup1300-GFP and the virus-induced gene silencing VIGS vector (pTRV1) were obtained from the Shandong Academy of Forestry Sciences, and are publicly available from the Crop Research Institute of the Academy of Forestry Sciences. These biological materials are for the sole purpose of replicating experiments related to this invention and should not be used for other purposes. Agrobacterium competent cells (GV3101, catalog number: DLC301; EHA105, catalog number: DLC303) were purchased from Tsingke Biotechnology (Beijing).

[0041] Example 1: Tissue-specific expression analysis of the PsPILS1b gene The peony bloom is divided into five periods, such as Figure 1 As shown, the outermost three petals are loosened in period S1, the petals are half open in period S2, the petals are fully open in period S3, the petals begin to soften in period S4, and the petals fall off in period S5.

[0042] The PILS gene expression matrix was displayed as a heatmap using the R package heatmap, and z-scores were normalized for each gene.

[0043] The analysis of the petal transcriptomes of seven gene members of the peony PILS family at five different stages revealed that each member had different expression levels at different flowering stages. In stage S2, PsPILS1b was highly expressed, followed by PsPILS1a. In stage S4, PsPILS5b was highly expressed. In stage S5, PsPILS2a was significantly highly expressed, followed by PsPILS6. It is speculated that the auxin-like transporter family plays a role at different stages of flower opening.

[0044] Ten tissues were selected from peony: root, stem, leaf, bract, sepal, petal, receptacle, stamen, pistil, and bud. Samples were rapidly frozen and ground in liquid nitrogen. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and reverse transcribed into cDNA (TransScript One-Step gDNA Remover and cDNA Synthesis SuperMix, TransGenBiotech, China). Quantitative primers were designed, and their sequences are shown in Table 1. Tissue expression of PsPILS1b was detected using real-time quantitative PCR (SYBR FAST qPCR Kit, KAPA Biosystems, USA).

[0045] Table 1 Primers for PsPILS1b gene amplification

[0046] like Figure 2 As shown, PsPILS1b expression is specific in different tissues of peony. The highest expression level is found in the sepals, followed by the leaves. It is also expressed in the roots, petals, receptacle, and bracts, while the expression level is lower in the stem, pistil, stamen, and bud.

[0047] Example 2: Ectopic expression of the PsPILS1b promoter Primers were designed based on the genome sequence to amplify the promoter sequence of the PsPILS1b gene (2000 bp upstream of ATG) and constructed into the pSoup1300-GUS modified vector. The primer sequences are shown in Table 2.

[0048] Table 2 Primers for vector construction

[0049] Transgenic Arabidopsis plants were obtained using the flower dip transformation method. T3 generation Arabidopsis were used as GUS staining material. Based on the growth stage of Arabidopsis, samples from 7-week-old flowering plants were selected for whole-plant GUS staining. The staining was performed in the dark at 37°C using a staining solution (50 mM phosphate buffer (pH 7.2), 0.1% (v / v) Triton™ X-100, 0.5 mM K4Fe(CN)6H2O, 0.5 mM K3Fe(CN)6, and 0.5 mM X-Gluc). The tissues were then destained with 95% ethanol and examined under a ZEISSV11 stereomicroscope (Nikon Japan). The distribution of GUS signals was observed to infer the site of action of PsPILS1b. Inflorescence organs were selected for focused observation of fluorescence signals. The dynamic distribution of PsPILS1b in various tissues of the floral organs during flowering was then analyzed.

[0050] By observing the distribution of staining signals in flowers at different stages of opening, the location and timing of gene expression can be inferred, such as... Figure 3 a and Figure 3 As shown in b, when the petals at the top of the inflorescence are not fully open, expression is only present at the tip of the pedicel and in the sepals; no obvious signal distribution is observed in other tissues of the flower. When the flower first opens, the signal in the sepals strengthens, and expression is present at the tip of the stigma. As the flower gradually opens... Figure 3 c- Figure 3 d), the signal is stronger in the sepals, and the expression level is also higher in the stamen filaments. The signal gradually increases in the petals until the flower is fully open. Figure 3 e- Figure 3 f) The signal in the petals is relatively strong, mainly distributed on the outer edge of the petals and in the main veins. As the petals age, the signal in the sepals and petals gradually weakens, and the petals fall off. It is speculated that PsPILS1b is involved in regulating the transport of auxin in the petals, promoting petal blooming.

[0051] Example 3: Study on the response relationship between PsPILS1b and auxin in petals during flowering. To analyze whether the expression of PsPILS1b during peony petal shedding responds to auxin, exogenous IAA was applied to measure changes in PsPILS1b expression levels. For ease of experimental treatment and to observe the senescence and shedding of peony petals, 25 cm long stems of the 'Snowy Peach Blossom' peony variety with slightly loosened outermost two whorls of petals were cut and transported fresh to the laboratory. The lower ends were obliquely cut and inserted horizontally into distilled water, placed in an incubator (14 hours light, 10 hours darkness, 40% humidity). Peony cut flower samples cultured for 12 hours were selected and inserted into deionized water as a control group. The group receiving 100 μM IAA was the experimental group. Samples were taken every 12 hours to measure the expression level of PsPILS1b in the outermost two whorls of petals.

[0052] The expression levels of PsPILS1b in the outermost two whorls of petals in distilled water and the IAA-added experimental group were measured, and the results are as follows: Figure 4 and Figure 5 As shown, PsPILS1b expression initially increased and then decreased throughout the flowering process, peaking at 36h and 48h, with almost no expression at 84h. However, the addition of IAA significantly increased its expression level, especially after 48h, after which PsPILS1b expression remained stable. Combined with phenotypic results, it was found that after IAA addition, flowers began to wilt at 84h due to water loss, with continuous transport of outer IAA from the flower stalk to the petals, maintaining flower opening. PsPILS1b continued its function of transporting auxin at this time. The positive response of PsPILS1b to auxin in the petals suggests that this tissue may play a role in auxin transport.

[0053] Example 4 Subcellular localization of the PsPILS1b gene The coding sequence of PsPILS1b (SEQ ID NO:2) was constructed into the pSoup1300-GFP vector using the target protein fusion GFP method, and the subcellular localization of the PsPILS1b-GFP fusion protein was observed. Primer sequences are shown in Table 3.

[0054] Table 3 Primer sequences for vector construction

[0055] For details on the specific steps of vector construction, Agrobacterium transformation, and tobacco infection, please refer to the following references: "Li Chiyu. Genetic and biochemical analysis of the response of Arabidopsis transcription factor EBP1 to negative regulation of RALF1 by RALF1-FERONIA signal [D], 2018. Li Lan. Genetic and biochemical analysis of the interaction between Arabidopsis receptor protein kinase TMK and FER and its mediating ABA signal [D], 2020. Kong Lingyao. Analysis of the regulatory mechanism of Arabidopsis protein phosphatase ABI1 [D]. 2014."

[0056] After infection, tobacco leaves were cultured under light for 3 days. Temporary sections were prepared, and the lower epidermis of the tobacco leaves was peeled off with tweezers, sprinkled with water, and spread evenly on a glass slide. Fluorescence signals were detected using a laser confocal fluorescence microscope. The excitation and detection spectra were as follows: GFP excitation light was 488 nm, and detection light was 510 nm; mCherry excitation light was 552 nm, and detection light was 568 nm.

[0057] Subcellular localization, such as Figure 6 As shown, the green fluorescent protein expressed by PsPILS1b fusion is located on the endoplasmic reticulum membrane. According to previous reports and software predictions, most PsPILS gene families are located on the endoplasmic reticulum membrane or vacuolar membrane, which is consistent with the results reported in previous studies. Therefore, it is speculated that PsPILS1b in peony mainly transports auxin in the endoplasmic reticulum and coordinates the transport of auxin in the cell.

[0058] Example 5 Virus-induced gene silencing (VIGS) of PsPILS1b VIGS primers for PsPILS1b were designed using NCBI (Primer designing tool (nih.gov)) and the specific fragment was constructed on the VIGS vector (pTRV1). The primer sequences are shown in Table 4.

[0059] Table 4 Primer sequences for vector construction

[0060] Take peony buds from the outermost two whorls of petals that are beginning to loosen (sampling method as above), and prick a small hole at the very tip of the bud beforehand. Place the treated peony buds upside down in a beaker containing the inoculum. Immerse the entire bud in the inoculum, place the beaker in a vacuum pump to remove air at 0.1 atmospheres for 20 minutes, then slowly release the air, repeating this process 3 times. Rinse twice with tap water and once with sterile water. Incubate at 8℃ in the dark for 3 days, then adjust the temperature in the climate chamber to 20℃ (16 h light / 8 h dark) and the relative humidity to 50%. Randomly select samples to be transferred into 100 μM IAA as the experimental group, while the remaining samples remain in deionized water. Take photos every 12 hours to observe changes in petal morphology and record the time of petal shedding. The pTRV1 empty vector serves as the control group. Each control and experimental group contains 30 samples.

[0061] RNA was extracted from petals of qTRV-PsPILS1b plants and control plants 48 h after they were transferred to a 20℃ climate chamber and placed in deionized water. The silencing status of the target gene in the samples was determined by qRT-PCR. Primer sequences are shown in Table 1. The effect of PsPILS1b gene silencing on senescence and petal drop in peony was analyzed by combining the changes in phenotype and gene expression levels after silencing.

[0062] Petal abscission rate was investigated in pTRV-PsPILS1b plants and control plants. Starting from the temperature of 20℃, the number of hours required for complete petal fall in both the silent group and the control group was recorded. The abscission rate was calculated separately, with 30 samples from each group. Senescence-related physiological indicators were detected in isolated samples, including proline (Pro), superoxide dismutase (SOD), and the osmotic regulator malondialdehyde (MDA). Specific measurement methods followed the procedures outlined in the Solarbio assay kit. Changes in these indicators were used to further analyze the petal senescence process.

[0063] like Figure 7 and Figure 8 It can be seen that the silenced gene significantly accelerated the senescence and shedding time of peony flowers. Compared with the control group (shedding time 121.45h), the silenced group had a shedding time of 95.55h, which was shortened by 21.32%. After applying exogenous IAA treatment, the shedding time of the control group was 135.75h, while that of the silenced group was 108.35h, which was significantly shorter than that of the control group and not much different from the silenced group without exogenous IAA.

[0064] Measurement of physiological indicators of aging, such as Figure 9As shown, proline levels in the silent group increased rapidly at 48 hours, peaked at 72 hours, and then declined rapidly. In contrast, the control group showed significantly lower levels in petals at 48 hours compared to the silent group. Proline levels only increased after 84 hours following the application of exogenous IAA, indicating that the samples had just entered the senescence stage. SOD results similarly showed that the silent group accelerated petal tissue senescence. SOD levels were high at 24 hours, as the flowers needed to maintain their opening. Petals began to enter the senescence stage at 48 hours, with high SOD levels. In contrast, the control group reached its highest level at 48 hours, when the flowers were fully open, and then gradually decreased after 72 hours. Malondialdehyde (MDA) measurements revealed that the control group had high levels at 72 hours, but the levels declined rapidly at the end of senescence (84 hours). The silent group peaked at 48 hours and then declined significantly, indicating that intracellular osmotic regulators almost ceased to function at the end of petal shedding.

[0065] In summary, our results indicate that PsPILS1b participates in auxin transport in petals, promoting and maintaining flower blooming. Silencing auxin transport affects the transport of auxin in petals. Despite continuous IAA supplementation, the lack of related transport proteins affects the dynamic transport of IAA in petals, leading to accelerated petal senescence.

[0066] Example 6 35S: Identification and Phenotypic Observation of PsPILS1b Transgenic Plants The coding sequence of the PsPILS1b gene was constructed into the pSoup1300-GFP vector, and the primer sequences are shown in Table 5.

[0067] Table 5 Primer sequences for vector construction

[0068] Transgenic Arabidopsis plants were obtained using the flower dip transformation method. The T3 generation of transgenic Arabidopsis was used as observation material. Sixty days after transplanting, leaves were collected, ground, and DNA was extracted from wild-type and 35S:PsPILS1b transgenic plants using the Solarbio Plant Genomic DNA Extraction Kit for identification. RNA was also extracted to determine the expression level of PsPILS1b. Using the flower with white petals on the Arabidopsis inflorescence as position 1 (attachment point), 10 positive lines and a control group were selected. The status of flowers at positions 1-13 on the inflorescence was recorded, and petal abscission was compared to determine the exact location of complete petal abscission. Flowers at positions 4, 5, and 6 on the inflorescence axis were selected to measure the expression levels of the transgenic 35S:PsPILS1b and control flowers, and the time to complete petal abscission was measured to analyze the differences in abscission time. Simultaneously, the pulling force (petal tearing force) of petals at the same position was measured to analyze the ease of petal abscission.

[0069] The results are as follows Figure 10 , Figure 11 and Figure 12 As shown, the control group experienced complete petal detachment at position 6 on the inflorescence axis, while the transgenic lines experienced complete detachment at position 11, significantly delaying the time required for complete petal detachment. Petal tearing force measurements revealed a significant difference in equivalence between the experimental and control groups at position 4, with the difference increasing progressively downwards. At position 6, the control group's petal tearing force was 0.63, while the transgenic line still required 1.76, indicating that the transgenic lines may not have yet entered the petal detachment initiation stage. Abscission indicator gene assays showed the greatest difference in expression levels between the experimental and control groups at position 5. The control group exhibited lower expression levels at position 6, suggesting that the petals were already in the detachment stage. The 35S:PsPILS1b group showed little change in expression levels at positions 4, 5, and 6. Combining the phenotypic diagram and petal tearing force measurements, we conclude that the transgenic line significantly delayed petal detachment and prolonged the flowering period of Arabidopsis thaliana.

[0070] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of PsPILS1b gene or its related biological material in prolonging the flowering period of Arabidopsis thaliana, characterized in that, The nucleotide sequence of the PsPILS1b gene is shown in SEQ ID NO:1; The relevant biological material is a nucleic acid molecule capable of expressing the PsPILS1b gene, or an expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line containing the nucleic acid molecule; The application prolongs the flowering period of Arabidopsis thaliana by overexpressing the PsPILS1b gene in Arabidopsis thaliana.

2. Use according to claim 1, wherein The PsPILS1b gene encodes any one of the following proteins: (A1) A protein with the amino acid sequence SEQ ID NO:2; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. Application of silencing PsPILS1b gene in shortening the flowering period of Paeonia suffruticosa, characterized in that, The nucleotide sequence of the PsPILS1b gene is shown in SEQ ID NO:

1.

4. A method for prolonging the flowering period of plants, characterized in that, The method includes: using genetic engineering techniques to upregulate the expression of the PsPILS1b gene in plants; the sequence of the PsPILS1b gene is shown in SEQ ID NO:1; the plant is Arabidopsis thaliana.

5. The method according to claim 4, characterized in that, The upregulation of PsPILS1b gene expression in plants is achieved by overexpressing the PsPILS1b gene in plants.

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