Application of popils1a gene in regulating plant seed development

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the PoPILS1a gene in regulating plant seed development. The coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1. This invention is the first to discover that PoPILS1a is specifically expressed in peony seed tissues, and the expression level varies significantly at different developmental stages. The PoPILS1a protein is located in the endoplasmic reticulum. In Arabidopsis thaliana positive lines with overexpression of PILS1a, the attachment point between pods is significantly shortened, and the number of pods distributed within a limited inflorescence length is greater. Simultaneously, the length and weight of pods at corresponding positions show significant differences compared to the control, clarifying that PILS1a can promote fruit development and has good practical application value.

Description

Application of the PoPILS1a gene in regulating plant seed development Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the PoPILS1a gene in regulating plant seed development. Background Technology

[0002] Auxin, the earliest discovered plant hormone, plays a crucial role in regulating many aspects of plant growth and development and in responding to signals from the internal and external environment. Currently, several protein families with auxin transport functions have been identified. Among them, the PIN protein family is located in the plasma membrane and endoplasmic reticulum membrane, responsible for mediating auxin transport between cells and organelles. PILS (PIN-LIKES) proteins share some similarity in protein sequence with auxin-exporting PIN proteins. However, most PILS proteins are located in the endoplasmic reticulum, assisting in regulating intracellular auxin homeostasis and subsequently affecting auxin signaling transduction in the cell nucleus.

[0003] Currently, the PILS gene family has been identified and studied in various plants, including Arabidopsis thaliana, rice (Oryza sativa), soybean (Glycine max), potato (Solanum tuberosum L.), and sugarcane, with 7, 7, 19, 8, and 11 PILS gene members found in these species, respectively. Studies have confirmed that these genes are involved in auxin transport, maintaining intracellular auxin homeostasis, and regulating physiological processes such as root development, tillering, and branching. For example, Arabidopsis plants overexpressing AtPILS5 have shortened root hairs and fewer lateral roots, while AtPILS2 and AtPILS5 double mutants have elongated root hairs and more lateral roots. These phenotypic differences are mainly related to changes in the strength of the auxin nuclear signal transduction pathway: that is, the signal is weakened in overexpressing plants and strengthened in double mutants.

[0004] However, systematic research on the PILS gene family in peony is still lacking. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to provide the application of the PoPILS1a gene in regulating plant seed development.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the invention provides the application of the PoPILS1a gene in regulating plant seed development; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0008] A second aspect of the present invention provides a method for increasing the weight of plant pods, the method comprising: overexpressing the PoPILS1a gene in a plant using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0009] A third aspect of the present invention provides a method for increasing the length of plant pods, the method comprising: overexpressing the PoPILS1a gene in a plant using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0010] A fourth aspect of the present invention provides a method for shortening the distance between the attachment points of plant pods, the method comprising: overexpressing the PoPILS1a gene in a plant using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0011] A fifth aspect of the invention provides the application of the methods described in the second to fourth aspects in plant breeding.

[0012] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0013] This invention is the first to discover that the expression of the peony PoPILS1a gene varies significantly at different stages of seed development. In Arabidopsis thaliana positive lines with overexpression of PoPILS1a, the attachment point between pods is significantly shortened, and the number of pods distributed within a limited inflorescence length is greater. Simultaneously, the length and weight of pods at corresponding positions show significant differences compared to the control. This clearly demonstrates that PILS1a promotes fruit development and provides a basis for further molecular-level analysis of the peony seed development process and the regulatory patterns of lipid biosynthesis and metabolism. Attached Figure Description

[0014] Figure 1 shows the expression analysis results of PoPILS1a in 11 tissues, including roots, stems, leaves, petals, receptacles, bracts, sepals, pistils, stamens, buds, and seeds, in this embodiment of the invention. The tissue names are represented by rectangles of different colors, and the error range was determined by three measurements.

[0015] Figure 2 shows the expression analysis results of PoPILS1a at different seed development stages in the embodiments of the present invention.

[0016] Figure 3 shows the results of subcellular localization analysis of PoPILS1a in tobacco in an embodiment of the present invention.

[0017] Figure 4 shows the expression localization results of PoPILS1a in seed in situ hybridization in the embodiments of the present invention, where A is the negative control and B is the experimental group.

[0018] Figure 5 shows the gene expression levels of different 35S:PoPILS1a transgenic lines PoPILS1a in the embodiments of the present invention; in the figure, 1 to 10 represent 10 35S:PoPILS1a transgenic lines.

[0019] Figure 6 shows the inflorescence and pod phenotypes of the 35S:PoPILS1a transgenic plant in this embodiment of the invention, where A and B are the inflorescence and pod phenotypes of the transgenic plant; C and D are the inflorescence and pod phenotypes of the control group plant.

[0020] Figure 7 shows the detailed phenotypic diagrams of the pods at positions 5 to 15 on the inflorescence axis of the control plant and the 35S:PoPILS1a transgenic plant in the embodiments of the present invention.

[0021] Figure 8 is a statistical diagram showing the distance between the attachment points of the pods on the inflorescence axis of the 35S:PoPILS1a transgenic plant in the embodiment of the present invention; in the figure, This indicates that P < 0.01. P < 0.001, mock represents control group plants, and OE represents 35S:PoPILS1a transgenic plants.

[0022] Figure 9 is a statistical chart of pod length in 35S:PoPILS1a transgenic plants in an embodiment of the present invention; in the figure, P < 0.01, mock represents control group plants, and OE represents 35S:PoPILS1a transgenic plants.

[0023] Figure 10 is a statistical chart of pod weight of 35S:PoPILS1a transgenic plants in an embodiment of the present invention; in the figure, This indicates that P < 0.01. This indicates that P < 0.001. P < 0.0001, mock represents control group plants, and OE represents 35S:PoPILS1a transgenic plants. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] In a typical embodiment of the present invention, the application of the PoPILS1a gene in regulating plant seed development is provided; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0027] ATGGAGGGAAGAGCTACACCCGTAGCCAAACAAAGCCTGAAGGCATGGATCATAGGAAATTTGGGGAACTTGCCTCTTATTATCATCCCGGCAGTCTGTAAAGAGAAAGGCAGTCCATTTGGAGCACCTGATGTTTGCCATACACATGGAATGGCTTATGCTTCACTTTCTATGGCGGTTGGAGCCGTTTATTTGTGGTCTTATGTTTACAACATTGTACGGATTTCTTCAAGCAAGATTGCAACAGAAGTCAACATAGATGACTCTACAATTAATATGAAGCACAATGGGGAAACATCAAAATTACTTGATGGGAGTTGCACAGAAACTCTTCTGCCTTCAAAAGATTGCTTCATCTGTTTGGGTAACAAATATCAGGCGCCAATTTCAGCTAAGATTAAGCAACATTTTAGAAGGATTTTAAGAAATGTTAACCTGAAGGCATTGTTTGCACCTTCAACTATTGGAGCGATTGTTGGGTTTATCATCGGAGTTTTCCCCCTGACCCGAAAGCTTATCATCGGTAGCAGTGCTCCGCTTCATGTTGTTCAAGACTCGGCTTCCTTGTTGGGGGATGCAGCTATCCCAACCGTCACCCTGATAGTTGGAGCAAACCTTCTTAAAGGTTTAAAAGGCTCGGGTATTCAGCCATCACTTATTGTTGGAGTTGTAGCAGTTCGCTACATATTCTTGCCTCTGTCGGGCATTCTTGTTGTTAAAGGTGCTATTCACATGGGGCTGGTGCAGTCAGATCCATTGTATCAGTTTGTTCTTCTACTACAGTATGCACTTCCACCGGCAATGAACATAGGCACGATTACCCAATTGTTCGGAGCTGGAGAGAGTGAATGCTCTGTTATTATGCTGTGGACGTATGCTTTGGCATCGGTTGCCCTAACGCTTTGGTCAACCTTCTTCATGTGGCTAATAGCTTGA(SEQ ID NO:1)。

[0028] The PoPILS1a gene encodes the protein shown in either (a1) or (a2):

[0029] (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO:2 in the sequence listing;

[0030] (a2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1).

[0031] MEGRATPVAKQSLKAWIIGNLGNLPLIIIPAVCKEKGSPFGAPDVCHTHGMAYASSLSMAVGAVYLWSYVYNIVRISSSKIATEVNIDDSTINMKHNGETSKLLDGSCTETLLPSKDCFICLGNKYQAPISAKIKQHFRRILRNVNLKALFAPSTIGA IVGFIIGVFPLTRKLIIGSSAPLHVVQDSASSLGDAAIPTVTLIVGANLLKGLKGSGIQPSLIVGVVAVRYIFLPLSGILVVKGAIHMGLVQSDPLYQFVLLLQYALPPAMNIGTITQLFGAGESECSVIMLWTYALASVALTLWSTFFMWLIA (SEQ ID NO:2).

[0032] The application has any of the following functions:

[0033] (b1) Increase the weight of plant pods;

[0034] (b2) Increase the length of plant pods;

[0035] (b3) Shorten the distance between the pod attachment points.

[0036] The regulation of plant seed development refers to: increasing pod weight and length and shortening the distance between attachment points by overexpressing the PoPILS1a gene, thereby promoting seed development; or increasing pod weight and length and shortening the distance between attachment points by increasing the activity or content of the PoPILS1a protein, thereby promoting seed development.

[0037] In another typical embodiment of the present invention, a method for increasing the weight of plant pods is provided, the method comprising: overexpressing the PoPILS1a gene in a plant using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0038] In another typical embodiment of the present invention, a method for increasing the length of plant pods is provided, the method comprising: overexpressing the PoPILS1a gene in plants using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0039] In another typical embodiment of the present invention, a method for shortening the distance between the attachment points of plant pods is provided, the method comprising: overexpressing the PoPILS1a gene in plants using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1.

[0040] In another typical embodiment of the present invention, the application of the above method in plant breeding is provided.

[0041] In this invention, the plants include, but are not limited to, peony or Arabidopsis thaliana.

[0042] In any or any other embodiment of the foregoing methods, there are alternative methods for introducing a recombinant DNA construct comprising a polynucleotide operatively linked to at least one regulatory sequence into a regenerative plant cell. For example, a regulatory sequence (such as one or more enhancers, optionally as part of a transposon element) may be introduced into a regenerative plant cell, followed by screening for events in which the regulatory sequence is operatively linked to an endogenous gene encoding the PoPILS1a protein disclosed herein.

[0043] A "recombinant DNA construct" refers to a combination of nucleic acid fragments that do not normally exist together in nature. Therefore, recombinant DNA constructs can contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from those found in nature.

[0044] "Operationally ligated" refers to the linking of nucleic acid fragments into a single fragment, such that the function of one nucleic acid fragment is regulated by the other. For example, when a promoter can regulate the transcription of a nucleic acid fragment, the promoter is operably ligated to that nucleic acid fragment.

[0045] It should be understood that those skilled in the art will recognize that this invention is not limited to specific exemplary sequences. Changes to nucleic acid fragments that produce chemically equivalent amino acids at a given site without affecting the functional properties of the encoded polypeptide are well known in the art. For example, the codon for the hydrophobic amino acid alanine may be replaced by a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue such as valine, leucine, or isoleucine. Similarly, it is contemplated that changes such as replacing one negatively charged residue with another, such as aspartic acid with glutamic acid, or replacing one positively charged residue with another, such as lysine with arginine, will produce functionally equivalent products. It is also contemplated that nucleotide changes altering the N-terminal and C-terminal portions of a polypeptide molecule will not change the activity of the polypeptide. Each of the recommended changes is within the conventional art, as is the preservation of the biological activity of the encoded product.

[0046] The recombinant DNA constructs disclosed herein can be introduced into plants using any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector-mediated DNA transfer, bombardment, or Agrobacterium-mediated transformation.

[0047] Furthermore, methods for modifying or altering the host's endogenous genomic DNA are available. This includes altering the host's natural DNA sequence or a pre-existing transgenic sequence, which includes regulatory elements, coding and non-coding sequences. These methods are also used to enable nucleic acids to target pre-engineered target recognition sequences in the genome.

[0048] The development or regeneration of plants containing isolated, foreign nucleic acid fragments encoding proteins of interest is well known in the art. Regenerated plants can be self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated plants can be hybridized with seeds from agronomically important lines. In turn, the regenerated plants are pollinated with pollen from these important lines. Transgenic plants containing the desired PoPILS1a are then cultured using methods well known to those skilled in the art.

[0049] Those skilled in the art will also understand that changes can be introduced through mutations in the nucleic acid sequence, thereby causing alterations in the expression of the encoded mRNA or the amino acid sequence of the encoded polypeptide, resulting in changes in the biological activity of the mRNA or protein, or both, respectively. Therefore, variant nucleic acid molecules can be generated by introducing one or more nucleotide substitutions, additions, and / or deletions into the corresponding nucleic acid sequence or surrounding sequences disclosed herein. Such variant nucleic acid sequences are also covered by this invention.

[0050] Variant nucleic acid sequences can be prepared by randomly introducing sequence changes along all or part of a gene region, including but not limited to chemical or radiation mutagenesis and oligonucleotide-mediated mutagenesis (OMM). Alternatively or otherwise, sequence changes can be introduced using double-strand break techniques at specific selected sites, such as ZNF, custom homing endonucleases, TALEN, CRISPR / CAS (also known as guide RNA / Cas endonuclease systems), or other protein and / or nucleic acid-based mutagenesis techniques. The resulting variants can be screened for altered activity. It should be understood that the techniques are generally not mutually exclusive. In fact, the various methods can be used alone or in combination, in parallel or sequentially, to produce or obtain a variety of sequence variants.

[0051] It should be noted that "35S:" is an overexpression promoter. Adding this promoter to the plasmid containing the target gene fragment and then transferring it into the plant can ensure the overexpression of the target gene, which can be used to identify and analyze gene function.

[0052] In this invention, the terms "35S: PoPILS1a transgenic plant", "35S:PoPILS1a", and "transgenic plant overexpressing the PoPILS1a gene" have the same meaning.

[0053] 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.

[0054] The main sources of experimental materials used in the following examples are as follows:

[0055] The experimental plant materials were robust peony plants of the cultivar 'Xueyingtaohua' (Paeonia suffruticosa 'Xueyingtaohua') and the tobacco cultivar 'Nicotiana benthamiana'. The subcellular localization vector pCAMBIA1300-GFP and the Arabidopsis transformation vector pCAMBIAI1300 were obtained from the Shandong Academy of Forestry Sciences, which is publicly available from the Crop Research Institute of the Academy of Forestry Sciences. These biological materials were used solely for replicating the relevant experiments of this invention and should not be used for other purposes. Escherichia coli competent cells (DH5α, catalog number: DLC114) and Agrobacterium competent cells (GV3101, catalog number: DLC301, EHA105, catalog number: DLC303) were purchased from Tsingke Biotechnology (Beijing).

[0056] Example 1: Screening of candidate genes for peony PoPILS

[0057] The hmmsearch tool was used with default parameters to search for PF03547-related genes in three species: Arabidopsis thaliana, rice (Oryzasativa), and peony (Paeonia ostii, CNGBdb Assembly ID CNA0050666). At the same time, the PINS sequences reported in the article were used to search for similar genes using blastp. The genes screened by hmmsearch and blastp were combined as candidate genes, and all PIN and PIN-like genes in the three species were identified.

[0058] Based on the above analysis, seven PoPILS members were identified in peony and named PoPILS1a, PoPILS1b, PoPILS5a, PoPILS5b, PoPILS6, PoPILS2a, and PoPILS2b. Information on these PoPILS genes and their encoded proteins is shown in Table 1.

[0059] Table 1. Physicochemical properties of the PoPILS family

[0060] Gene Name | Gene ID | Chromosomal Location | Exon | Amino Acid Count | Relative Molecular Mass | Isoelectric Point | Subcellular Location | PoPILS1a | Pos.gene | 81279 | Chr | 04831 | 1332 | 84.45 | 9.19 | Vacuole | PoPILS1b | Pos.gene | 81280 | Chr | 04621 | 924 | 094.39 | 8.3 | Vacuole | PoPILS5a | Pos.gene | 3062 | Chr | 04619 | 1208 | 969.18 | Vacuole | PoPILS5b | Pos.gene | 75828 | Chr | r04726128972.815.37 Cytoplasm plasPoPILS6Pos.gene49961Chr041240944366.068.76 Cytoplasm plasPoPILS2aPos.gene54238unchr_scaffold_2144148390.865.26 Cytoplasm plasPoPILS2bPos.gene13258unchr_scaffold_267349454827.917.63 Cytoplasm plas surface

[0061] Table 1 shows that the lengths of the seven PoPILS proteins vary greatly, ranging from 191 amino acids (PoPILS5a) to 494 amino acids (PoPILS2b), and their molecular weights range from 20896 Da (PoPILS5a) to 54827.91 Da (PoPILS2b). Subcellular localization prediction indicates that PoPILS proteins are mainly distributed in vacuoles and cytoplasm. Among the seven PoPILS members, PoPILS2a and PoPILS2b are not spliced ​​onto chromosomes but are spliced ​​in scaffold form and cannot be displayed. All five PoPILS genes are located on chromosome 4. Furthermore, we investigated fragment duplication and tandem duplication gene pairs throughout the peony genome, confirming that no duplication events occurred in the PoPILS gene family members. The expression of the PoPILS gene family in peony tissues exhibits tissue specificity. PoPILS1a is concentrated in seeds and pistils, while PoPILS5a and PoPILS5b are highly expressed in most tissues, especially stamens, pistils, and sepals. The remaining members are hardly expressed in seeds. Therefore, it is speculated that PoPILS1a in peony is involved in seed development.

[0062] Example 2: Analysis of the expression pattern of the peony PoPIL1a gene

[0063] Eleven tissues were selected from peony: roots, stems, leaves, bracts, sepals, petals, receptacle, stamens, pistils, buds, and seeds. 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 2. The relative expression level of the PILS family gene PoPILS1a in different tissues was detected using real-time quantitative PCR (SYBR FAST qPCR Kit, KAPA Biosystems, USA).

[0064] Table 2 Primers for Real-Time Fluorescence Quantitative Analysis

[0065]

[0066] As shown in Figure 1, PoPILS1a is specifically expressed in seeds and pistils, while its expression level is very low in other peony tissues. The specific expression of this gene suggests that it may be involved in auxin transport in specific pathways such as pistil and posterior ovary development and seed development.

[0067] Seeds at different developmental stages were selected for PoPILS1a expression analysis. The results are shown in Figure 2. It was found that the gene expression varied greatly at different stages. PoPILS1a expression was high in the early stage of seed development, and then increased sharply at 100 days, followed by a decrease. At the seed maturity stage, the expression level was low, suggesting that PoPILS1a may be involved in the development and formation of certain specific seed structures.

[0068] Example 3 Subcellular localization of PoPILS1a

[0069] The PoPILS1a ORF sequence was constructed into the pCAMBIA1300-GFP vector using the target protein fusion with GFP fluorescent protein method, and the subcellular localization of the PoPILS1a:GFP fusion protein was observed. Primer sequences are shown in Table 3.

[0070] Table 3. Primers for subcellular localization vectors

[0071]

[0072] 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."

[0073] After tobacco leaves were stained, they 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. The fluorescence signal was detected by laser confocal fluorescence microscopy. 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.

[0074] The subcellular localization results are shown in Figure 3. The green fluorescent protein expressed by the PoPILS1a:GFP fusion is located on the endoplasmic reticulum membrane in the cell. Previous studies have reported that plant PILS transporters are mostly located on the endoplasmic reticulum membrane or vacuolar membrane in cells, and the results of this experiment are consistent with previous reports. Therefore, it is believed that PoPILS1a in peony mainly participates in auxin transport on the endoplasmic reticulum and regulates the dynamic balance of auxin in the cell.

[0075] Example 4: In situ hybridization of PoPILS1a in seeds

[0076] Fresh peony seeds (120 days old) were used as tissue samples. They were fixed in FAA fixative and paraffin sections were prepared. Detailed paraffin sectioning procedures were performed according to the literature "Shi Z, Jiang Y, Han X, et al. SlPIN1 regulates auxin efflux to affect flower abscission process [J]. Scientific Reports, 2017, 7:14919.". Changes in PoPILS1a expression in peony seeds were observed. Needle labeling was performed according to the protocols provided by the DIG RNA labeling kit (SP6 / T7) and the DIG nucleic acid detection kit. The concentration of PILS1a and the probe was 211.7 ng / μL. The samples were stained overnight with NBT / BCIP at 37°C. Subsequently, the samples were decomposed and examined under a Nikon Eclipse ci optical microscope.

[0077] The PILS1a probe (156 bp) sequence is as follows:

[0078] TGATGGGATGCCAAGGGTTCCCGGCGAGGCACTCTAATTTTGGTCCGGCGGATTTGTATTCTGTTCAGTCATCAAGGGGTCCAACGCCAAGGCCGTCGAATTTCGAGGAAAATTGCGCACCGCCGCCGCCACCACCACCACCTCAAACAACCACGT (SEQ ID NO: 9).

[0079] The scanning results are shown in Figures A and B in Figure 4. The expression of PoPILS1a in the seeds is similar, and it is expressed in both the embryo and the endosperm. The signal in the endosperm is relatively strong, and it is mainly distributed in the tissues surrounding the embryo. The expression pattern of the gene in the seeds suggests that it may be involved in the accumulation of nutrients in the endosperm and the development of the embryo. Since the sample measured was a seed at 100 days of development, the expression distribution of the PoPILS1a gene may be different at other developmental stages, which requires further analysis and verification.

[0080] Example 5 35S: Identification and Phenotypic Observation of PoPILS1a Transgenic Plants

[0081] The coding sequence of the PoPILS1a gene (SEQ ID NO:1) was constructed into the pCAMBIA1300 vector, and the primer sequences are shown in Table 4. 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 the T3 generation Arabidopsis seedlings, leaves were collected, ground, and DNA was extracted from the wild-type control group and the 35S:PoPILS1a transgenic plants using the Solarbio Plant Genomic DNA Extraction Kit for positive identification. RNA was also extracted to determine the expression level of PoPILS1a. The position 1 was defined as the flower with white petals on the Arabidopsis inflorescence, with subsequent positions designated as 2, 3, 4, etc.

[0082] Table 4 35S: Primers for constructing the PoPILS1a transgenic vector

[0083]

[0084] Ten 35S:PoPILS1a transgenic lines were selected for PoPILS1a expression level determination, as shown in Figure 5. Among the ten lines, lines 4, 1, and 3 showed higher PoPILS1a expression levels. Further, 60-day-old seedlings were selected, and seed development at positions 5-15 was recorded. Pod length, weight, and the distance between pod attachment points were measured to comprehensively analyze the effect of PoPILS1a overexpression on Arabidopsis seed development. As shown in Figures 6 (A, B, C, D) and 7, compared to the control, the 35S:PoPILS1a positive plants had significantly shorter distances between pod attachment points, a greater number of pods distributed within the limited inflorescence length, and thus a larger seed yield. As shown in Figures 8, 9, and 10, the pod weight and length of the transgenic plants were significantly increased compared to the control group, showing highly significant differences at positions 13-15. The average pod length of the transgenic plant at position 15 reached 12.43 mm, and the weight was 6.77 μg. Therefore, it can be concluded that PoPILS1a can promote the development of Arabidopsis fruit, and may have a similar function in peony.

[0085] 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 application of the PoPILS1a gene in regulating plant seed development, characterized by, The coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1; the plant is Arabidopsis thaliana.

2. The application as described in claim 1, characterized in that, The PoPILS1a gene encodes a protein as shown in (a1) or (a2) below: (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO:2 in the sequence listing; (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. The application as described in claim 1, characterized in that, The regulation of plant seed development refers to: increasing pod weight and length and shortening the distance between attachment points by overexpressing the PoPILS1a gene, thereby promoting seed development.

4. The application as described in claim 1, characterized in that, The application has any of the following functions: (b1) increasing the weight of plant pods; (b2) increasing the length of plant pods; (b3) shortening the distance between the pod attachment points.

5. A method for increasing the weight of plant pods, characterized in that, The method includes: overexpressing the PoPILS1a gene in a plant using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1; the plant is Arabidopsis thaliana.

6. A method for increasing the length of plant pods, characterized in that, The method includes: overexpressing the PoPILS1a gene in a plant using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1; the plant is Arabidopsis thaliana.

7. A method for shortening the distance between the attachment points of plant pods, characterized in that, The method includes: overexpressing the PoPILS1a gene in a plant using genetic engineering techniques; the coding sequence of the PoPILS1a gene is shown in SEQ ID NO:1; the plant is Arabidopsis thaliana.

8. The application of the method according to any one of claims 5 to 7 in plant breeding; wherein the plant is Arabidopsis thaliana.

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