Application of a birch BpSPL2 gene in promoting pollen abortion and reducing pollen allergenicity

Abstract

The application relates to the technical field of genetic engineering, in particular to a white birch BpSPL2 application of a gene in promoting pollen abortion and reducing pollen allergenicity. BpSPL2 application of a gene in promoting pollen abortion and reducing pollen allergenicity. The application provides a new gene resource for regulating pollen development of forest trees by using a transgenic technology, and provides a theoretical basis for promoting pollen abortion and reducing pollen allergenicity by a genetic engineering method, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to a birch species. BpSPL2 Application of genes in promoting pollen abortion and reducing pollen allergenicity. Background Technology

[0002] SPL (SQUAMOSA promoter-binding protein-like) SPL transcription factors are a class of plant-specific transcription factors found in all green plants, including single-celled green algae, mosses, gymnosperms, and angiosperms. SPL transcription factors were first cloned by Huijser et al. from snapdragon (Antirrhinum majus), at which time they discovered two closely related... AmSBP1 and AmSBP2 Genes that can bind to the promoter of the floral meristem characteristic gene SQUAMOSA (SQUA) have been named Squamosa promoter binding proteins (SBPs). Subsequently, cDNA cloning of the SBP transcription factor family was performed in Arabidopsis and mosses, revealing multiple copies of SBP transcription factor genes in both plants. In recent years, similar genes have also been found in birch, maize, tomato, and strawberry. Existing research indicates that SBPs are involved in regulating several important biological processes, such as developmental stage transitions, flower development, leaf growth, plant height and tillering, plant stress responses, and copper ion homeostasis, among other developmental and physiological biochemical processes.

[0003] Many trees, including birch, spruce, and hemlock, produce allergenic pollen. In Europe, exposure to birch pollen allergens is a major trigger for allergic rhinitis and often induces respiratory illnesses, adversely affecting the health of many people. The wide distribution, long reproductive cycle, and large pollen production of birch trees over long distances have raised concerns about genetic pollution caused by the spread of pollen from genetically modified (GM) trees. In recent years, GM technology has been applied to birch trees, making it crucial to prevent GM birch pollen from contaminating native species. Regulating the flowering process of trees and cultivating male-sterile varieties can effectively address issues such as genetic pollution caused by the spread of GM tree pollen and pollen-induced allergic reactions.

[0004] Studies have reported that in Arabidopsis thaliana... ,ABORTED MICROSPORES(AMS) As a transcription factor, it directly regulates genes related to callose breakdown (A6) and fatty acid hydroxylation (A6). CYP703A2, CYP704B1, CYP86C3 Pollen sheath formation-related genes (extracellular lipase genes) EXLs Genes rich in glycine protein GRPs ) and lipid transport-related genes ( ABCG26 The expression of these genes is involved in the regulation of pollen wall development. Some flower development-related genes have also been verified in woody plants. Genes isolated from birch... BpAP1 When the gene is overexpressed in birch, phenotypes such as premature development of male inflorescences, anther shrunkenness, and a significant decrease in pollen dispersal ability occur. In willow, the gene was cloned... pSgPG1-pSgPG4、pSgPME1 and pSgGN1 A series of genes, and in situ hybridization analysis showed SgPGs and SgPME1 Its specific expression in mature pollen grains after microspore mitosis suggests that it may be involved in pollen development.

[0005] Currently, research on pollen development mainly focuses on model plants and crops, while research on long-lived perennial woody plants such as birch is less common. SPLs There are few reports on gene function, and none involve birch. BpSPL2 Functional study of genes in promoting pollen abortion and reducing pollen allergenicity. Summary of the Invention

[0006] The purpose of this invention is to provide a birch tree. BpSPL2 Application of genes in promoting pollen abortion and reducing pollen allergenicity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides birch BpSPL2 Application of genes in promoting pollen abortion and reducing pollen allergenicity.

[0008] Preferably, the promotion of pollen abortion and reduction of pollen allergenicity is achieved by increasing the concentration of pollen in birch. BpSPL2 This is achieved through gene expression.

[0009] Preferably, the improvement BpSPL2 Gene expression includes... BpSPL2 Gene overexpression vectors were transformed into birch.

[0010] Preferably, the overexpression vector is pGWB5- BpSPL2 -GFP.

[0011] This invention provides a method for promoting pollen abortion and reducing pollen allergenicity in birch, including increasing the concentration of pollen in birch flowers. BpSPL2 Gene expression.

[0012] Preferably, the improvement BpSPL2 Gene expression includes... BpSPL2 Gene overexpression vectors were transferred into birch.

[0013] This invention provides a recombinant expression vector containing birch bark. BpSPL2 The gene was used to overexpress the gene in birch.

[0014] This invention provides a transgenic birch cell containing the aforementioned recombinant expression vector.

[0015] This invention provides a method for cultivating birch plants with low pollen abortion allergenicity, comprising transferring the recombinant expression vector into birch and screening for the obtained... BpSPL2 Plants with overexpressed genes.

[0016] Preferably, the birch described in this invention BpSPL2 The nucleotide and amino acid sequences of the gene can be found in the patent document (CN111690664 A).

[0017] It is important to note that there is a direct causal relationship between pollen abortion and reduced pollen allergenicity. Pollen is an important carrier of male reproductive cells in plants and also one of the main sources of airborne allergens. Mature pollen grains produced by trees such as birch contain various allergenic proteins. When pollen is released into the air and inhaled by humans, it can trigger respiratory diseases such as allergic rhinitis and asthma. Pollen abortion refers to the process during pollen development where, due to genetic regulation or external environmental influences, pollen fails to mature normally, lacks viability, or cannot be released. When pollen abortion occurs, its development process stops before the microsporocyte or tetrad stage, preventing the formation of mature pollen grains with dispersal capabilities and allergenic protein expression functions. This reduces the release of allergenic pollen at the source, significantly lowering the risk of pollen allergy in the environment.

[0018] In this invention, birch is overexpressed. BpSPL2 The gene can significantly inhibit the normal development process of pollen. Experimental results show that... BpSPL2 In plants that overexpress pollen, the anthers only develop to the microsporocyte stage, failing to complete meiosis to form tetrads, let alone mature pollen grains. This pollen abortion phenotype directly prevents the plant from producing pollen with dispersive and allergenic activity, thus effectively reducing the likelihood of pollen-induced allergic reactions.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes four-year-old children. BpSPL2 Inflorescences of transgenic birch with overexpression and suppressed expression, as well as wild-type birch, were collected, and the morphological characteristics of the inflorescences and anthers were recorded. Simultaneously, anther anatomical structure analysis was performed to study... BpSPL2 The function of genes in pollen development regulation. Anther cross-section observation results showed: BpSPL2 The anthers of the overexpressing plants only developed to the microsporocyte stage and had not yet entered meiosis; while the wild-type plants had completed meiosis and developed to the tetrad stage. In contrast, BpSPL2The meiotic initiation of the suppressed expression plants was earlier than that of the wild type, having already passed the tetrad stage and entered the overwintering preparation period. These results indicate that... BpSPL2 Overexpression of this substance can effectively promote pollen abortion.

[0020] The present invention provides BpSPL2 Overexpression and repression of transgenic birch plants can be used to identify downstream regulatory genes related to pollen development, enriching the regulatory network of plant pollen development. Among these, overexpression... BpSPL2 35S:: obtained BpSPL2 Birch plants can significantly promote pollen abortion, making them suitable for cultivating tree varieties with desirable traits related to pollen abortion, thereby reducing pollen allergenicity. This invention further confirms that... BpSPL2 Genes involved in the regulation of birch pollen development can provide important reference resources for research on forest tree pollen development and the functional analysis of the plant SBP gene family in pollen development.

[0021] This invention utilizes transgenic technology to provide new gene resources for the regulation of pollen development in forest trees, and provides a theoretical basis for promoting pollen abortion and reducing pollen allergenicity through genetic engineering, which has broad application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 for BpSPL2 Statistics on inflorescence phenotype, inflorescence length, and diameter of overexpression, suppressed expression transgenic plants, and wild-type plants.

[0024] Figure 2 for BpSPL2 Statistical analysis of anther phenotype, anther length, and width in overexpression, suppressed expression transgenic plants, and wild-type plants.

[0025] Figure 3 for BpSPL2 Pollen development characteristics of anther cross sections in overexpression, suppressed expression transgenic plants and wild-type plants. Detailed Implementation

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 1. Using the leaf disc transformation method to construct BpSPL2 Gene overexpression vector pGWB5- BpSPL2 -GFP and the repressor expression vector pROKII- BpSPL2 -SRDX is converted into Birch to obtain pGWB5- BpSPL2 -GFP transgenic birch plants 35S:: BpSPL2 and pROKII- BpSPL2 -SRDX transgenic birch plants 35S::BpSPL2 -SRDX. (Overexpression vector pGWB5- BpSPL2 -GFP and the repressor expression vector pROKII- BpSPL2 -The method for constructing SRDX and converting birch to obtain pGWB5- BpSPL2 -GFP transgenic birch plants and pROKII- BpSPL2 -The method for producing SRDX transgenic birch plants is described in patent document CN 111690664 A).

[0028] 2. Cultivation of transgenic birch: (1) Transgenic birch was subcultured using WPM rooting medium (the growth conditions were a temperature of about 24±2℃, a relative humidity of 65-75%, and a light intensity and photoperiod of 46 μmol / m). -2 s -1 (and 16 h light / 8 h dark), the culture medium system is as follows: WPM powder 2.14 g / L, sucrose 20 g / L, calcium salt 0.56 g / L, IBA 0.4 mg / L, plant agar 8 g / L, pH adjusted to 5.8 with 5 M NaOH, dispensed into 100 mL / bottle.

[0029] (2) Wild-type birch, 35S::BpSPL2 -3、 35S::BpSPL2 -5、 35S::BpSPL2 -SRDX-1 35S:: BpSPL2 -SRDX-4 (see patent document CN 111690664 A) transgenic birch tissue culture seedlings. Cut stem segments with apical buds, about 3 cm long, and insert them into WPM rooting medium and culture for 40 days (consistent with subculture conditions).

[0030] (3) Transplant the transgenic seedlings that have grown for 40 days under normal culture conditions (subculture conditions) into a soil substrate of vermiculite: potting soil: peat moss = 1:1:1 (Note: the culture medium on the roots of the tissue culture seedlings must be rinsed clean when transplanting into the soil substrate) and culture them indoors until the seedlings are stable (they can survive and grow normally after being transplanted from the culture medium into the soil substrate).

[0031] 3. The inflorescences used for transgenic verification were collected on September 20, 2025, from four-year-old plants in Fuyu Forest Farm, Fuyu County, Qiqihar City, Heilongjiang Province, China (47°47′N, 124°28′E). 35S::BpSPL2 , 35S::BpSPL2 -SRDX transgenic birch and wild-type birch. The collected samples were preserved in formalin-acetic acid-alcohol fixative (FAA) for morphological recording of inflorescences and anthers and structural analysis of anther anatomy.

[0032] 4. Inflorescence morphology record: for four-year-old plants 35S::BpSPL2 , 35S::BpSPL2 - Color records, length and diameter measurements were taken of inflorescences of SRDX transgenic birch and wild-type birch, with at least 27 inflorescences measured for each genotype. Data are expressed as mean ± standard deviation.

[0033] 5. Anther morphology recording: Using a stereomicroscope, the anther morphology of four-year-old anthers was recorded. 35S::BpSPL2 , 35S::BpSPL2 - The inflorescences of SRDX transgenic birch and wild-type birch were dissected. After the complete anthers were dissected, the color was recorded and the length and width were measured. At least 30 anthers were measured for each genotype. The data are expressed as mean ± standard deviation.

[0034] 6. Anther anatomical structure analysis: BpSPL2 The inflorescences of transgenic and wild-type plants were fixed in FAA, embedded in paraffin, and then 10 μm thick transverse sections were prepared. After toluidine blue staining, the structure of the connective tissue, tapetum, microsporocytes, tetrads, and mature pollen grains was observed under an optical microscope.

[0035] This invention utilizes four-year-old children. BpSPL2 Inflorescences of transgenic birch with overexpression and repression vectors, as well as wild-type birch, were collected. Morphological records of the inflorescences and anthers were performed, and structural analysis of the anthers was conducted to study their structure. BpSPL2 The function of genes in regulating pollen development. Phenotypic observation revealed that wild-type (WT) and BpSPL2 Overexpression ( 35S::BpSPL2-3、35S::BpSPL2-5 The inflorescences are similar in size and color intensity, while BpSPL2 Inhibit inflorescence ( 35S:: BpSPL2-SRDX-1、35S::BpSPL2-SRDX-4 The flowers exhibited brown pigmentation and enlargement, and similar results were obtained from the observation and statistical analysis of the anthers obtained from the dissection of the inflorescence (e.g.) Figure 1 , 2 Pollen development was observed through cross-sectional views of the anthers. BpSPL2 The anthers of the overexpressing plants only developed to the microsporocyte stage and had not yet undergone meiosis, while the wild-type plants had already undergone meiosis and developed to the tetrad stage; conversely, BpSPL2The inhibited plants undergo meiosis earlier than the wild type, having already passed through the tetrad stage in preparation for overwintering (e.g. Figure 3 Based on the above results, it can be concluded that... BpSPL2 Overexpression promotes pollen sterility.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Birch BpSPL2 Application of genes in promoting pollen abortion and reducing pollen allergenicity.

2. The application according to claim 1, characterized in that, The method of promoting pollen abortion and reducing pollen allergenicity is achieved by increasing the concentration of pollen in birch. BpSPL2 This is achieved through gene expression.

3. The application according to claim 2, characterized in that, The improvement BpSPL2 Gene expression includes... BpSPL2 Gene overexpression vectors were transformed into birch.

4. The application according to claim 3, characterized in that, The overexpression vector is pGWB5- BpSPL2 -GFP.

5. A method for promoting pollen abortion and reducing pollen allergenicity in birch, characterized in that, Including improving birch trees BpSPL2 Gene expression.

6. The method according to claim 5, characterized in that, The improvement BpSPL2 Gene expression includes... BpSPL2 Gene overexpression vectors were transferred into birch.

7. A recombinant expression vector, characterized in that, Contains birch BpSPL2 The gene was used to overexpress the gene in birch.

8. A transgenic birch cell, characterized in that, Contains the recombinant expression vector as described in claim 7.

9. A method for cultivating birch plants with low pollen abortion allergenicity, characterized in that, This includes transferring the recombinant expression vector described in claim 7 into birch, and then screening to obtain... BpSPL2 Plants with overexpressed genes.

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