Nitrate ion transport channel gene PbrSLAH3b and application thereof in promoting growth of pear pollen tube

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly discloses a nitrate ion transport channel gene PbrSLAH3b and application of the nitrate ion transport channel gene PbrSLAH3b in promoting growth of pear pollen tubules. The method comprises the following steps: extracting total RNA (Ribonucleic Acid) of Dangshan pears from pollen of Dangshan pears; carrying out reverse transcription on the RNA to obtain cDNA (complementary deoxyribonucleic acid); and by taking the cDNA as a template, carrying out amplification to obtain the pear pollen tube nitrate transport channel gene PbrSLAH3b, the PbrSLAH3b gene provided by the invention belongs to a slow anion channel SLAC / SLAH family member, the PbrSLAH3b is remarkably up-regulated in expression in the early growth stage of a pollen tube, and the PbrSLAH3b is positioned in a plasma membrane. The invention verifies that PbrSLAH3b can form a functional anion channel with NO3 <-> selectivity under the condition of not depending on proton gradient or phosphorylation state, and the PbrSLAH3b has relatively strong selectivity on NO3 <->. Therefore, the PbrSLAH3b gene can mediate the absorption and transport of the pollen tube to NO3 <-> and maintain the polarity growth directivity of the pollen tube, and has important application value for revealing the ion regulation mechanism of the growth of the fruit tree pollen tube and improving the reproductive development of the fruit tree.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for participating in the production of nitrate ions (NO3). - The transmembrane transport anion channel gene PbrSLAH3b and its application in regulating pollen tube growth and polarity development in pear flowers. Background Technology

[0002] Polar growth of pollen tubes is a crucial step in the sexual reproduction process of angiosperms. It determines whether pollen can successfully reach the ovule after germination from the stigma to complete fertilization, thus directly affecting the plant's reproductive efficiency and fruit formation (Cheungand Wu, 2008. Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annual Review of Plant Biology 59, 547–572.). Directional growth of pollen tubes depends on the synergistic action of various ion channels on the plasma membrane. Among them, the transmembrane transport of anions (Cl⁻, NO⁻, etc.) plays a vital role in regulating pollen tube cell membrane potential, cell turgor pressure, and ion homeostasis (Michard et al., 2009. Ion dynamics and Ca²⁺ signaling in pollen tubes. Plant Physiology 150, 1732–1744.). Previous studies have shown that members of the slow anion channel (SLAC / SLAH) family are widely involved in physiological processes such as stomatal movement, stress response, and ion transport in plants (Hedrich, 2012. Ion channels in plants. Physiological Reviews 92, 1777–1811.).

[0003] SLAC1 (Slow Anion Channel-Associated 1) was first identified in Arabidopsis as an anion channel that regulates stomatal opening and closing. Its homologous gene SLAH (SLAC1 Homologs) family members include SLAH1–SLAH4, which have different expression characteristics and functions in tissues such as roots, leaves and pollen (Negi et al., 2008. CO2regulator SLAC1 and its homologs in plant anion transport. Nature 452, 483–486.). SLAH3 has been shown to form NO3⁻-selective anion channels in Arabidopsis, interacting with calcium-dependent protein kinases (CPKs) or the CIPK / CBL complex to regulate their phosphorylation state and channel activity (Geiger et al., 2011. Guard cell anion channel SLAC1 is regulated by CDPK proteinkinases with distinct Ca²⁺ affinities. Proceedings of the National Academy of Sciences USA 108, 8028–8033.). Furthermore, SLAH3 has also been found to be closely related to Ca²⁺ dynamics and plasma membrane polarity maintenance in pollen tubes (Gutermuth et al., 2013. Pollen tube growth regulation by the CPK17 and CPK34 protein kinases in Arabidopsis. Plant Cell 25, 646–661.).

[0004] However, no research has been reported on the SLAH3 gene in the pollen of woody fruit trees, especially pear. As an important economic fruit tree, the polar growth of pollen tubes in pear is crucial for pollen fertilization and fruit development. Abnormal pollen tube growth directly leads to pollination and fertilization failure and a decrease in fruit set, thus affecting yield and quality (Deng Xiuxin et al., 2018. A Review of a Century of Development in Pomology. Journal of Agronomy 8, 24–34.; Wang Wenhui et al., 2019. 70 Years of Pomology Research in New China—Pear. Journal of Pomology 36, 1273–1282.). Currently, the compositional characteristics, ion selectivity, and functional mechanisms of anion channels in pear pollen tubes during polar growth remain unclear. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a nitrate ion transport channel gene, PbrSLAH3b, which clarifies the role of NO3 in pear pollen tubes. - Transmembrane transport and its regulatory mechanism on pollen tube polar growth are crucial for elucidating the NO3-dependent processes in pear pollen tubes. - This invention provides a theoretical basis for the molecular mechanisms of absorption and signal transduction, and also offers new gene resources for improving pollen viability, promoting pollination and fertilization, and increasing fruit set rate in fruit trees. It has significant application value for high-quality and stable pear yield and molecular breeding. Furthermore, it provides a new molecular basis for elucidating the ion regulation mechanism of pollen tube growth in woody fruit trees, and also provides important theoretical support for improving the reproductive development characteristics of fruit trees and increasing fruit set rate. Another objective of this invention is to provide the application of the channel gene PbrSLAH3b in promoting pollen tube growth and regulating polarity development in pear.

[0006] This invention is achieved through the following technical solution: A type of NO3 - The transport pathway gene PbrSLAH3b is a DNA molecule having the nucleotide sequence shown in SEQ ID No. 1, or having more than 90% homology with the nucleotide sequence shown in SEQ ID No. 1 and encoding NO3- that regulates pear pollen tubes. - DNA molecules that undergo transport and polar growth.

[0007] Furthermore, a type of NO3 mentioned above - The protein encoded by the transport channel gene PbrSLAH3b.

[0008] Furthermore, the protein is a protein having the amino acid sequence shown in SEQ ID No. 2, or a protein having the amino acid sequence shown in SEQ ID No. 2 modified by substitution, deletion, or insertion of one or more amino acid residues while retaining NO3. - The channel function is a protein derived from the amino acid sequence shown in SEQ ID No. 2.

[0009] Furthermore, containing the aforementioned NO3 - Recombinant expression vectors, expression cassettes, transgenic cell lines, or recombinant bacteria for the transport pathway gene PbrSLAH3b.

[0010] Furthermore, it is used to amplify the NO3 - Primer pairs for the full length or any fragment of the transport channel gene PbrSLAH3b, wherein the forward and reverse primers of the primer pairs have nucleotide sequences as shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0011] A further improvement to the present invention is as follows: The NO3- The transport pathway gene PbrSLAH3b or biological material associated with the PbrSLAH3b gene is used in at least one of the following (1)-(8): (1) Application in promoting the growth of pollen tubes in pear flowers; (2) Application in the preparation of products that promote the growth of pear pollen tubes; (3) Application in regulating NO3⁻ absorption, translocation and polar growth in pear pollen tubes; (4) Application in the preparation of products that regulate the absorption, transport and polar growth of NO3⁻ in pear pollen tubes; (5) Application in improving the germination rate of pear pollen; (6) Application in the preparation of products that improve the germination rate of pear pollen; (7) Application in enhancing the fruit set rate of pear trees; (8) Application in the preparation of products that enhance the fruit setting rate of pear trees; Furthermore, the biomaterial associated with the PbrSLAH3b gene is at least one of the following (1)-(5): (1) The protein encoded by the PbrSLAH3b gene; (2) An expression cassette containing the PbrSLAH3b gene; (3) A recombinant vector containing the PbrSLAH3b gene, or a recombinant vector containing the expression cassette described in (2); (4) Recombinant bacteria containing the PbrSLAH3b gene, or recombinant bacteria containing the expression cassette of (2), or recombinant bacteria containing the recombinant vector of (3); (5) A transgenic cell line containing the PbrSLAH3b gene, or a transgenic cell line containing the expression cassette described in (2), or a transgenic cell line containing the recombinant vector described in (3); Furthermore, the PbrSLAH3b gene was stably overexpressed in pear pollen tubes to promote pollen tube growth, increase pollen germination rate, or enhance reproductive development efficiency of fruit trees.

[0012] A further improvement of the present invention is as follows: A method for promoting polar growth of pear pollen tubes and improving pear pollen germination rate or fruit setting efficiency involves stably overexpressing the PbrSLAH3b gene in pear pollen tubes to promote polar growth of pear pollen tubes and improve pear pollen germination rate or fruit setting efficiency.

[0013] Furthermore, this includes the following steps: (1) The PbrSLAH3b gene was ligated to the expression vector to obtain a recombinant vector; (2) The recombinant vector was transferred into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; (3) Using the recombinant Agrobacterium tumefaciens to infect target plant tissues or pollen, transgenic plants or transient expression systems are screened.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes plant gene cloning technology to clone the functional NO3⁻ transporter gene PbrSLAH3b from the pollen tube of 'Dangshan Crisp Pear'. PbrSLAH3b is located in the plasma membrane and possesses NO3⁻ channel activity, positively regulating pollen tube polar growth. The PbrSLAH3b gene, its encoded protein, or related recombinant materials can be used to enhance the responsiveness of pollen tubes to NO3⁻, maintain intracellular anion homeostasis, and promote pollen tube polar growth, thereby improving the pollination and fertilization efficiency and fruit set rate of pears, demonstrating significant potential for industrial application. Attached Figure Description

[0015] Figure 1 This invention relates to the NO3⁻ transport channel gene for pear pollen tubes. PbrSLAH3b Amplification electrophoresis image; Figure 2 This invention relates to the NO3⁻ transport channel gene for pear pollen tubes. PbrSLAH3b A schematic diagram of the cloning, positioning, and functional verification process; Figure 3 The construction process and structure diagram of the recombinant expression vector are shown below: A is the construction process of the recombinant expression vector, B is the structure diagram of the BS-35S-GFP vector, and C is the structure diagram of the pT7TS vector. Figure 4 The NO3⁻ transport channel gene for pear pollen tubes in Example 2 of this invention PbrSLAH3b Relative expression levels at different time points during pollen tube growth; Figure 5 The NO3⁻ transport channel gene for pear pollen tubes in Example 3 of this invention PbrSLAH3b Subcellular localization map; Figure 6 Electrophysiological characteristics of the pear pollen tube NO3⁻ transport channel gene PbrSLAH3b heterologously expressed in Example 4; A and B. Typical whole oocyte recordings of PbrSLAH3b anion current in oocytes injected with control (deionized water) and PbrSLAH3b cRNA using standard nitrate-based bath solution; C. Instantaneous average amplitude of PbrSLAH3b anion current at -100 mV.

[0016] Figure 7A. Ion selective absorption characteristics of the pear pollen tube NO3⁻ transport channel gene PbrSLAH3b heterologously expressed in Example 4; A. At 50 mM NO3⁻ − Cl − SO4 2− and malic acid 2− A. Typical whole oocyte anionic currents recorded in the bath solution. B. Average voltage-current curves of whole oocyte currents.

[0017] Figure 8 To inhibit the antisense oligonucleotide (ODN) technology in Example 5 PbrSLAH3b Pollen tube growth characteristics were analyzed using expression analysis. A. Control; B. Pollen tubes treated with transfection reagent showed normal growth; C. S-ODN served as a control, showing normal pollen tube growth; D. AS-ODN treatment inhibited pollen tube growth; E. Pollen tube length statistics; F. qRT-PCR detection of antisense oligonucleotide silencing. PbrSLAH3b Gene expression levels. Detailed Implementation

[0018] The following description, in conjunction with the accompanying drawings and specific embodiments, illustrates the pear pollen tube nitrate ion transport channel gene provided by this invention. PbrSLAH3b The application of pollen tubes in regulating their growth capacity is described in detail, but it should not be construed as limiting the scope of protection of this invention.

[0019] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all obtained through commercial purchase.

[0020] In this invention, the biomaterials used to verify the function of PbrSLAH3b or its encoded protein include tobacco ( Nicotiana benthamiana ), pear pollen in vitro culture system and Xenopus laevis oocytes ( Xenopus laevis oocytes).

[0021] When the biological material is tobacco, the experimental procedure includes the following steps: (1) Amplification of the pear pollen tube nitrate transport channel gene PbrSLAH3b ; (2) The above PbrSLAH3b The gene was ligated into the plant expression vector BS-35S-GFP to obtain the recombinant vector PbrSLAH3b-BS-35S-GFP; (3) The recombinant vector was transferred into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium tumefaciens; (4) Tobacco leaves were infected using Agrobacterium-mediated transient transformation to obtain transient expression samples of the PbrSLAH3b gene in tobacco leaves.

[0022] Laser confocal microscopy revealed that the PbrSLAH3b-GFP signal was mainly distributed in the plasma membrane, indicating that this protein is a typical membrane-localized anion channel protein.

[0023] When the biological material is pear pollen tubes, the experimental procedure includes the following steps: (1) Design and synthesis PbrSLAH3b Gene ODN sequence primers, which are then thiolated; (2) PbrSLAH3b-AS-ODN was mixed with cell transfection reagent (15 μg / mL) and liquid culture medium in a certain ratio (4:1:95) and incubated at room temperature for 15 min, and then pollen was treated. Microscopic observation revealed that PbrSLAH3b-ODN interference treatment significantly inhibited pollen tube growth, indicating that PbrSLAH3b has the function of promoting polar growth during pollen tube growth.

[0024] When the biological material is Xenopus laevis oocyte, it is obtained PbrSLAH3b The process of heterologous gene expression in Xenopus laevis oocytes includes the following steps: (1) Amplification of the pear pollen tube nitrate transport channel gene PbrSLAH3b The full-length sequence; (2) The above PbrSLAH3b The gene was ligated to the expression vector pT7TS to obtain the recombinant vector PbrSLAH3b-pT7TS; (3) The recombinant plasmid was transcribed in vitro using an in vitro transcription kit to synthesize PbrSLAH3b cRNA; (4) The synthesized cRNA was injected into Xenopus oocytes by microinjection to obtain the PbrSLAH3b heterologous expression system.

[0025] In the heterologous expression system, channel current was measured using the two-electrode voltage-clamp (TEVC) technique. The results showed that PbrSLAH3b generated a slow anion current on the oocyte membrane with NO3⁻ selectivity and membrane potential dependence, and the channel activity was independent of exogenous proton concentration and phosphorylation state, indicating that PbrSLAH3b is a functional anion channel protein with nitrate-specific conduction capability.

[0026] The cloning, localization, and functional verification process of the pear pollen tube nitrate transport channel gene PbrSLAH3b in this invention is as follows: Figure 2 As shown, the experimental steps in the following embodiments will be carried out using this process.

[0027] Example 1: Pear PbrSLAH3bCloning of full-length cDNA A gene regulating nitrate transport in pear pollen tubes was identified by screening a pear whole-genome cDNA library. PbrSLAH3b Cloning was performed based on the characteristics of homologous recombination expression vectors and PbrSLAH3b The gene sequence was determined, primers were designed using Primer 5.0, and full-length PCR amplification was performed using "Dangshan Crisp Pear" pollen cDNA as a template. Detailed steps are as follows: The research material used was pollen from the 'Dangshan Crisp Pear' planted at the Nanjing Agricultural University Pear Orchard Experimental Base. Pollen was collected at the peak of pollen germination and immediately flash-frozen in liquid nitrogen at -80℃ for later use. Approximately 200 mg of sample was randomly weighed and immediately flash-frozen in liquid nitrogen for total RNA extraction. RNA was extracted using the CTAB method. Before the experiment, RNA-free blue, yellow, and white pipette tips and 1.5 mL centrifuge tubes were prepared. The mortar, pestle, and small key were sterilized with alcohol at high temperature beforehand and then flash-frozen in liquid nitrogen after cooling. The CTAB extraction buffer consisted of: 2% CTAB (w / v, g / 100 mL), 2% PVP K-30 (w / v, g / 100 mL), 10 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, and 0.5 g / L spermidine.

[0028] After extraction, the quality of the extracted RNA was detected by 1% agarose gel electrophoresis, and the concentration and quality of the RNA were detected by NanoDrop 2000 spectrophotometer.

[0029] After obtaining pollen RNA from Dangshan pear, cDNA was obtained by reverse transcription; the synthesis of the first strand of cDNA was performed according to the operating manual of ThermoScientific RevertAid First Strand cDNA Synthesis Kit.

[0030] The obtained first-strand cDNA was used for PbrSLAH3b Gene amplification was performed using forward primer F1 and reverse primer R1 to obtain the pear pollen tube nitrate transport channel gene. PbrSLAH3b The full-length sequence.

[0031] The amplification primer sequences are as follows: F1: 5'-ATGATGGAGAACAGCAAAAATCTG-3' (SEQ ID No. 3) R1: 5'-TAGCTTGGCATCTTCTAAAATCTTT-3' (SEQ ID No. 4) The amplification system includes: Template cDNA approximately 100 ng; I-5™ 2× High-Fidelity Master Mix (Molecular Cloning Laboratories, USA); 10.0 μM forward primer F1; 10.0 μM reverse primer R1; Add RNase-free water to bring the total volume to 50 μL.

[0032] PCR was performed according to the following procedure: pre-denaturation at 98°C for 2 min; 35 amplification cycles, including denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 3 min, extension at 72°C for 5 min after each cycle, followed by incubation at 15°C.

[0033] After amplification, PCR products with a single target band were detected by 1% agarose gel electrophoresis (e.g., Figure 1 As shown in the figure, the specific target band was recovered by following the extraction steps in the instructions of the gel recovery kit (purchased from Kangwei Century, China).

[0034] After recovery, purification, and sequencing verification, the pear pollen tube nitrate transport channel gene was obtained. PbrSLAH3b The nucleotide sequence of the gene is shown in SEQ ID No. 1, containing an open reading frame of 1824 bp.

[0035] SEQ ID No.1 The present invention also provides a protein encoded by the pear pollen tube nitrate transport channel gene PbrSLAH3b, the amino acid sequence of which is shown in SEQ ID No. 2: MMENSKNLDSGNQNSPELPSLIKYISSNEVVGFDNVEESIFVQSSDHQPISPAEGIEIAALESVVDESELPHHQRVYSISISMPPSPMEIHLQNSKRMLFGGETSSNNEIPNSSSPTEISGSKQPKAVKFHSQPMPKRAAIEQATINGHFS YHPNIERLKDKRFDSFKTWSGKLERQMTLLRGKTPRETETENDNAQNAEVERLPADRYFDALEGPELDTLKASEKILLPEDKQWPFLLRYPVSAFSICLGVSSQAILWKTIPTSASTKYLHLNLIPNLVLWCISVALLVVVACIYLLKVTLY FEAVRREYYHPIRVNFFFAPWIALLFLALGVPPSFANKLHPALWYILMTPILCLELKIYGQWMSGGQRRLSKVANPVNHISIVGNFVGALLGASMGLKEGPIFFFGVGMAHYLVLFVTLYQRLPTNETVIPKDLHPVFFLFVAAPSVASMAW ARIQGSFGCGSRILYFIALFLYLSLAVRVNFFRGFKFSLAWWAYTFPMTGAAIATIRYSNEVTNAVTQAMAVILSVVATIIVTILLIVTVLHAFVLQDLFPNDIAIAISDRKPKAQRKWFHLRHGSSDSRDIDKFLKSATSLENKDLEDAKL The protein encoded by PbrSLAH3b comprises approximately 607 amino acid residues. This protein is located in the cell membrane, belongs to the membrane protein family, and exhibits typical transmembrane domain characteristics of the SLAH family. This protein can form anion channels selective for nitrate ions (NO3⁻) and mediate transmembrane transport of nitrate ions in the pollen tube, thereby regulating the polar growth and elongation development of the pollen tube.

[0036] The present invention provides PbrSLAH3b Genes or their encoded proteins can be used to regulate the absorption, transport, and polar growth of nitrate ions during pollen tube growth in plants. This regulation can be either promoting or inhibiting; when applied to promote nitrate absorption or pollen tube elongation, it can be achieved through heterologous expression or transgenic technology. PbrSLAH3bGene function verification; when applied to repression, gene expression can be inhibited through RNA interference or ODN oligonucleotide technology. The aforementioned heterologous expression, transgenic, and gene silencing can all be achieved using molecular biology methods commonly used in this field.

[0037] This invention obtains PbrSLAH3b After gene expression, the gene was ligated into expression vectors BS-35S-GFP and pT7TS, respectively, to obtain two recombinant vectors. Figure 3 (A, 3B). In the specific implementation of this invention, the homologous recombination ligation kit from Novizan is preferably used for the ligation reaction, and the operation steps are performed in accordance with the instruction manual.

[0038] Example 2: Pollen tube growth process of Dangshan crisp pear PbrSLAH3b qRT-PCR analysis of genes To analyze the nitrate ion transport channel genes in Dangshan pear PbrSLAH3b The expression patterns during pollen germination and pollen tube growth were investigated using real-time quantitative PCR (qRT-PCR) to analyze pollen tube expression patterns at different time points (0h, 1h, 3h, 5h, 7h, and 9h after pollen germination). PbrSLAH3b The transcriptional level of the gene was detected.

[0039] according to PbrSLAH3b The coding region sequence of the gene was used to design upstream and downstream PCR primers for amplifying the gene by 200-300 bp using Primer 5.0 software, following general primer design principles. RNA was extracted using a kit, and the synthesis of the first strand of cDNA was performed according to the operating manual of the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit.

[0040] The 20 µL reaction system consisted of: 10 µL SYBR Green, 5 µL sterile ultrapure water, 1 µL cDNA, 2 µL forward primer F2, and 2 µL reverse primer R2.

[0041] F2: 5'-CAGTCCAGCTGAAGGAATTGAGA-3' (SEQ ID No.5) R2: 5'-CTTTTTGGCATTGGCTGGGA-3' (SEQ ID No. 6) Using UBQ as an intrinsic parameter, the sequence is as follows: UBQ-F3: 5'- CCCTTCACTTGGTTCTCCGT-3' (SEQ ID No. 7) UBQ-R3: 5'-TAATCAGCAAGCGTGCGACC-3' (SEQ ID No. 8) The qRT-PCR program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 3 s, 60℃ annealing for 10 s, 72℃ extension for 30 s, 45 cycles; 72℃ extension for 3 min, 20℃ incubation for 30 s.

[0042] Pollen from Dangshan pears was immediately inoculated into a basic pollen culture medium under aseptic conditions after collection (the formula of the pollen liquid culture medium (100 ml): 10 g sucrose, 20 g polyethylene glycol 4000 (PEG4000), 0.01 g boric acid (H3BO3), 0.03 g calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), 30 mM 2-(N-morpholino)ethanesulfonic acid (MES), and the pH was adjusted to 6.2 using tris(hydroxymethyl)aminomethane (Tris)). The medium was then cultured at a constant temperature of 25℃. PbrSLAH3b The relative expression levels of genes showed different expression levels at different time points during pollen tube growth, such as... Figure 4 As shown, the results indicate that... PbrSLAH3b The gene expression level is highest 5 hours after pollen germination.

[0043] Example 3: Subcellular localization of PbrSLAH3b protein, a nitrate ion channel in pear pollen tubes according to PbrSLAH3b Nucleotide sequences and expression vector maps of genes ( Figure 3 Xba I and BamHI restriction sites are added before and after the gene sequence, respectively, to facilitate ligation with the expression vector. The restriction site sequences are shown below: Xba I: TCTAGA (SEQ ID No.9) BamH I: GGATCC (SEQ ID No. 10) Using cDNA from Dangshan pear pollen as a template, amplification was performed using specific primers containing the aforementioned restriction enzyme sites. The primer sequences are as follows: F1: 5'-ATGATGGAGAACAGCAAAAATCTG-3' (SEQ ID No. 3) R1: 5'-TAGCTTGGCATCTTCTAAAATCTTT-3' (SEQ ID No. 4) The stop codon TGA was removed from the 3' end of the gene to allow for gene fusion with GFP. After PCR product electrophoresis on a 1% agarose gel, the target band was recovered using a gel electrophoresis kit. The purified amplified fragment was recombined with an expression vector and transformed into *E. coli* competent cells DH5α using a heat shock method. The transformed bacterial culture was detected by PCR, and the PCR-positive cultures were sequenced to obtain the correct recombinant target vector, named PbrSLAH3b-BS-35S-GFP. The recombinant vector PbrSLAH3b-BS-35S-GFP was introduced into *Agrobacterium* GV3101 using a freeze-thaw method (referring to Sambrook, *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, 2002).

[0044] The induction solution was prepared using the instantaneous conversion method of tobacco leaves, as shown in Table 1 below: Table 1. Preparation method of induction solution for instantaneous conversion of tobacco leaves reagents Mother liquor Required amount / 10 mL <![CDATA[MgCl2]]> 200 mM 500 μL MES (pH 6.2) 200 mM 500 μL <![CDATA[ddH2O]]> - 9 mL Acetosyringone 200 μM 20 μL The specific operating method is as follows: (1) Take out the Agrobacterium tumefaciens solution stored in the -80℃ refrigerator, streak it on the LB solid medium with the corresponding resistance (usually kanamycin and rifampin), and place it upside down in a 30℃ incubator to activate the bacterial solution.

[0045] (2) Once single clones have grown, expand them in LB liquid medium containing kanamycin and rifampicin antibiotics, and culture overnight at 28-30°C on a shaker at 220-240 rpm.

[0046] (3) Collect bacterial culture. Generally, two collections are sufficient using a 10 mL centrifuge tube. The amount of bacteria at the bottom of the tube may vary. More can be collected.

[0047] (4) Discard the supernatant, drain the liquid, and add 2 mL of induction solution.

[0048] (5) Vortex mixing, and induce shaking on a shaker for more than 3 hours.

[0049] (6) Infect tobacco by injection, inject from the back of the tobacco plant; observe the fluorescence 2-3 days later using a laser confocal scanning microscope. Before observation, use a vacuum pump or syringe to evacuate the leaves for clearer observation.

[0050] The plasmids PbrSLAH3b-BS-35S-GFP and the control empty vector BS-35S-GFP were injected into tobacco epidermal cells, respectively. The localization of the PbrSLAH3b protein was determined by detecting the GFP fluorescence location in the leaf epidermal cells. The results are as follows: Figure 5As shown, 35S-GFP represents the localization of the control empty vector, with fluorescence covering the cell membrane, cytoplasm, and nucleus; PbrSLAH3b-GFP represents the transient expression of PbrSLAH3b-BS-35S-GFP in tobacco epidermal cells, with green fluorescence distributed on the cell membrane, and no fluorescence detected elsewhere. These results indicate that PbrSLAH3b protein is localized to the cell membrane and is a membrane-localized protein.

[0051] Example 4: Study on the absorption characteristics of nitrate ions by heterologous expression of PbrSLAH3b 1. In vitro synthesis of cRNA (1) Extraction of high-purity plasmids Using a high-purity plasmid extraction kit (Vigrass), plasmids containing... PbrSLAH3b DNA from the gene expression vector pT7TS was extracted from E. coli to obtain the plasmid of the recombinant vector PbrSLAH3b-pT7TS. Refer to the kit instructions for specific procedures.

[0052] (2) Linearization of plasmids Take 6–7 μg of plasmid, add 4 µL of restriction endonuclease Xba I and 2 µL of 10× buffer, and adjust the volume to 50 µL with ddH2O. Digest at 37°C for 5 h. Then, use 1% agarose gel electrophoresis to check whether linearization is complete.

[0053] (3) Purification of linearized plasmids Add 20 µL of 1% SDS (g / 100 mL) and 0.4 µL of proteinase K (20 mg / mL) to the linearized plasmid, then incubate at 50 °C for 30 min. Make up the volume to 100 µL with RNase-free water, then add 100 µL of phenol / chloroform solution. Gently mix and centrifuge at room temperature for 5 min at 13000 rpm. Transfer the supernatant to a new RNase-free centrifuge tube, add 10 µL of sodium acetate (3M) solution and 200 µL of 95% ethanol, and freeze at -20 °C for 30 min. Then centrifuge at 4 °C and 13000 rpm for 30 min, discard the supernatant, wash the precipitated linearized plasmid twice with 70% ethanol, air dry the ethanol in the centrifuge tube, and dissolve it in RNase-free water to achieve a final concentration of approximately 0.5 µg / µL.

[0054] (4) In vitro synthesis of cRNA Add the linearized plasmid and reaction solution according to the system shown in Table 2 below: Table 2 Reaction System

[0055] Next, add 115 µL of RNase-free water, 15 µL of sodium acetate (3M), and 150 µL of phenol / chloroform solution. Vortex to mix, centrifuge at room temperature for 3 min at 13000 rpm. Transfer the supernatant to a new RNase-free centrifuge tube, being careful not to aspirate the lower layer. Add an equal volume of isopropanol to the supernatant, precipitate at -20°C for 30 min, then centrifuge for another 30 min at 4°C and 13000 rpm. Aspirate the supernatant, retain the precipitate, and elute twice with 70% ethanol. Dissolve the cRNA in RNase-free water to a final concentration of 1 µg / µL, and assess the cRNA quality using a freshly prepared 1% agarose gel electrophoresis. Finally, aliquot the cRNA and store at -80°C for later use.

[0056] 2. Isolation and digestion of Xenopus oocytes Select a mature, robust African clawed frog with a darker color and a robust body. Bury it in ice for 1 hour to induce dormancy, then dissect it to retrieve the eggs. Place the oocytes in Ca... 2+ The oocytes were washed 5-6 times with a 1 mg / mL collagenase (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4) solution. Then, 1 mg / mL collagenase (Roche) was added, and the cells were digested at 20-24°C for 1-2 hours until most oocytes were single-celled. The oocytes were then washed 6-8 times with ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.4) containing 1 mg / mL gentamicin. Oocytes of uniform size and with smooth surfaces were selected under a microscope using a pipette for later use.

[0057] 3. Microinjection First, use a microelectrode drawing instrument to draw a glass capillary tube to a tip diameter of 20 μm. Inject mineral oil into the capillary tube, ensuring no air bubbles are generated. Then, attach the microinjection tube to the microinjector (PLI-100, made in the USA). Draw 1 μL of RNase-free water and adjust the injection time to inject 19-21 times with 1 μL of RNase-free water, repeating the calibration 2-3 times. Because cRNA is more viscous, the number of injections for the same 1 μL of cRNA will be 1-2 fewer than for 1 μL of water. Injection should be performed in the white area of ​​the oocyte, using Xenopus oocytes injected with water as a control.

[0058] 4. Culture of Xenopus oocytes Xenopus oocytes injected with cRNA and water were cultured in ND96 containing 100 mg / L streptomycin and 60 mg / L penicillin, and then aliquoted into 48-well plates with 2-3 oocytes per well. The plates were then incubated at 18°C ​​for 2-3 days, with the ND96 solution changed twice daily, and any damaged Xenopus oocytes removed promptly.

[0059] 5. Voltage clamp recording and data analysis After expression, oocyte currents were recorded using a two-electrode voltage-clamp technique, with an Axoclamp 900A (Axon Instruments, Foster City, CA, USA) voltage-clamp amplifier. First, 3 mol / L KCl was injected into both electrode glass tubes to remove air bubbles from the tips. The tubes were then attached to the two probes of the voltage and current clamps, respectively. The cells were placed in the recording chambers, and the voltage and current electrodes were inserted into the cells. Oocyte condition was assessed by reading the voltage electrodes. A clamp value above -39.6 mV indicated good cell condition, allowing for pulse activation and current recording. The standard solution contained 5 mM Tris / MES (pH 7.5), 1 mM Ca(gluconate)₂, 1 mM Mg(gluconate)₂, 100 mM NaNO₃, and 1 mM LaCl₃. The osmotic pressure of the buffer solution was adjusted to 220 mos-mol / kg using sorbitol. Finally, the data acquisition and analysis were performed using pClampfit 10.3 and Sigmaplot 12.5 software, respectively.

[0060] The electrophysiological properties of the PbrSLAH3b channel were detected by injecting PbrSLAH3b-cRNA and ddH2O into Xenopus laevis oocytes. Results are as follows: Figure 6 As shown, PbrSLAH3b possesses typical anion channel functions, and its channel activity exhibits stable characteristics independent of proton or phosphorylation states. Figure 6 A and 6B. Typical whole-oocyte recordings of PbrSLAH3b anion currents in oocytes injected with control (deionized water) and PbrSLAH3b-cRNA using standard nitrate-based bath solution. C. Instantaneous average amplitude of PbrSLAH3b anion current at -100 mV.

[0061] The ion selectivity of the PbrSLAH3b channel was investigated to further understand its functional specificity. Electrophysiological measurements were performed after perfusing Xenopus laevis oocytes with different anion solutions (NO3⁻, Cl⁻, SO4²⁻, and malate). The results are as follows: Figure 7As shown, PbrSLAH3b exhibits high selectivity for nitrate and tends to preferentially mediate NO3⁻ transmembrane transport. Figure 7 A. at 50 mM NO3 − Cl − SO4 2− and malic acid 2− Typical whole oocyte anionic currents recorded in the bath solution. 7B. Average voltage-current curves of whole oocyte currents.

[0062] Example 5: Application of PbrSLAH3 nitrate ion channel in pear pollen tube regulation To determine whether pollen tube growth is related to knockdown PbrSLAH3b Related to gene expression levels, control and knockdown PbrSLAH3b The pollen tubes expressing the gene were cultured normally in a shaker at a temperature of 25-27 ℃ and a rotation speed of 100-120 rpm. After 2 hours of culture, the growth status of the pollen tubes was observed under a microscope and photographed to record the growth status.

[0063] Under normal culture conditions, PbrSLAH3b The lengths of pollen tubes from gene knockdown and control were statistically analyzed, and the results are as follows: Figure 8 As shown. Figure 8A is the control group; Figure 8 B represents pollen tubes treated with transfection reagent, and the pollen tubes grow normally. Figure 8 C represents S-ODN as a control, and pollen tubes grow normally. Figure 8 D represents pollen tubes treated with AS-ODN, which inhibits pollen tube growth; Figure 8 E represents the pollen tube length statistics; Figure 8 F represents the silencing effect of antisense oligonucleotides in qRT-PCR detection. PbrSLAH3b Gene expression levels. Results showed that under normal culture conditions, knocking down the nitrate ion channel in pear pollen tubes... PbrSLAH3b The gene significantly inhibits pollen tube growth. Figure 8 ).

[0064] 1. The steps for transient transformation of pollen tubes mediated by the cell transfection reagent Lipofectamine 2000 are as follows: (1) The synthesized antisense oligonucleotide primers and sense oligonucleotide primers were diluted with distilled water to 1 mm / L. The appropriate volume of pollen was cultured according to the requirements, and observed after hydration for 30-45 min. The transfection system was prepared as follows. After mixing, it was placed in an incubator at 25 ℃ for 15 min. Table 3. Transfection System Scheme Total system (600 μL) Distilled water (μL) Lipofectamine 2000 (μL) Antisense oligonucleotide primers (μL) Positive oligonucleotide primers (μL) Comparison 1 60 - - - Comparison 2 55.5 4.5 - - Comparison 3 37.5 4.5 - 18 Processing group 37.5 4.5 18 - (2) Add 540 μL of pre-cultured pollen culture medium and continue culturing in a shaker at 25 ℃ and 120 rpm for 2 h; (3) Observe and photograph the images using a NiKON ECLIPSE E100 microscope (Figures 8A-8D). (4) Centrifuge at 12000 rpm for 3 min, gently pour out the supernatant, freeze quickly with liquid nitrogen, and store the sample at -80 ℃.

[0065] 2. PbrSLAH3b Gene relative expression level detection Experimental samples were obtained according to the above method. RNA was extracted using a kit (Chengdu Fuji, China). The synthesis of the first strand of cDNA was performed according to the operating manual of the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The 20 µL reaction system included: 10 µL SYBR Green, 5 µL sterile ultrapure water, 1 µL cDNA, 2 µL forward primer, F2: 5'-CAGTCCAGCTGAAGGAATTGAGA-3' (SEQ ID No. 5), and 2 µL reverse primer, R2: 5'-CTTTTTGGCATTGGCTGGGA-3' (SEQ ID No. 6). PbrUBQ The intrinsic parameters are arranged in the following sequence: PbrUBQ -F: 5'-CCCTTCACTTGGTTCTCCGT-3' (SEQ ID No. 7); PbrUBQ -R:5'-TAATCAGCAAGCGTGCGACC-3' (SEQ ID No. 8)).

[0066] The qRT-PCR program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 3 s, 60℃ annealing for 10 s, 72℃ extension for 30 s, 45 cycles; 72℃ extension for 3 min, 40℃ incubation for 30 s. Detection... PbrSLAH3b Relative gene expression levels were found PbrSLAH3b The expression level of decreased significantly (Figure 8E).

[0067] 3. Statistics on the length of pear blossom pollen tubes IPWin32 software is used to count the length of pollen tubes, and GraphPad Prism 9 is used to plot the graph. Figure 8 F).

[0068] The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A type of NO3 - The transport pathway gene PbrSLAH3b is characterized by, The gene is a DNA molecule having the nucleotide sequence shown in SEQ ID No. 1, or having more than 90% homology with the nucleotide sequence shown in SEQ ID No. 1 and encoding NO3, which regulates pear pollen tubes. - DNA molecules that undergo transport and polar growth.

2. A NO3 according to claim 1 - The protein encoded by the transport channel gene PbrSLAH3b.

3. The protein according to claim 2, characterized in that: The protein is a protein having the amino acid sequence shown in SEQ ID No. 2, or a protein having the amino acid sequence shown in SEQ ID No. 2 modified by substitution, deletion, or insertion of one or more amino acid residues while retaining NO3. - The channel function is a protein derived from the amino acid sequence shown in SEQ ID No.

2.

4. Containing the NO3 as described in claim 1 - Recombinant expression vectors, expression cassettes, transgenic cell lines, or recombinant bacteria for the transport pathway gene PbrSLAH3b.

5. For amplifying the NO3 described in claim 1 - Primer pairs for the full length or any fragment of the transport pathway gene PbrSLAH3b, characterized in that... The forward and reverse primers of the primer pair have nucleotide sequences as shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

6. The NO3 as described in claim 1 - The transport pathway gene PbrSLAH3b or biological material associated with the PbrSLAH3b gene is used in at least one of the following (1)-(8): (1) Application in promoting the growth of pollen tubes in pear flowers; (2) Application in the preparation of products that promote the growth of pear pollen tubes; (3) Application in regulating NO3⁻ absorption, translocation and polar growth in pear pollen tubes; (4) Application in the preparation of products that regulate the absorption, transport and polar growth of NO3⁻ in pear pollen tubes; (5) Application in improving the germination rate of pear pollen; (6) Application in the preparation of products that improve the germination rate of pear pollen; (7) Application in enhancing the fruit set rate of pear trees; (8) Application in the preparation of products that enhance the fruit setting rate of pear trees.

7. The application according to claim 6, characterized in that: The biomaterial associated with the PbrSLAH3b gene is at least one of the following (1)-(5): (1) The protein encoded by the PbrSLAH3b gene; (2) An expression cassette containing the PbrSLAH3b gene; (3) A recombinant vector containing the PbrSLAH3b gene, or a recombinant vector containing the expression cassette described in (2); (4) Recombinant bacteria containing the PbrSLAH3b gene, or recombinant bacteria containing the expression cassette of (2), or recombinant bacteria containing the recombinant vector of (3); (5) A transgenic cell line containing the PbrSLAH3b gene, or a transgenic cell line containing the expression cassette described in (2), or a transgenic cell line containing the recombinant vector described in (3).

8. The application according to claim 6, characterized in that: The PbrSLAH3b gene was stably overexpressed in pear pollen tubes to promote pollen tube growth, increase pollen germination rate, or enhance reproductive development efficiency of fruit trees.

9. A method for promoting polar growth of pear pollen tubes and increasing pear pollen germination rate or fruit setting rate, characterized in that, The PbrSLAH3b gene described in claim 1 is stably overexpressed in pear pollen tubes to promote polar growth of pear pollen tubes and increase the pollen germination rate or fruit setting rate.

10. The method according to claim 9, characterized in that, Includes the following steps: (1) The PbrSLAH3b gene was ligated to the expression vector to obtain a recombinant vector; (2) The recombinant vector was transferred into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; (3) Using the recombinant Agrobacterium tumefaciens to infect target plant tissues or pollen, transgenic plants or transient expression systems are screened.

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