Application of pear transcription factor PbrWHY2 gene in promoting pear pollen tube growth

Abstract

The application discloses application of a pear transcription factor PbrWHY2 gene in promoting pear pollen tube growth and belongs to the technical field of plant genetic engineering. The application clones a gene PbrWHY2 from 'Dangshan pear' pollen by using a plant gene cloning technique, the nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence coded by the gene is shown in the sequence table SEQ ID No. 2. The PbrWHY2 can promote pear pollen tube growth, the change of PbrWHY2 expression is related to the change of ROS level and cellulose content at the top of the pollen tube, meanwhile, the PbrWHY2 also participates in the regulation mechanism of self-incompatibility reaction, and targets to regulate downstream genes PbrAGP16 / PbrRALF6 / PbrGIS1. The pollen magnetic transfection technology is used to study the function of the gene in the pollen tube, and provides a wide application prospect for improving pollination efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to the function and application of the pear transcription factor PbrWHY2 gene. The present application clones the transcription factor PbrWHY2 gene from 'Dangshan Quli' pollen. Through antisense oligonucleotide transfection (ODN) and pollen magnetic transfection overexpression, it is shown that PbrWHY2 promotes the change of reactive oxygen species (ROS) level and cellulose content at the top of the pollen tube, and further promotes the growth of the pear pollen tube. Further analysis shows that PbrWHY2 responds to self S-RNase and targets to activate the transcription of PbrAGP16, PbrRALF6 and PbrGIS1. BACKGROUND

[0002] Self-incompatibility (SI) is a complex mechanism developed by plants in the long-term evolution process to protect genetic diversity (McClure et al., 2011). So far, many non-S factors have been found to be involved in SI response. Choline oxidase (CgMIOX3) responds to SI signals emitted by S-RNase enzymes entering the pollen tube by increasing expression level and enhancing activity, and then promotes AsA synthesis by catalyzing choline degradation (Xu et al., 2024). PrMPK9-1 is a key regulator of SI in pollen, which acts upstream of programmed cell death, involving actin and DEVDase activation (Chai et al., 2017). The physical interaction between S-RNase and soluble inorganic pyrophosphatase (MdPPa) inhibits its activity, resulting in reduced pollen tube growth (Li et al., 2018). Similarly, many transcription factors are also involved in SI response. Self S-RNase can prevent pollen tube growth by limiting PbABF.D.2-PbLRXA2.1 / PbLRXA2.2 signal cascade (Wu et al., 2023a). S-RNase reduces the expression of PbCOB, which prevents pollen tube growth by reducing the expression of its upstream factor PbC2H2.K16.2 (Wu et al., 2023b).

[0003] The WHY family contains a characteristic "swivel" secondary structure and a conserved "KGKAAL" DNA-binding domain. WHY was first identified as a factor named p24 that functions as a transcriptional activator during infection by fungal pathogens (Despres et al., 1995; Desveaux et al., 2000). Most plants have two WHY proteins, WHY1 and WHY2. WHY1 is a nuclear localization protein. WHY2 targets mitochondria, but there is evidence that it localizes to the nucleus. A third protein has been found in Arabidopsis, which localizes to chloroplasts (Huang et al., 2020; Krause et al., 2005). WHY2 plays a crucial role in maintaining the integrity of mitochondrial DNA and is also involved in protecting mitochondrial DNA from degradation during pollen development (Cai et al., 2015). In the nucleus, WHY2 binds to the promoter of the SWEET11 / 15 gene, which encodes a sucrose transporter protein. It also increases the expression of genes involved in jasmonic acid signaling and related defense responses (Huang et al., 2020). Therefore, WHY2 is crucial for signal transduction and transcriptional regulation of plant organelles and the nucleus. However, the potential role of WHY2 in pollen tube growth and SI response has not been extensively explored.

[0004] Pears are a favorite fruit among the people, and successful pollination and fertilization is the key to ensuring pear yield. In this study, the transcription factor PbrWHY2 gene was obtained from pear pollen, and its effect on pear pollen tube growth and role in pear self-incompatibility were studied. Exploring and applying pollen magnetic transfection technology to practice can greatly reduce the cost of artificial pollination, and has important theoretical and practical significance for agricultural production. SUMMARY

[0005] The purpose of the present application is to provide the application of PbrWHY2 gene or biological material related to the PbrWHY2 gene in promoting pear pollen tube growth.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] In a first aspect, the present application claims the application of PbrWHY2 gene or biological material related to the PbrWHY2 gene in at least one of the following (a1)-(a9):

[0008] (a1) in promoting pear pollen tube growth;

[0009] (a2) in the preparation of a product for promoting pear pollen tube growth;

[0010] (a3) use in increasing the level of ROS at the tip of the pear pollen tube;

[0011] (a4) use in the preparation of a product for increasing the level of ROS at the tip of the pear pollen tube;

[0012] (a5) use in increasing the content of cellulose at the tip of the pear pollen tube;

[0013] (a6) use in the preparation of a product for increasing the content of cellulose at the tip of the pear pollen tube;

[0014] (a7) use in alleviating the self-S-RNase toxicity of pear;

[0015] (a8) use in increasing the efficiency of in vitro pollination of pear;

[0016] (a9) use in the preparation of a product for increasing the efficiency of in vitro pollination of pear;

[0017] The PbrWHY2 gene is any one of the following (b1)-(b3) DNA molecules:

[0018] (b1) a DNA molecule whose coding region comprises the nucleotide sequence shown as SEQ ID NO. 1;

[0019] (b2) a DNA molecule whose nucleotide sequence is shown as SEQ ID NO. 1;

[0020] (b3) a DNA molecule which hybridizes to the DNA sequence defined in (b1) or (b2) under stringent conditions and encodes a protein associated with promoting the growth of pear pollen tubes.

[0021] Further, the biological material associated with the PbrWHY2 gene in the above-mentioned applications is at least one of the following (c1)-(c6):

[0022] (c1) a protein encoded by the PbrWHY2 gene;

[0023] (c2) an expression cassette containing the PbrWHY2 gene;

[0024] (c3) a recombinant vector containing the PbrWHY2 gene, or a recombinant vector containing the expression cassette of (c2);

[0025] (c4) a recombinant microorganism containing the PbrWHY2 gene, or a recombinant microorganism containing the expression cassette of (c2), or a recombinant microorganism containing the recombinant vector of (c3);

[0026] (c5) a transgenic plant cell line containing the PbrWHY2 gene, or a transgenic plant cell line containing the expression cassette of (c2), or a transgenic plant cell line containing the recombinant vector of (c3);

[0027] (c6) a magnetofection reagent containing the PbrWHY2 gene, or a magnetofection reagent containing the expression cassette of (c2), or a magnetofection reagent containing the recombinant vector of (c3).

[0028] Further, the PbrWHY2 gene encodes a protein as follows (d1) or (d2) or (d3):

[0029] (d1) a protein with an amino acid sequence as shown in SEQ ID NO. 2;

[0030] (d2) a protein derived from SEQ ID NO. 2 with substitution and / or deletion and / or addition of one or several amino acid residues and related to promoting pollen tube growth of Pyrus;

[0031] (d3) a fusion protein with N-terminal or / and C-terminal of (d1) or (d2) connected with a protein tag.

[0032] Further, the PbrWHY2 gene is stably overexpressed in the pollen tube of Pyrus, promotes the growth of the pollen tube of Pyrus or / and increases the ROS level or / and the cellulose content at the tip of the pollen tube of Pyrus. Further, the PbrWHY2 gene is stably overexpressed in the pollen tube of Pyrus by using magnetofection overexpression technology to treat the pollen of Pyrus in vitro.

[0033] In a second aspect, the present application claims to protect a method for promoting the growth of the pollen tube of Pyrus, which stably overexpresses the aforementioned PbrWHY2 gene in the pollen tube of Pyrus to promote the growth of the pollen tube of Pyrus.

[0034] In a third aspect, the present application claims to protect a method for increasing the ROS level or / and the cellulose content at the tip of the pollen tube of Pyrus, which stably overexpresses the aforementioned PbrWHY2 gene in the pollen tube of Pyrus to increase the ROS level or / and the cellulose content at the tip of the pollen tube of Pyrus.

[0035] In a fourth aspect, the present application claims to protect a method for alleviating the self-S-RNase toxicity of Pyrus, which stably overexpresses the aforementioned PbrWHY2 gene in the pollen tube of Pyrus to alleviate the characteristics of the pollen tube growth inhibition, the ROS level reduction at the tip of the pollen tube, and the cellulose content decrease of the pollen tube caused by the self-S-RNase of Pyrus.

[0036] Further, in the above method, the PbrWHY2 gene is stably overexpressed in the pollen tube of pear pollen by using magnetic transfection overexpression technology to treat the pear pollen in vitro. Further, the magnetic transfection overexpression technology specifically comprises the following steps:

[0037] (1) Designing primers to PCR-amplify the PbrWHY2 gene, inserting the PbrWHY2 gene into the enzyme digestion sites of XbaI and BamHI of the LAT52::GFP vector, and constructing a recombinant plasmid PbrWHY2-LAT52::GFP;

[0038] (2) Mixing the transfection reagent and the recombinant plasmid PbrWHY2-LAT52::GFP to prepare a PbrWHY2-LAT52::GFP transfection reagent; transfecting the pollen cells by using the PbrWHY2-LAT52::GFP transfection reagent.

[0039] The present application belongs to the technical field of genetic engineering, and specifically relates to the application of a functional gene PbrWHY2 identified and cloned from pear pollen in promoting the growth of pear pollen tubes and the application of PbrWHY2 in relieving self S-RNase toxicity. The present application uses plant gene cloning technology to clone the gene PbrWHY2 from 'Dangshan Quli' pollen, and the gene belongs to the Whirly type transcription factor. The 'Dangshan Quli' pollen is treated in vitro through ODN and pollen magnetic transfection tests, and the results show that PbrWHY2 can actively promote the growth of pollen tubes, and the change in the expression of PbrWHY2 is related to the change in the ROS level and the cellulose content at the top of the pollen tube. In addition, PbrWHY2 also participates in the regulation mechanism of the self-incompatibility reaction, and targets to activate the transcription of downstream genes PbrAGP16, PbrRALF6 and PbrGIS1. The present application uses pollen magnetic transfection technology to study the function of genes in pollen tubes, and provides a broad application prospect for improving pollination efficiency.

[0040] Compared with the prior art, the present application has the advantages and effects that:

[0041] (1) The discovery of the PbrWHY2 gene provides a new idea for improving the in-vitro pollination efficiency of pear, reduces labor costs, and provides a new way for implementing green agriculture.

[0042] (2) Compared with the traditional pollen gene gun technology, the method of overexpressing pollen tube genes through pollen magnetic transfection technology has the advantages of high transformation efficiency, simple operation, cost saving, and directional improvement of traits. BRIEF DESCRIPTION OF DRAWINGS

[0043] ​​Figure 1 The test results of PbrWHY2 affecting pear pollen tube growth; wherein a is the phenotype image of as-ODN treated pollen tube, and the red line indicates a scale (50 μm); b is the length of pollen tube treated by as-ODN, s-ODN, cell transfection and control; c is the expression level of PbrWHY2 in pollen tube treated by different methods.

[0044] Figure 2 The test results of PbrWHY2 promoting pollen tube growth by magnetic transfection; wherein a is the phenotype image of pollen tube transferred by PbrWHY2-LAT52::GFP fusion vector and LAT52::GFP vector, and the red line indicates a scale (10 μm); b is the length of pollen tube determined by pollen magnetic staining experiment; c is the expression level of PbrWHY2 in pollen tube detected by pollen magnetic staining experiment.

[0045] Figure 3 The test results of PbrWHY2 positively mediating the ROS level and cellulose content of pollen tube tip; wherein a is the fluorescence value of ROS in pollen tube treated by as-ODN, s-ODN, cell transfection and control by CM-H2DCFDA staining, b is the fluorescence value of cellulose in pollen tube treated by as-ODN, s-ODN, cell transfection and control; c is the ROS level in pollen tube detected by pollen magnetic staining experiment; d is the image of cellulose content in pollen tube induced by lat52::GFP vector and PbrWHY2-lat52::GFP fusion vector.

[0046] Figure 4 The test results of PbrWHY2 alleviating self S-RNase toxicity; wherein a is the expression level of PbrWHY2 in pollen tube treated by self S-RNase, non-self S-RNase and control; b is the length of pollen tube treated by magnetic transfection and S-RNase; c is the change of cellulose fluorescence intensity after magnetic transfection and S-RNase co-treatment; d is the change of ROS level after magnetic transfection and S-RNase co-treatment.

[0047] Figure 5 The subcellular localization map of PbrWHY2.

[0048] Figure 6 The dual luciferase result map of PbrWHY2.

[0049] Figure 7 The gel shift experiment result map of PbrWHY2. DETAILED EMBODIMENT

[0050] The present application is described in detail below with reference to specific embodiments. Upon consideration of the following description and embodiments, one skilled in the art can ascertain the essential characteristics of the application, and without departing from the spirit and scope of the application, can make various changes and modifications to the application to adapt it to various uses and conditions.

[0051] Example 1 Identification of PbrWHY2 on pollen tube growth

[0052] The antisense oligodeoxynucleotide sequence (as-ODN) and the sense oligodeoxynucleotide sequence (s-ODN) of PbrWHY2 were designed by the RNAfold website (http: / / rna.tbi.univie.ac.at). At the same time, the primer sequence was modified by thiol and purified by HPLC.

[0053] The PbrWHY2-ODN primers are as follows:

[0054] PbrWHY2-as-ODN: 5'-CCCAACCCATGATGTGGGGC-3' (SEQ ID No. 5)

[0055] PbrWHY2-s-ODN: 5'-GCCCCACATCATGGGTTGGG-3' (SEQ ID No. 6)

[0056] The specific steps are as follows: pear pollen was added to 2 mL of culture medium (5 mM 2-morpholinoethanesulfonic acid (MES), 440 mM sucrose, 0.55 mM calcium nitrate, 1.60 mM magnesium sulfate, 1.60 mM boric acid, 1.00 mM potassium nitrate, pH = 6.2-6.3) and incubated on a shaker for 60 min; 12.5 μL of liquid culture medium, 1.5 μL Lipofectamine 2000 and 6 μL ODN primers were incubated at room temperature for 15 min; the premixed ODN primers were added to 180 μL of incubated pollen culture medium; the pollen was further cultured at 25°C for 3 h; observation and photography were performed using a Nikon Eclipse E100 microscope (Tokyo, Japan), and the pollen tube length (as shown in Fig. 1a) was measured using ImageJ. Figure 1 The results showed that after 3 hours of treatment (HAT), the pollen tube treated with as-ODN was shorter than that treated with s-ODN, transfection reagent and buffer (as shown in Fig. 1b). Figure 1

[0057] ​3000rpm, 10 min to collect the pollen, remove the liquid medium, and store in -80 °C refrigerator. Extract the pollen RNA, and get the first strand cDNA by reverse transcription, which is used for qRT-PCR experiment of PbrWHY2 gene. The RNA extraction uses the total RNA extraction kit (purchased from Beijing Tiangen Biotech Co., Ltd., and the operation is according to the operation instruction provided by the kit). The reverse transcription of RNA to cDNA uses TransScript One-Step RT-PCR SuperMix (purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., and the operation is according to the instruction provided by the kit). The specific quantitative primer of PbrWHY2 is designed as follows:

[0058] PbrWHY2-qPCR-F: 5'-CACCAGATTTTCAACTGCCACTC-3' (SEQ ID No. 7)

[0059] PbrWHY2-qPCR-R: 5'-AACAGGAGTCAAAGAGAGGGCAG-3' (SEQ ID No. 8)

[0060] The specific quantitative primer of pear UBQ gene as the internal reference is as follows:

[0061] PbUBQ-F: 5'-CCCTTCACTTGGTTCTCCGT-3' (SEQ ID No. 9)

[0062] PbUBQ-R: 5'-TAATCAGCAAGCGTGCGACC-3' (SEQ ID No. 10)

[0063] The qRT-PCR experiment uses LC480 SYBR Green Mix kit (purchased from Roche Company), and the operation is according to the instruction of the kit. The 20 μL qRT-PCR reaction system includes: 10 μL 2x SYBR Green Mix, 0.4 uM forward primer and reverse primer, 20 ng cDNA, and the rest is supplemented with sterilized water. The 96-well qRT-PCR plate (purchased from Roche Company) is used, and the qRT-PCR instrument (model: LightCycler 480, Roche Company) is used for PCR. The qRT-PCR reaction program is as follows: 95 °C pre-denaturation for 10 minutes; 95 °C denaturation for 3 seconds, 62 °C annealing for 10 seconds, 72 °C extension for 30 seconds, 45 cycles. Each cDNA sets three biological repeats and three technical repeats, calculates the average Ct value of each cDNA sample, and calculates 2 -ΔΔCtThe relative expression of PbrWHY2 gene was obtained. The standard error was based on three repeated qRT-PCR analysis and at least 90 pollen tube growth analysis. The variance analysis used Student's t test, and the letters (a and b) represented P<0.05. As shown in c, the expression level of PbrWHY2 in the as-ODN treated pollen tube was reduced. These results showed that the reduction of PbrWHY2 expression inhibited the growth of pear pollen tube. Figure 1

[0064] Example 2 Application of magnetic transfection PbrWHY2 in promoting pollen tube growth

[0065] In the present application, the plasmid vector is LAT52::GFP vector (Qian et al., 2020). The resistance tag of this vector is kanamycin, and the double enzyme digestion is preferably XbaI and BamHI (purchased from NEB company). The optimal reaction system and conditions of enzyme digestion: 800 ng LAT52::GFP empty plasmid, 5 μL 10×Cutsmart Buffer, 1 μL BamHI endonuclease, 1 μL XbaI endonuclease, ddH2O to 50 μL, 37°C reaction for 4 h.

[0066] The primer pair for PCR amplification of the gene is:

[0067] PbrWHY2-F: 5'-ATGTTGAAGGTGTTGCGCG-3' (SEQ ID No. 3)

[0068] PbrWHY2-R: 5'-TCTATCCCACTCCTCCAAAAGC-3' (SEQ ID No. 4)

[0069] The amplification system of PCR: 2 μL cDNA, 2.5 μL of forward and reverse primers, 25 μL 2×PhantaMax Buffer, 1 μL dNTP Mix, 1 ul PhantaMax Super-Fidelity DNA Polymerase (purchased from Nanjing Novozyme Biotech Co., Ltd.), and ddH2O to 50 μL. The amplification program is: 94°C, pre-denaturation for 3 min, 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 140 s, 35 cycles of thermal cycling, 72°C extension for 10 min, and 4°C storage. After amplification, the PCR product with a single target band was detected by 1.5% agarose gel electrophoresis, and the specific band was recovered according to the gel recovery kit (purchased from Nanjing Novozyme Biotech Co., Ltd.) according to the instructions.

[0070] ​The double enzyme-digested vector was connected with the purified DNA, and the ligase Exnase II was purchased from Nanjing Novizen Biotech Co., Ltd. The reaction system was 20 μL: 150 ng LAT52::GFP linear vector, 50 ng gene fragment, 4 μL 5×CE II Buffer, 2 μL Exnase II, and the rest was supplemented with ddH2O, 37°C for 30 min. Then the ligation product was transferred into E. coli DH5a (purchased from Nanjing Novizen Biotech Co., Ltd.), ice bath for 30 min, heat shock for 45 s, then ice bath for 2 min, 37°C 220 rpm shaker activation for 60 min. After activation, it was coated on the LB solid plate with 100 μg / ml kanamycin, and after 14 hours, 5 positive clones were picked for sequencing (completed by Shanghai Biotech Co., Ltd.). The recombinant plasmid PbrWHY2-LAT52::GFP with successful sequencing was extracted using an endotoxin-free plasmid large extraction kit (purchased from Nanjing Novizen Biotech Co., Ltd.).

[0071] The optimal method of pollen magnetic transfection is as follows: put the pollen cells of ‘Dangshan pear’ into a tissue culture dish placed on a magnetic marker plate (purchased from Nanjing Dongna Co., Ltd.), the volume of the medium containing cells depends on the specification of the culture dish, preferably the transfection volume is 0.5 mL, and incubate for 15 minutes. Mix the transfection reagent (purchased from Nanjing Dongna Co., Ltd.) and the PbrWHY2-LAT52::GFP recombinant plasmid, preferably 0.5-2 μL, and the DNA amount is 0.5-3 μg. The LAT52::GFP empty plasmid is used as a control; the prepared / PbrWHY2-LAT52::GFP transfection reagent and the control reagent are added to the cells, and at this time the cell culture plate is still placed on the magnetic marker plate for continuous incubation for 20 minutes; carefully remove the supernatant pollen medium from the cells, and add fresh medium, and at this time the culture plate is still placed on the magnetic marker plate. Be careful not to suck away the cells that sink due to magnetic force; remove the culture plate from the magnetic marker plate; after standard condition culture at 25°C for 120 rpm for 3 h, observe whether there is GFP fluorescence in the magnetically transduced pollen by laser confocal microscope LSM800 (Zeiss, Germany) to determine whether the MNP / DNA complex is transferred into the pollen. Use Nikon Eclipse E100 microscope (Tokyo, Japan) to count the pollen tube length and perform data analysis. Extract the RNA of the magnetically transduced pollen, obtain the first strand cDNA by reverse transcription, and use it for qRT-PCR experiment of PbrWHY2 gene. The pollen RNA extraction, cDNA reverse transcription and qRT-PCR experiment are the same as in Example 1. The standard error is based on 3 repeated qRT-PCR analysis and at least 90 pollen tube growth analysis. The variance analysis uses Student's t-test, and the letters (a and b) represent P < 0.05.

[0072] The results showed that GFP fluorescence was detected in pollen tubes with the introduction of LAT52::GFP or PbrWHY2-LAT52::GFP fusion vectors, indicating the applicability of pollen magnetization technology in the study of gene overexpression in pear pollen tubes (as shown in Figure 2 a of FIG. 6). The pollen tubes with the introduction of PbrWHY2-LAT52::GFP fusion vectors were longer than those with the introduction of LAT52::GFP vectors (as shown in Figure 2 b of FIG. 6), and the expression level of PbrWHY2 in the pollen tubes with the introduction of PbrWHY2-LAT52::GFP fusion vectors was increased compared with that in the pollen tubes with the introduction of LAT52::GFP vectors (as shown in Figure 2 c of FIG. 6). These results indicated that the increase in the expression level of PbrWHY2 promoted the growth of pear pollen tubes.

[0073] Example 3 Identification of the effect of PbrWHY2 on ROS and cellulose content in pollen tubes

[0074] Pollen culture was the same as in Examples 1 and 2.

[0075] The present application used 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo Fisher Scientific, USA) and nitroblue tetrazolium (NBT, Merck, Germany) fluorescence staining method to determine the ROS level at the tip of the pollen tube, and the optimal method was as follows: after adding H2DCFDA fluorescent dye with a final concentration of 20 mM to the as-ODN treated pollen sample for 20 min, the sample was washed three times with liquid medium; after adding NBT fluorescent dye with a final concentration of 1 mg / ml to the magnetofection treated pollen sample, the sample was washed three times with liquid medium; observation and photography were performed using a laser confocal microscope LSM800 (Zeiss, Germany), and the fluorescence intensity of the pollen tube tip was counted using Zeiss software. Calcofluor White (Merck, Germany) was used for cellulose staining of the pollen tube, and the final concentration was 1 mg / mL.

[0076] The results showed that the ROS fluorescence at the tip of the pollen tube treated with as-ODN decreased compared with that of the pollen tubes treated with s-ODN, transfection reagent and buffer (as shown in Figure 3 a of FIG. 7). In contrast, the pollen tube tip with the introduction of PbrWHY2-LAT52::GFP vectors showed higher ROS fluorescence compared with that of the pollen tube with the introduction of LAT52::GFP vectors (as shown in Figure 3PbrWHY2 gene plays a positive role in the ROS level at the pollen tube tip of pear. In addition, Calcofluor White fluorescence staining showed that the cellulose fluorescence decreased after as-ODN treatment (as shown in FIG. 3c), and the cellulose fluorescence of the pollen tube with PbrWHY2-LAT52::GFP was enhanced (as shown in FIG. 3d). These results indicate that PbrWHY2 also positively mediates the cellulose content at the pollen tube tip of pear. Figure 3 PbrWHY2 gene plays a positive role in the ROS level at the pollen tube tip of pear. In addition, Calcofluor White fluorescence staining showed that the cellulose fluorescence decreased after as-ODN treatment (as shown in FIG. 3c), and the cellulose fluorescence of the pollen tube with PbrWHY2-LAT52::GFP was enhanced (as shown in FIG. 3d). These results indicate that PbrWHY2 also positively mediates the cellulose content at the pollen tube tip of pear. Figure 3 PbrWHY2 gene plays a positive role in the ROS level at the pollen tube tip of pear. In addition, Calcofluor White fluorescence staining showed that the cellulose fluorescence decreased after as-ODN treatment (as shown in FIG. 3c), and the cellulose fluorescence of the pollen tube with PbrWHY2-LAT52::GFP was enhanced (as shown in FIG. 3d). These results indicate that PbrWHY2 also positively mediates the cellulose content at the pollen tube tip of pear.

[0077] Example 4 Application of PbrWHY2 to alleviate self-S-RNase toxicity

[0078] The full-length coding sequences of PbrS1-RNase and PbrS2-RNase genes were amplified from the style of “Yellow Flower Pear”, and the full-length coding sequences of PbrS7-RNase and PbrS34-RNase genes were amplified from the style of “Dangshan Pear”; four S-RNases were inserted into the pCold-TF expression vector to generate his-tagged recombinant proteins; BamHI and XbaI were selected as endonucleases. The primer sequences are as follows:

[0079] PbrS1-RNase-F: 5’-ATGTACGATTATTTTCAATTTACGCAGCAAT-3’ (SEQ ID No. 11)

[0080] PbrS1-RNase-R: 5’-ATACTGAACACTGGAGGGGCAGG-3’ (SEQ ID No. 12)

[0081] PbrS2-RNase-F: 5’-ATGGCGAGATACGATTATTTTCAATTTACGC-3’ (SEQ ID No. 13)

[0082] PbrS2-RNase-R: 5’-ATACTGAATATCATCAATGGGGCAGAA-3’ (SEQ ID No. 14)

[0083] PbrS7-RNase-F: 5’-ATGTACGATTATTTTCAATTTACGCAGCAAT-3’ (SEQ ID No. 15)

[0084] PbrS7-RNase-R: 5’-ATACTTAACATCGGCCGGGCAG-3’ (SEQ ID No. 16)

[0085] PbrS34-RNase-F: 5'-ATGTACGATTATTTTCAATTTACGCAGCAAT-3' (SEQ ID No. 17)

[0086] PbrS34-RNase-R: 5'-ATACTGAATACTATTGTTTGGGCAAAAATG-3' (SEQ ID No. 18)

[0087] The preferred procedure of PCR, enzyme digestion system and recombination vector construction are the same as those in Example 2.

[0088] 500 ng of the recombination plasmid was transformed into E. coli Rosetta (DE3) respectively, and coated on an ampicillin-containing plate with 100 μg / mL to screen the recombination gene, and cultured in a 37°C incubator for 14 h. The pCold-TF vector plasmid was also transformed into Rosetta (DE3) as a control. Single colonies were selected for identification. The E. coli Rosetta (DE3) transformed with the recombination plasmid was inoculated in a liquid screening medium for activation, and cultured at 37°C and 220 rpm overnight, then transferred to a new liquid screening medium for culture with an OD 600 = 0.4-0.6, and the inoculation amount of the activation culture and the expansion culture was preferably 1:50; 2 ml of the bacterial solution was taken as a negative control. The expansion culture conical flask was quickly placed on ice for 45 min, then IPTG inducer was added, preferably with a final concentration of 0.5 mmol / L, and induced for expression at 16°C, 220 rpm for 18-24 h.

[0089] After the expression was completed, the bacterial cells were collected by centrifugation at 4°C and 12000 rpm. The bacterial cells were resuspended with 15 mL of PBS buffer (140 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L disodium hydrogen phosphate, 1.8 mmol / L potassium dihydrogen phosphate, pH 7.4), and then ultrasonically broken with a power of 240 W for 4 s and a stop of 6 s until the solution was clear. After the ultrasonic breaking was completed, the supernatant was collected by centrifugation at 4°C and 12000 rpm for 20 min. The control protein pCold-TF was also expressed by the above method.

[0090] The recombinant protein was purified by Ni-NTA agarose affinity chromatography column (purchased from Shanghai Gener Biotech Co., Ltd.). The specific operation was as follows: the column was equilibrated with PBS buffer at a flow rate of 1 ml / min; the protein supernatant after crushing was added to the column at a flow rate of 0.5 ml / min; the column was washed with 20 mmol / L imidazole-containing buffer (500 mmol / L NaCl, 50 mmol / L Tris (hydroxymethyl) aminomethane, 20 mM imidazole, pH = 7.4) at a flow rate of 1 ml / min; the column was eluted with 400 mmol / L imidazole-containing elution buffer (500 mmol / L NaCl, 50 mmol / L Tris (hydroxymethyl) aminomethane, 400 mM imidazole, pH = 7.9) at a flow rate of 1 ml / min, and the eluate was collected to obtain the purified protein. The protein was concentrated and desalted by using a 30 kDa ultrafiltration tube. 10 μL of the purified protein was taken, 2 μL of 5x protein loading buffer (purchased from Shanghai Gener Biotech Co., Ltd.) was added, and 10 μL was taken for 12% regular SDS-PAGE electrophoresis. The recombinant protein was detected by Coomassie blue staining and destaining. Finally, the purified protein was dialyzed at 4°C and 5000 rpm using pollen culture medium, and then stored at -80°C.

[0091] The "Dangshan pear" pollen was pre-cultured in the basic medium for 1 h, and then treated with recombinant PbrS1-RNase and PbrS2-RNase proteins or recombinant PbrS7-RNase and PbrS34-RNase proteins (final concentration of 0.15 U). Then, it was cultured at 25°C for 30 min on a 120 rpm shaker. The RNA extraction, cDNA reverse transcription, and qRT-PCR experiments were the same as in Example 1.

[0092] To verify whether PbrWHY2 is involved in GSI, we treated the Dangshan pear pollen tubes with non-self (PbrS1-RNase and PbrS2-RNase) and self S-RNase (PbrS7-RNase and PbrS34-RNase) for 30 min. Real-time fluorescent quantitative PCR analysis showed that the expression level of PbrWHY2 in the pollen tubes treated with self SI was lower than that in the pollen tubes treated with non-self SC and pCold (as shown in a of Figure 4 Therefore, PbrWHY2 participates in pear GSI by responding to self S-RNase.

[0093] To investigate the effect of PbrWHY2 on self S-RNase, the pollen tubes with PbrWHY2-LAT52::GFP vector were treated with self and non-self S-RNases. The pollen magnetofection technique was the same as Example 2. The results showed that the self S-RNases-treated pollen tubes were shorter than the non-self S-RNases-treated pollen tubes in the pollen tubes with LAT52::GFP vector, supporting the theory that self S-RNase inhibited the growth of pollen tubes (as shown in b of Figure 4 ). The same results were observed in the pollen tubes overexpressing PbrWHY2 (as shown in b of Figure 4 ). Notably, the pollen tubes overexpressing PbrWHY2 were longer than the control LAT52 under the treatment of self S-RNases (as shown in b of Figure 4 ). This indicates that PbrWHY2 may weaken the inhibition of S-RNase on the growth of pollen tubes.

[0094] To clarify the changes of cellulose in the pollen tube tip, the pollen tubes magnetofected with S-RNases were stained with Calcofluor White, and the staining method was the same as Example 3. The results showed that the fluorescence intensity of cellulose in the self S-RNases-treated pollen tubes was lower than that in the non-self S-RNases-treated pollen tubes, supporting the depolymerization of self S-RNase on the cellulose of pollen tubes (as shown in c of Figure 4 ). The same results were observed in the pollen tubes overexpressing PbrWHY2 (as shown in c of Figure 4 ). Notably, the cellulose content in the pollen tubes overexpressing PbrWHY2 was higher than that in the control LAT52 under the treatment of self S-RNases (as shown in c of Figure 4 ). This indicates that PbrWHY2 may depolymerize the cellulose of pollen tubes through self S-RNase.

[0095] To study the changes of ROS in the pollen tube tip, NBT staining was performed on each treated pollen tube, and the staining method was the same as Example 3. The results showed that the ROS gray level of the self S-RNases-treated pollen tubes was lower than that of the non-self S-RNases-treated pollen tubes, supporting that S-RNase could cause the decrease of ROS level in the pollen tube tip (as shown in d of Figure 4 ). Surprisingly, the ROS level of the pollen tubes with PbrWHY2-LAT52::GFP vector was higher than that of the pollen tubes with LAT52::GFP vector under the treatment of self S-RNases (as shown in d of Figure 4 ). This result indicates that PbrWHY2 compensates for the toxic effect of self S-RNase on the ROS in the pollen tube tip.

[0096] Example 5 Subcellular localization identification of transcription factor PbrWHY2

[0097] In the present application, the plasmid vector is 1300::GFP vector (Tang et al., 2023). The resistance tag of this vector is kanamycin, and the double enzyme digestion is preferably XbaI and BamHI (purchased from NEB company). The optimal reaction system and conditions of enzyme digestion: 800 ng of 1300::GFP empty plasmid, 5 μL of 10×Cutsmart Buffer, 1 μL of BamHI endonuclease, 1 μL of XbaI endonuclease, supplemented with ddH2O to 50 μL, 37°C reaction for 4 h.

[0098] The optimal operation of constructing the vector is the same as that of Example 2. 500 ng of the recombinant plasmid with correct sequencing was transferred into GV3101 competent cells, ice bath for 5 min, liquid nitrogen for 5 min, 37°C for 5 min, ice bath for 5 min again, 30°C incubation for 2 h on a shaking bed, and then plated on plates containing 50 mg / mL kanamycin and rifampicin. After two days, the correct Agrobacterium monoclonal strain was identified in a 10 ml centrifuge tube containing 2 ml of liquid LB medium (containing 50 mg / mL rifampicin and kanamycin), and cultured at 28°C on a shaking bed for 24 h. 2 mL of the above bacterial cells were transferred into 20 mL of liquid LB medium, and cultured at 28°C on a shaking bed at 220 rpm for 8-12 h until the OD600 of the bacterial solution was 0.8-1.0. Centrifugation at 5000 rpm for 5 min, and the culture medium was discarded. Resuspend with induction solution containing (10 mM MES, 10 mM MgCl2 and 200 μM acetosyringone) until OD600 = 0.6-0.8, and then incubate at 25°C on a horizontal shaking bed at 80 rpm in the dark for 3 h. After that, use a 1 ml syringe to inject the induction solution into the growing leaves of tobacco, and first incubate in the dark for 24 h, and then transfer to light conditions for further treatment for 48 h before observing under a fluorescence microscope.

[0099] As shown in Figure 5 , the tobacco cells transformed with empty 1300::GFP vector showed green fluorescence in the cell membrane, cytoplasm and nucleus, while the tobacco cells transformed with PbrWHY2 showed green fluorescence in the nucleus, thus the transcription factor PbrWHY2 was localized in the nucleus of tobacco cells Figure 5 ).

[0100] Example 6 Dual luciferase assay of transcription factor PbrWHY2

[0101] The DNA of Dangshan pear was extracted by DNA kit FastPure Plant DNA Isolation Mini Kit, the promoter sequences of PbrAGP16, PbrRALF6 and PbrGIS1 were found in the genome of pear, the upstream 2000 bp sequences were cloned and connected to pGreenII 0800-LUC vector to obtain recombinant plasmids PbrAGP16pro-LUC, PbrRALF6pro-LUC and PbrGIS1pro-LUC, and the double digestion was preferably HandIII and BamHI (purchased from NEB company). The method of cloning and constructing vector was the same as that of Example 2.

[0102] PbrAGP16-F: 5'-TAGGTATCAAACTCAAGCACAAATATAATA-3' (SEQ ID No. 19)

[0103] PbrAGP16-R: 5'-TCTTTTTTCTATCTATTAAACTCTAAAAGCA-3' (SEQ ID No. 20)

[0104] PbrRALF6-F: 5'-AAATGTATCCGTTTTCTAATGTAAGACC-3' (SEQ ID No. 21)

[0105] PbrRALF6-R: 5'-GTGTTGCATGTAAATATTGTTTAAACG-3' (SEQ ID No. 22)

[0106] PbrGIS1-F: 5'-TGTAGTATTTTAAATATTTCATGCATTGTTA-3' (SEQ ID No. 23)

[0107] PbrGIS1-R: 5'-TCTTTTTTCTATCTATTAAACTCTAAAAGCA-3' (SEQ ID No. 24)

[0108] The pSAk277-PbrWHY2 expression vector (Wu et al. 2023) was used as the Effector plasmid. The antibiotic resistance tag for this vector was spectinomycin. The preferred double enzyme digestion was HindIII-F and XbaI (purchased from NEB). The expression vector construction method was the same as in Example 2. Recombinant plasmids PbrAGP16pro-LUC, PbrRALF6pro-LUC, and PbrGIS1pro-LUC were used as Reporter plasmids. Agrobacterium transformation was performed as in Example 5. Agrobacterium resuspension carrying the promoter and Agrobacterium resuspension carrying the transcription factor were mixed at a concentration ratio of 1:9. A 1 ml sterile syringe (without the needle) was used to gently inject the resuspension onto the back of tobacco leaves. Each combination was injected into 4-6 leaves. After 12 hours in the dark, the leaves were cultured normally in a culture room. Dual-luciferase detection was performed 2 days later. This experiment was performed in triplicate. The Dual-luciferase reporter gene detection kit was used. The ReporterAssay System (Cat#E1910, Promega) was used to detect LUC and REN values, and the experimental results were repeated 6 times. The instrument used was an MDiD5 microplate reader (Spectramax ID5, Molecular, USA).

[0109] The results showed that the LUC / REN ratio detected by simultaneous addition of the promoter and transcription factor pSAk277-PbrWHY2 was significantly higher than that in the control group. Figure 6 ).

[0110] Example 7: Gel Migration Analysis (EMSA)

[0111] The probe is a biotin-labeled probe manufactured by Sangon Biotech (Shanghai) Co., Ltd.

[0112] The biotin-labeled PbrAGP16 probe sequence is: AATATGAGTAGATTAATGACTCTATTAGAC; the mutant probe sequence is: AATATGAGTCCCCCCCCCCCCTATTAGAC;

[0113] The biotin-labeled PbrRALF6 probe sequence is: ATCTTCAAGTGAGTAATTGGCAATTTGGAA; the mutant probe sequence is: ATCTTCAAGCCCCCCCCCCCCAATTTGGAA.

[0114] The biotin-labeled PbrGIS1 probe sequence is: TTATATTTACAAGTAATGGAGGTTGCTAGT;

[0115] Mutant probe sequence: TTATATTTACCCCCCCCCCCCGTTGCTAGT.

[0116] The probe primer was diluted to 10 mM with ddH2O, and a portion was diluted to 200 nM. The total annealing system was 50 mL, and the specific components were as follows: 20 mL of forward primer, 20 mL of reverse primer, 10 mL of annealing buffer, and water to 50 mL. The annealing reaction conditions were set as follows: 95°C pre-denaturation for 2 min, slow cooling to 25°C by reducing 1°C every 30 s, and the product could be stored at -20°C for a long time.

[0117] The full-length PbrWHY2 was constructed into the pCold-TF vector, and the method was the same as that in Example 2. The protein was expressed in prokaryotes, and the protein purification was the same as that in Example 4.

[0118] Polyacrylamide gel electrophoresis: 100 V electrophoresis for 30-40 min, and bromophenol blue was stopped at 2 / 3 or 3 / 4 of the bottom of the gel. The nylon membrane was cut to the same size or slightly smaller than the protein gel, 380 mA, 4°C, and the membrane was transferred for 30-60 min. The membrane was cross-linked with UV-light cross-linker, blocked with blocking buffer, and incubated in a shaker for 30 min. The blocking buffer was discarded, and Conjugate / Blocking Buffer was added and gently incubated for 15 min. 30 mL of 4x Wash Buffer was added to 90 mL of pure water to prepare 1x Wash Buffer. The membrane was transferred to a new container and rinsed with 30 mL of 1x Wash Buffer for 5 min. The membrane was gently washed with 1x Wash Buffer solution for 3 times, 10 min each time. The membrane was moved to a new container, 30 mL of equilibration solution was added, and gently incubated on a shaker for 5 min. 4 mL of BeyoECL Plus Reagent A and 4 mL of BeyoECL Plus Reagent B were mixed, protected from light, and prepared into BeyoECL Plus Reagent Working Buffer. The prepared Working Buffer was covered on the entire surface of the membrane, and developed and imaged on a ChemiDoc MP after 5 min of light reaction.

[0119] The results are as follows: Figure 7As shown, DNA-protein complex can be detected after co-incubation of PbrWHY2-His and PbrAGP16 / PbrRALF6 / PbrGIS1 probes. With the increase of cold probe concentration, less and less complex can be detected, and the highest concentration of cold probe can even completely compete off the binding of fusion protein and labeled probe. However, no binding can be detected when PbrWHY2 promoter probe is co-incubated with His negative control. After mutating the elements of PbrAGP16 / PbrRALF6 / PbrGIS1 probe, no binding can be detected after co-incubation of mutant probe and recombinant protein PbrWHY2-His, only free probe is detected. Thus, it is shown that PbrWHY2 and PbrAGP16 / PbrRALF6 / PbrGIS1 promoters can specifically bind in vitro.

[0120] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

[0121] SEQUENCE LISTING

[0122] SEQ ID NO: 1

[0123] ATGTTGAAGGTGTTGCGCGTCTTGTCCTCTTCCACCGCGAAATTCAGGAGCCATTTCTGC

[0124] ACTAGAGATGCTTTATCAATGTATGCTTTTACTCATATCACCAGATTTTCAACTGCCACTC

[0125] AGAAATTTTCTGTTAAAGGTCCCAGTTCTCATCAAGTATATGCTTCTTTTGATATCTTCAA

[0126] AGGCAAAGCTGCCCTCTCTTTGACTCCTGTTCTTCCAACATTCACTAAATTGGAATCTGG

[0127] AAGTCTTGTAGTTGATCGACGTGGTTCTGTCATGTTGAAGTTCACTCCTGCCATTGGTGA

[0128] ACGTAAGTATGACTGGGAAAAGAGACAGATGTTTGCTTTATCAGCTACAGAGGTTGGCG

[0129] CTCTGATAAGCTTGGGTCCCAATGATTCTTGTGAACTATTTCATGATCCCTCAATGAAATC

[0130] AAGTAATGCTGGTCAAGTGAGGAAGAGCTTATCAATTAAGCCTCATGCGGATGGCAGTG

[0131] GCTACTTTGTTTCCTTAACTGTTGTCAACAACCTGCTAAAGACCAGAGAGAGTTTCTCA

[0132] GTTCCTGTCACGACTGCTGAATTTGCTGTTATGAAGACTGCTTGCAGTTTTGCATTGCCC

[0133] CACATCATGGGTTGGGATAGATTGACGAACAAGATGCCAGCAGGTGGTGGTGGTGGAG

[0134] GAGGGCAAGAATCAAAGGCGGTTCCACAGCTTTTGGAGGAGTGGGATAGATGA

[0135] SEQ ID NO:2

[0136] MLKVLRVLSSSTAKFRSHFCTRDALSMYAFTHITRFSTATQKFSVKGPSSHQVYASFDIFKGK

[0137] AALSLTPVLPTFTKLESGSLVVDRRGSVMLKFTPAIGERKYDWEKRQMFALSATEVGALISL

[0138] GPNDSCELFHDPSMKSSNAGQVRKSLSIKPHADGSGYFVSLTVVNNLLKTRESFSVPVTTAEFAVMKTACSFALPHIMGWDRLTNKMPAGGGGGGGQESKAVPQLLEEWDR*

[0139] SEQ ID NO:3

[0140] 5’-ATGTTGAAGGTGTTGCGCG-3’

[0141] SEQ ID NO:4

[0142] 5'-TCTATCCCACTCCTCCAAAAGC-3'

[0143] SEQ ID NO: 5

[0144] 5'-CCCAACCCATGATGTGGGGC-3'

[0145] SEQ ID NO: 6

[0146] 5'-GCCCCACATCATGGGTTGGG-3'

[0147] SEQ ID NO: 7

[0148] 5'-CACCAGATTTTCAACTGCCACTC-3'

[0149] SEQ ID NO: 8

[0150] 5'-AACAGGAGTCAAAGAGAGGGCAG-3'

[0151] SEQ ID NO: 9

[0152] 5'-CCCTTCACTTGGTTCTCCGT-3'

[0153] SEQ ID NO: 10

[0154] 5'-TAATCAGCAAGCGTGCGACC-3'

[0155] SEQ ID NO: 11

[0156] 5'-ATGTACGATTATTTTCAATTTACGCAGCAAT-3' SEQ ID NO: 12

[0157] 5'-ATACTGAACACTGGAGGGGCAGG-3'

[0158] SEQ ID NO: 13

[0159] 5'-ATGGCGAGATACGATTATTTTCAATTTACGC-3' SEQ ID NO: 14

[0160] 5'-ATACTGAATATCATCAATGGGGCAGAA-3'

[0161] SEQ ID NO: 15

[0162] 5'- ATGTA CGATTATTTTCAATTTACGCAGCAAT -3' SEQ ID NO: 16

[0163] 5'- ATACTTAACATCGGCCGGGCAG -3'

[0164] SEQ ID NO: 17

[0165] 5'- ATGTA CGATTATTTTCAATTTACGCAGCAAT -3' SEQ ID NO: 18

[0166] 5'- ATACTGAATACTATTGTTTGGGCAAAAATG -3' SEQ ID NO: 19

[0167] 5'- TAGGTATCAAACTCAAGCACAAATATAATA -3' SEQ ID NO: 20

[0168] 5'- TCTTTTTTCTATCTATTAAACTCTAAAAGCA -3' SEQ ID NO: 21

[0169] 5'- AAATGTATCCGTTTTCTAATGTAAGACC -3'

[0170] SEQ ID NO: 22

[0171] 5'- GTGTTGCATGTAAATATTGTTTAAACG -3'

[0172] SEQ ID NO: 23

[0173] 5'- TGTAGTATTTTAAATATTTCATGCATTGTTA -3' SEQ ID NO: 24

[0174] 5'- TCTTTTTTCTATCTATTAAACTCTAAAAGCA -3'.

Claims

1. PbrWHY2 application of the gene in at least one of the following (al) - (a9): (a1) use in promoting pear pollen tube growth; (a2) use in preparing a product for promoting pear pollen tube growth; (a3) use in increasing ROS level at the tip of pear pollen tube; (a4) use in preparing a product for increasing ROS level at the tip of pear pollen tube; (a5) use in increasing cellulose content at the tip of pear pollen tube; (a6) use in preparing a product for increasing cellulose content at the tip of pear pollen tube; (a7) use in alleviating self S-RNase toxicity of pear; (a8) use in increasing in vitro pollination efficiency of pear; (a9) use in preparing a product for increasing in vitro pollination efficiency of pear; The PbrWHY2 The gene nucleotide sequence is shown as SEQ ID NO.

1.

2. The method of claim 1 wherein the biological material is a gene associated biological material. PbrWHY2 The gene associated biological material is used in at least one of (al) - (a9): (a1) use in promoting pear pollen tube growth; (a2) use in preparing a product for promoting pear pollen tube growth; (a3) use in increasing ROS level at the tip of pear pollen tube; (a4) use in preparing a product for increasing ROS level at the tip of pear pollen tube; (a5) use in increasing cellulose content at the tip of pear pollen tube; (a6) use in preparing a product for increasing cellulose content at the tip of pear pollen tube; (a7) use in alleviating self S-RNase toxicity of pear; (a8) use in increasing in vitro pollination efficiency of pear; (a9) use in preparing a product for increasing in vitro pollination efficiency of pear; The biological material is at least one of the following (c1)-(c6): (c1 ) the protein encoded by the gene PbrWHY2 gene; (c2) a vector comprising the expression cassette of (c1); and PbrWHY2 a gene; (c3) a recombinant vector comprising the nucleic acid molecule of (c2) or (c1 ), or a recombinant vector comprising the expression cassette of (c2); and PbrWHY2 (c3) a recombinant vector comprising the nucleic acid molecule of (c2) or (c1 ), or a recombinant vector comprising the expression cassette of ( (c4) a recombinant microorganism comprising the PbrWHY2 gene, or a recombinant microorganism comprising the expression cassette of (c2), or a recombinant microorganism comprising the recombinant vector of (c3); (c5) a transgenic plant cell line comprising said PbrWHY2 a transgenic plant cell line comprising (c2) said expression cassette, or a transgenic plant cell line comprising (c3) said recombinant vector; (c6) a magnetic transfection reagent containing said PbrWHY2 gene, or a magnetic transfection reagent containing (c2) said expression cassette, or a magnetic transfection reagent containing (c3) said recombinant vector.

3. Use according to claim 2, characterized in that, The PbrWHY2 The protein encoded by the gene is as follows (d1) or (d2): (d1) a protein with an amino acid sequence as shown in SEQ ID NO. 2; (d2) a fusion protein with (d1) connected with a protein tag at N terminal or / and C terminal.

4. Use according to claim 1 or 2, characterized in that, The method comprises the following steps: PbrWHY2 The gene is stably overexpressed in pear pollen tubes, promotes pear pollen tube growth, or / and increases the ROS level or / and the cellulose content of the top of the pear pollen tube.

5. Use according to claim 4, characterized in that, The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro. PbrWHY2 The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro.

6. A method of promoting pollen tube growth in pear, characterized by, The method of claim 1, wherein the compound is PbrWHY2 Stable overexpression of the gene in pear pollen tubes promotes pear pollen tube growth.

7. A method of increasing the level of ROS or / and the content of cellulose at the pollen tube tip of Pyrus, characterized in that, The method of claim 1, wherein the compound is PbrWHY2 The gene is stably overexpressed in pear pollen tube, and the ROS level or / and the cellulose content at the top of the pear pollen tube is increased.

8. A method of alleviating self-S-RNase toxicity in Pyrus, comprising, The method of claim 1, wherein the compound is PbrWHY2 The gene stably overexpressed in pear pollen tube alleviates the inhibition of pear pollen tube growth caused by self S-RNase, reduces the level of ROS at the tip of the pollen tube, and reduces the content of cellulose in the pollen tube.

9. The method according to any one of claims 6-8, characterized in that, The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro. PbrWHY2 The gene was stably overexpressed in pear pollen tubes by using magnetic transfection overexpression technology to treat pear pollen in vitro.

10. The method of claim 9, wherein, The magnetic transfection overexpression technology specifically comprises the following steps: (1) Design primers for PCR amplification PbrWHY2 Genes, will be described PbrWHY2 The gene was inserted into the XbaI and BamHI restriction sites of the LAT52::GFP vector to construct the recombinant plasmid PbrWHY2. - LAT52::GFP; (2) The MagTransf® transfection reagent was mixed with the recombinant plasmid PbrWHY2-LAT52::GFP to prepare MagTransf® / PbrWHY2 - The MagTransf® transfection reagent was mixed with the recombinant plasmid PbrWHY2-LAT52::GFP to prepare MagTransf® / PbrWHY2 - The MagTransf® transfection reagent was mixed with the recombinant plasmid PbrWHY2-LAT52::GFP to prepare MagTransf® / PbrWHY2

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