Application of rapid alkalization factor pbr RALF6 gene in promoting pear pollen tube growth

Abstract

The application discloses application of a rapid alkalization factor family gene PbrRALF6 in promoting pear pollen tube growth and belongs to the technical field of plant genetic engineering. The application clones the gene PbrRALF6 from 'Dangshan quxi pear' pollen by using plant gene cloning technology, 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. Research shows that PbrRALF6 can promote pear pollen tube growth, the change of PbrRALF6 expression is related to the change of the ROS level at the top of the pollen tube, and meanwhile, PbrRALF6 also participates in the regulation mechanism of self-incompatibility reaction. The pollen magnetic transfection technology is used to study the function of the gene in the pollen tube, thereby providing wide application prospects for improving pollination efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to application of a rapid alkalinization factor PbrRALF6 gene in promoting pear pollen tube growth. The present application clones a rapid alkalinization factor PbrRALF6 gene from 'Dangshan pear' pollen, and transiently knocks down or stably overexpresses the PbrRALF6 gene in pear pollen tubes through antisense oligonucleotide transfection (ODN) and pollen magnetic transfection. The results show that overexpression of the PbrRALF6 gene can promote the growth of pear pollen tubes. Further analysis shows that the change in PbrRALF6 gene expression is related to the change in the level of ROS at the tip of the pollen tube. BACKGROUND

[0002] Flowering plants have evolved pollen tubes to transport sperm cells deep into maternal reproductive tissues towards the embryo sac for double fertilization (Dresselhaus et al. 2016). During the process of pollen tube transmission from the stigma to the style and then to the ovule, it secretes many small proteins to communicate with various maternal tissues (Ogawa and Kessler 2023). These include rapid alkalinization factors (RALFs), which regulate water uptake and cell wall integrity during pollen germination, pollen tube growth and reception in the model plant Arabidopsis thaliana (Zhong et al. 2022). RALF genes were first discovered in tobacco (Nicotiana tabacum) leaves (Pearce et al. 2001). Functional studies have mainly been carried out in Arabidopsis: for example, overexpression of AtRALF1 or application of its peptide inhibited rapid root growth (Li et al. 2022), AtRALF23 / 33 regulated water uptake of pollen grains at the stigma (Liu et al. 2021), and incompatibility sRALFs (AtRALF1 / 22 / 23 / 33) and compatible pollen-derived pRALFs (AtRALF10 / 12 / 13 / 25 / 26 / 30) produced by the stigma regulate pollen tube penetration at the stigma (Lan et al. 2023). AtRALF4 / 19 redundantly regulates pollen germination and pollen tube growth, and this regulation occurs through binding to LRX cell wall proteins (Wang et al. 2018). RALF has also been reported to play an important role in reproduction: it was shown to inhibit pollen germination in tomato (Solanum lycopersicum) when applied in vitro with pollen-specific SlPRALF, which interacts with pollen-specific leucine-rich repeat (LRR) / extensin-like receptor kinase (LRX) (Covey et al. 2010).

[0003] Pears are a fruit that is deeply loved by the people, and successful pollination and fertilization is the key to guarantee the yield of pears. The present application obtains a rapid alkalization factor PbrRALF6, and studies the influence of the PbrRALF6 on the growth of pear pollen tubes and the role played in the process of self-incompatibility of pear. Exploring the pollen magnetic transfection technology and applying it to practice can greatly reduce the cost of artificial pollination, and has considerable theoretical and practical significance to agricultural production. SUMMARY

[0004] The present application aims to provide the application of PbrRALF6 gene or biological material related to the PbrRALF6 gene in promoting the growth of pear pollen tubes. The applicant screens the PbrRALF6 gene from 'Dangshan pear', and the gene belongs to the rapid alkalization factor family gene. The pollen magnetic transfection technology is applied to the pollen overexpression research, and combined with the pollen ODN transfection, the PbrRALF6 gene is identified to have the functions of affecting the ROS level at the top of the pollen tube and affecting the growth of pear pollen tubes.

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0006] In the first aspect, the present application claims the application of PbrRALF6 gene or biological material related to the PbrRALF6 gene in at least one of the following (a1)-(a6):

[0007] (a1) the application in promoting the growth of pear pollen tubes;

[0008] (a2) the application in preparing a product for promoting the growth of pear pollen tubes;

[0009] (a3) the application in improving the ROS level at the top of pear pollen tubes;

[0010] (a4) the application in preparing a product for improving the ROS level at the top of pear pollen tubes;

[0011] (a5) the application in improving the efficiency of pear in vitro pollination;

[0012] (a6) the application in preparing a product for improving the efficiency of pear in vitro pollination;

[0013] The PbrRALF6 gene is any one of the following (b1)-(b4) DNA molecules:

[0014] (b1) a DNA molecule comprising the nucleotide sequence as shown in SEQ ID NO. 1 in the coding region;

[0015] (b2) a DNA molecule with the nucleotide sequence as shown in SEQ ID NO. 1;

[0016] (b3) a DNA molecule hybridizing under stringent conditions with the DNA sequence defined in (bl) or (b2) and encoding a protein associated with promoting pollen tube growth in Pyrus;

[0017] Further, the biological material associated with the PbrRALF6 gene is at least one of the following (c1)-(c6):

[0018] (c1) a protein encoded by the PbrRALF6 gene;

[0019] (c2) an expression cassette containing the PbrRALF6 gene;

[0020] (c3) a recombinant vector containing the PbrRALF6 gene, or a recombinant vector containing the expression cassette of (c2);

[0021] (c4) a recombinant microorganism containing the PbrRALF6 gene, or a recombinant microorganism containing the expression cassette of (c2), or a recombinant microorganism containing the recombinant vector of (c3);

[0022] (c5) a transgenic plant cell line containing the PbrRALF6 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);

[0023] (c6) a magnetic transfection reagent containing the PbrRALF6 gene, or a magnetic transfection reagent containing the expression cassette of (c2), or a magnetic transfection reagent containing the recombinant vector of (c3).

[0024] Still further, the protein encoded by the PbrRALF6 gene is as follows (d1) or (d2) or (d3):

[0025] (d1) a protein having an amino acid sequence as shown in SEQ ID NO. 2;

[0026] (d2) a protein derived from SEQ ID NO. 2 by substitution and / or deletion and / or addition of one or several amino acid residues and associated with promoting pollen tube growth in Pyrus;

[0027] (d3) a fusion protein in which the N-terminal or / and C-terminal of (d1) or (d2) is linked to a protein tag.

[0028] Further, the above-mentioned application is to stably overexpress the PbrRALF6 gene in the pollen tube of Pyrus, promote the growth of the pollen tube of Pyrus or / and increase the level of ROS at the tip of the pollen tube of Pyrus. Still further, the PbrRALF6 gene is stably overexpressed in the pollen tube of Pyrus by using magnetic transfection overexpression technology to treat the pollen of Pyrus in vitro.

[0029] The PbrRALF6 gene is screened from 'Dangshan Quli' and belongs to the rapid alkalinization factor family gene; and the rapid alkalinization factor family gene PbrRALF6 is cloned from 'Dangshan Quli' (Pyrus) by using gene cloning technology. The PbrRALF6 nucleotide sequence is shown in SEQ ID No. 1, contains a 372 bp open reading window, encodes 123 amino acids, the amino acid sequence encoded by the PbrRALF6 is shown in SEQ ID No. 2, the isoelectric point is 10.088, and the molecular weight is 14.04 kDa.

[0030] The applicant designs a primer pair for cloning the cDNA sequence of the PbrRALF6 gene, and the base sequence is as follows:

[0031] PbrRALF6 F1: 5'-ATGGGATTCAAGCTCTACCTGATCACC-3', namely SEQ ID No. 3

[0032] PbrRALF6 R1: 5'-GCGTCTGGCGCAGCGGGT-3', namely SEQ ID No. 4.

[0033] The pollen of 'Dangshan Quli' is treated in vitro by using ODN and magnetic transfection overexpression technology, and it is verified that the PbrRALF6 has the functions of promoting pollen growth and affecting the ROS level at the top of the pollen tube.

[0034] In the second aspect, the application requests to protect a method for promoting the growth of pear pollen tubes, wherein the PbrRALF6 gene is stably overexpressed in the pear pollen tubes to promote the growth of the pear pollen tubes.

[0035] In the third aspect, the application requests to protect a method for improving the ROS level at the top of the pear pollen tubes, wherein the PbrRALF6 gene is stably overexpressed in the pear pollen tubes to improve the ROS level at the top of the pear pollen tubes.

[0036] In the fourth aspect, the application requests to protect a method for improving the in vitro pollination efficiency of pear, wherein the PbrRALF6 gene is stably overexpressed in the pear pollen tubes to promote the growth of the pear pollen tubes and improve the in vitro pollination efficiency of pear.

[0037] Further, in the above method, the PbrRALF6 gene is stably overexpressed in the pear pollen tubes by using magnetic transfection overexpression technology to treat the pear pollen in vitro. Further, the magnetic transfection overexpression technology specifically comprises the following steps:

[0038] (1) design primer PCR amplification PbrRALF6 gene, the PbrRALF6 gene is inserted into the XbaI and BamHI enzyme cutting site of LAT52::GFP carrier, and the recombinant plasmid PbrRALF6-LAT52::GFP is constructed;

[0039] (2) the PbrRALF6-LAT52::GFP recombinant plasmid is transfected into pollen cells of 'Dangshan pear' by pollen magnetic transfection technology. The PbrRALF6-LAT52::GFP transfection reagent is prepared by mixing the transfection reagent and the PbrRALF6-LAT52::GFP recombinant plasmid. The PbrRALF6-LAT52::GFP transfection reagent is used to treat pollen cells.

[0040] The application utilizes plant gene cloning technology to clone the gene PbrRALF6 from pollen of 'Dangshan pear', and the gene belongs to a member of the rapid alkalinization factor family gene. The pollen of 'Dangshan pear' is treated in vitro by ODN and pollen magnetic transfection test, and the results show that overexpression of the gene PbrRALF6 can actively promote the growth of pollen tubes, and the change of PbrRALF6 expression is related to the change of ROS level at the top of the pollen tube. 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.

[0041] The room temperature in the application is generally 25±10 DEG C, but is not limited to this.

[0042] Compared with the prior art, the application has the advantages and effects:

[0043] (1) The discovery of the PbrRALF6 gene provides a new idea for improving the in vitro pollination efficiency of pear, reduces the labor cost, and provides a new way for implementing green agriculture.

[0044] (2) Compared with the traditional pollen gene gun technology, the method of overexpressing the pollen tube gene 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

[0045] Figure 1 The test results of the influence of the PbrRALF6 gene on the growth of pollen tubes;

[0046] Wherein, a is the length of the pollen tube treated by as-ODN, s-ODN and cell transfection; b is the expression level of the PbrRALF6 gene in the pollen tube treated by as-ODN, s-ODN and cell transfection.

[0047] Figure 2 The test results of the PbrRALF6 gene promoting the growth of pollen tubes by magnetic transfection;​

[0048] a, the pollen magnetic transfection fluorescence picture, the red line indicates a scale (10 μm); b, the length of pollen tube determined by pollen magnetic dyeing experiment; c, the expression level of PbrRALF6 in pollen tube detected by pollen magnetic dyeing experiment.

[0049] Figure 3 The test results of PbrRALF6 gene positively mediating the ROS level of pollen tube tip;

[0050] a, the ROS level in pollen tube treated by as-ODN, s-ODN and cell transfection; b, the ROS level in pollen tube induced by lat52::GFP vector and PbrRALF6-lat52::GFP fusion vector.

[0051] Figure 4 The self S-RNase reduces the expression of PbrRALF6 gene. Specific embodiments

[0052] The present application is described in detail below in conjunction with specific examples. According to the following description and these examples, those skilled in the art can determine the basic features of the present application, and can make various changes and modifications to the present application without departing from the spirit and scope of the present application, so as to adapt the present application to various uses and conditions.

[0053] Example 1 Identification of PbrRALF6 on pollen tube growth

[0054] The antisense oligodeoxynucleotide sequence (as-ODN) and the sense oligonucleotide sequence (s-ODN) of PbrRALF6 are designed through the RNAfold website (http: / / rna.tbi.univie.ac.at). At the same time, the primer sequence is modified by sulfur, and purified by HPLC.

[0055] The PbrRALF6-ODN primers are as follows:

[0056] PbrRALF6-as-ODN: 5'-GCATTGCGTCTCAGGGCACC-3' (SEQ ID No. 5)

[0057] PbrRALF6-s-ODN: 5'-CGCAATGCTGTTCCATGCGG-3' (SEQ ID No. 6)

[0058] The specific steps are as follows: an appropriate amount of pear pollen is 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 to obtain the incubated pollen culture medium. 12.5 μL of the culture medium, 1.5 μL of Lipofectamine 2000 and 6 μL of ODN primer are incubated at room temperature for 15 min to obtain the premixed ODN primer; the premixed ODN primer is added to 180 μL of the incubated pollen culture medium; the pollen is further cultured at 25°C for 3 h. Observation and photography are performed using a Nikon Eclipse E100 microscope (Tokyo, Japan), and the pollen tube length is measured using Image J. The results show that, after 3 hours of treatment (HAT), the pollen tube treated with as-ODN is shorter than that treated with s-ODN and transfection reagent (as shown in a of FIG. 8). Figure 1

[0059] 3000 rpm, 10 min to collect the pollen, remove the liquid medium, and store in a -80°C refrigerator. The pollen RNA is extracted, and the first strand cDNA is obtained by reverse transcription for qRT-PCR experiment of the PbrRALF6 gene. The total RNA extraction kit (purchased from Beijing Tiangen Biotech Co., Ltd., and operated according to the operation instruction provided by the kit) is used for RNA extraction. The TransScript One-Step RT-PCR SuperMix (purchased from Beijing Zonnuo Biotech Co., Ltd., and operated according to the instruction provided by the kit) is used for reverse transcription of the RNA to cDNA. The specific quantitative primer of the PbrRALF6 gene is designed as follows:

[0060] PbrRALF6-qPCR-F: 5'-CGACTTGATCGGAGAGGAGAATG-3' (SEQ ID No. 7)

[0061] PbrRALF6-qPCR-R: 5'-GCCTTCCTTGGCAGTTGTAGTAA-3' (SEQ ID No. 8)

[0062] The pear UBQ gene is used as an internal reference, and the specific quantitative primer thereof is as follows:

[0063] PbUBQ-F: 5'-CCCTTCACTTGGTTCTCCGT-3' (SEQ ID No. 9)

[0064] ​PbUBQ-R: 5'-TAATCAGCAAGCGTGCGACC-3' (SEQ ID No. 10)

[0065] qRT-PCR experiments were performed using the LC480 SYBR Green Mix kit (purchased from Roche) according to the kit instructions. The 20 μL qRT-PCR reaction system included: 10 μL 2x SYBR Green Mix, 0.4 uM forward and reverse primers, 20 ng cDNA, and the rest was supplemented with sterile water. A 96-well qRT-PCR plate (purchased from Roche) was used, and a qRT-PCR instrument (model: LightCycler 480, Roche) was used for PCR. The qRT-PCR reaction program was: 95 °C pre-denaturation for 10 min; 95 °C denaturation for 3 s, 62 °C annealing for 10 s, 72 °C extension for 30 s, 45 cycles. Each cDNA was set up in three biological replicates and three technical replicates, and the average Ct value of each cDNA sample was calculated. The relative expression of PbrRALF6 gene was calculated by 2 -ΔΔCt The standard error was based on three repeated qRT-PCR analyses and at least 90 pollen tube growth analyses. The variance analysis used the Student's t-test, and the letters (a and b) indicated P < 0.05. As shown in Fig. 6b, the expression level of PbrRALF6 gene in the pollen tube treated with as-ODN decreased. These results indicated that the decrease in the expression level of PbrRALF6 inhibited the growth of pear pollen tubes. Figure 1

[0066] Example 2 Application of magnetic transfection of PbrRALF6 gene in promoting pollen tube growth

[0067] In the present application, the plasmid vector is the 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). The optimal reaction system and conditions for enzyme digestion: 800 ng LAT52::GFP empty plasmid, 5 μL 10x Cutsmart Buffer, 1 μL BamHI endonuclease, 1 μL XbaI endonuclease, supplemented with ddH2O to 50 μL, 37 °C reaction for 4 h.

[0068] The primer pair for PCR amplification of the gene is:

[0069] PbrRALF6-F: 5'-ATGGGATTCAAGCTCTACCTGATCACC-3' (SEQ ID No. 3)

[0070] ​PbrRALF6-R: 5'-GCGTCTGGCGCAGCGGGT-3' (SEQ ID No. 4)

[0071] PCR amplification system: 2 μL of 'Dangshan pear' pollen cDNA, 2.5 μL of forward and reverse primers, 25 μL of 2x PhantaMax Buffer, 1 μL of dNTP Mix, 1 ul of PhantaMax Super-Fidelity DNA Polymerase (purchased from Nanjing Novozyme Biotech Co., Ltd.), and ddH2O was added to 50 μL. The amplification program was as follows: 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 of the 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.

[0072] The double enzyme-digested vector was ligated with the purified DNA, and the ligase Exnase II was purchased from Nanjing Novozyme Biotech Co., Ltd. The reaction system was 20 μL: 150 ng of LAT52::GFP linear vector, 50 ng of gene fragment, 4 μL of 5x CE II Buffer, 2 μL of 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 Novozyme Biotech Co., Ltd.), ice bath for 30 min, heat shock for 45 s, then ice bath for 2 min, 37°C 220 rpm shaking bed 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 Sangon Biological Co., Ltd.). The recombinant plasmid PbrRALF6-LAT52::GFP with successful sequencing was extracted using an endotoxin-free plasmid large extraction kit (purchased from Nanjing Novozyme Biotech Co., Ltd.).

[0073] The optimal method of pollen magnetic transfection was as follows: the 'Dangshan pear' pollen cells were placed in a tissue culture dish placed on a magnetic marker plate (purchased from Nanjing Dongna Co., Ltd.), and the volume of the medium containing the cells was determined according to the specifications of the culture dish, and the optimal transfection volume was 0.5 mL, and the incubation time was 15 minutes. The transfection reagent (purchased from Nanjing Dongna Co., Ltd.) and PbrRALF6-LAT52::GFP recombinant plasmid were mixed, and the optimal amount was 0.5-2 μL, and the DNA amount was 0.5-3 μg. The LAT52::GFP empty plasmid was used as a control; the prepared / PbrRALF6-LAT52::GFP transfection reagent and control reagent were added to the cells, and the cell culture dishes were still placed on the magnetic marker plate for incubation for 20 minutes; the supernatant pollen culture medium was carefully removed from the cells, and fresh culture medium was added, and the culture plate was still placed on the magnetic marker plate. Care should be taken not to suck away the cells sinking due to magnetic force; remove the culture plate from the magnetic marker plate; after 3h standard condition culture at 25℃ 120rpm, whether there is GFP fluorescence in the magnetic transfection pollen was observed by laser confocal microscope LSM800 (Zeiss, Germany), to determine whether the MNP / DNA complex was transferred into the pollen. The pollen tube length was counted by Nikon Eclipse E100 microscope (Tokyo, Japan), and data analysis was performed. The RNA of the pollen after magnetic transfection was extracted, and the first strand cDNA was obtained by reverse transcription, which was used for qRT-PCR experiment of PbrRALF6 gene. The pollen RNA extraction, cDNA reverse transcription and qRT-PCR experiment were the same as in Example 1. The standard error was based on the qRT-PCR analysis of 3 repeats 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.

[0074] The results showed that GFP fluorescence was detected in the pollen tube with the introduction of LAT52::GFP or PbrRALF6-LAT52::GFP fusion vector, indicating the applicability of pollen magnetic staining technology in the study of gene overexpression in pear pollen tube (as shown in a of Figure 2 The three HATs (as shown in b of Figure 2 The pollen tube with the introduction of PbrRALF6-LAT52::GFP fusion vector was longer than that with the introduction of LAT52::GFP vector. At the same time, compared with the LAT52::GFP vector, the expression amount of PbrRALF6 in the pollen tube with the PbrRALF6 fusion vector was increased (as shown in c of Figure 2 These results showed that the increase of PbrRALF6 expression promoted the growth of pear pollen tube.

[0075] Example 3 Identification of PbrRALF6 affecting ROS level in pollen tube

[0076] The pollen culture was the same as in Examples 1 and 2.

[0077] This invention uses 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluoresceindiacetate (H2DCFDA, Thermo Fisher Scientific, USA) and nitroblue tetrazolium (NBT, Merck, Germany) fluorescence staining to determine the ROS level at the pollen tube tip. The optimal method is as follows: H2DCFDA fluorescent dye at a final concentration of 20 μM is added to as-ODN-treated pollen samples and stained for 20 min, followed by washing the samples three times with liquid culture medium; NBT fluorescent dye at a final concentration of 1 mg / ml is added to magnetically transfected pollen samples and stained, followed by washing the samples three times with liquid culture medium; observation and photography are performed using a laser confocal microscope LSM800 (Zeiss, Germany), and the fluorescence intensity at the pollen tube tip is statistically analyzed using Zeiss software.

[0078] The results showed that, compared with pollen tubes treated with s-ODN and transfection reagents, the ROS fluorescence at the tips of pollen tubes treated with as-ODN was reduced (e.g., Figure 3 (As shown in a). In contrast, the pollen tube tips of the PbrRALF6-LAT52::GFP vector showed higher ROS gray levels (e.g., as shown in a) compared to the LAT52::GFP vector. Figure 3 (As shown in b). The results indicate that the PbrRALF6 gene also positively mediates the ROS level at the tip of the pear pollen tube.

[0079] Example 4: Identification of the PbrRALF6 gene's role in self-incompatibility.

[0080] The full-length coding sequences of PbrS1-RNase and PbrS2-RNase genes were amplified from the style of *Pyrus pyrifolia*, and the full-length coding sequences of PbrS7-RNase and PbrS34-RNase genes were amplified from the style of *Pyrus pyrifolia*. Four S-RNases were inserted into the pCold-TF expression vector to generate his-tagged recombinant proteins. BamHI and XbaI were selected as restriction enzymes. The primer pair sequences are shown below:

[0081] PbrS1-RNase-F:5'-ATGTACGATTATTTTCAATTTACGCAGCAAT-3'(SEQ ID No.11)

[0082] PbrS1-RNase-R:5'-ATACTGAACACTGGAGGGGCAGG-3'(SEQ ID No.12)

[0083] PbrS2-RNase-F: 5'-ATGGCGAGATACGATTATTTTCAATTTACGC-3' (SEQ ID No. 13)

[0084] PbrS2-RNase-R: 5'-ATACTGAATATCATCAATGGGGCAGAA-3' (SEQ ID No. 14)

[0085] PbrS7-RNase-F: 5'-ATGTACGATTATTTTCAATTTACGCAGCAAT-3' (SEQ ID No. 15)

[0086] PbrS7-RNase-R: 5'-ATACTTAACATCGGCCGGGCAG-3' (SEQ ID No. 16)

[0087] PbrS34-RNase-F: 5'-ATGTACGATTATTTTCAATTTACGCAGCAAT-3' (SEQ ID No. 17)

[0088] PbrS34-RNase-R: 5'-ATACTGAATACTATTGTTTGGGCAAAAATG-3' (SEQ ID No. 18)

[0089] The preferred procedure of PCR, enzyme digestion system and recombination vector construction are the same as those in Example 2.

[0090] 500 ng of the recombination plasmid was transformed into E. coli Rosetta (DE3) respectively, and coated on LB plate containing 100 μg / mL ampicillin to screen the recombination gene, and cultured in 37°C incubator for 14 h. The pCold-TF vector plasmid was also transformed into Rosetta (DE3) as a control. Single colony was selected for identification. The E. coli Rosetta (DE3) transformed with the recombination plasmid was inoculated in LB liquid screening medium for activation, and cultured at 37°C, 220 rpm for overnight, then transferred to new liquid screening medium for culture with OD 600 = 0.4-0.6, and the inoculation amount of the activation culture and the expansion culture was preferably 1:50 by volume ratio; 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.

[0091] After expression, the bacteria were collected by centrifugation at 4°C, 12000 rpm. The bacteria were resuspended in 15 mL PBS buffer (140 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na2HPO4, 1.8 mmol / L KH2PO4, pH 7.4) and then broken by ultrasonication at 240 W, 4 s on and 6 s off until the solution was clear. After ultrasonication, the supernatant was collected by centrifugation at 4°C, 12000 rpm for 20 min. The control protein pCold-TF was also expressed by the above method.

[0092] The recombinant protein was purified by Ni-NTA agarose affinity chromatography (purchased from Shanghai Gener Biotech Co., Ltd.). Specifically, the above-mentioned filler was equilibrated with PBS buffer at a flow rate of 1 ml / min; the supernatant of the broken protein was added to the purification column at a flow rate of 0.5 ml / min; the column was washed with a 20-fold filler volume of a washing solution containing 20 mmol / L imidazole (500 mmol / L NaCl, 50 mmol / L Tris (hydroxymethyl) aminomethane, 20 mM imidazole, pH = 7.4) at a flow rate of 1 ml / min; and the purification column was eluted with an 8-fold column volume of an elution solution containing 400 mmol / L imidazole (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. Protein concentration and desalination were performed 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. After staining with Coomassie brilliant blue and destaining, the purification of the recombinant protein was detected. Finally, the purified protein was dialyzed against pollen medium at 4°C, 5000 rpm, and then stored at -80°C.

[0093] 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, 120 rpm for 30 min on a shaker. The RNA extraction, cDNA reverse transcription and qRT-PCR experiment were the same as in Example 1.

[0094] To verify whether PbrRALF6 gene is involved in GSI, we treated 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 PbrRALF6 in self SI-treated pollen tubes was lower than that in non-self SC-treated and pCold (as shown in Fig. 2). Therefore, PbrRALF6 gene is involved in pear GSI by responding to self S-RNase. Figure 4

[0095] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can also be made, which should be considered as the protection scope of the present application.

[0096] SEQ ID NO: 1

[0097] ATGGGATTCAAGCTCTACCTGATCACCCTCATGGTGGTCTTGGCCATGGTGGCTAGGTCA

[0098] TCGGCCAAGGTCCACGATGCCTCGTGGACTCTCAGCCACTTCGGCGATCACGATGATGA

[0099] GTCGTTGCTCGACAGCGCCAACAGCAAAGTCGGCGACTTGATCGGAGAGGAGAATGAG

[0100] ATGATGATGGAGTCAGAGAGCACTCGTAGGACACTCAGAGGCCGCGGCACGAGGTACA

[0101] TTGGCTATGGTGCCCTGAGACGCAATGCTGTTCCATGCGGACGCCGCGGTCGGTCTTACT

[0102] ACAACTGCCAAGGAAGGCAGAGGGCTAACCCTTACAAGCGCGGTTGCCGGTACATCAC

[0103] CCGCTGCGCCAGACGCTAA

[0104] SEQ ID NO: 2

[0105] ​MGFKLYLITLMVVLAMVARSSAKVHDASWTLSHFGDHDDESLLDSANSKVGDLIGEENEM

[0106] MMESESTRRTLRGRGTRYIGYGALRRNAVPCGRRGRSYYNCQGRQRANPYKRGCRYITRCARR*

[0107] SEQ ID NO: 3

[0108] 5'- ATGGGATTCAAGCTCTACCTGATCACC -3'

[0109] SEQ ID NO: 4

[0110] 5'- GCGTCTGGCGCAGCGGGT -3'

[0111] SEQ ID NO: 5

[0112] 5'- GCATTGCGTCTCAGGGCACC -3'

[0113] SEQ ID NO: 6

[0114] 5'- CGCAATGCTGTTCCATGCGG -3'

[0115] SEQ ID NO: 7

[0116] 5'- CGACTTGATCGGAGAGGAGAATG -3'

[0117] SEQ ID NO: 8

[0118] 5'- GCCTTCCTTGGCAGTTGTAGTAA -3'

[0119] SEQ ID NO: 9

[0120] 5'- CCCTTCACTTGGTTCTCCGT -3'

[0121] SEQ ID NO: 10

[0122] 5'- TAATCAGCAAGCGTGCGACC -3'

[0123] SEQ ID NO: 11

[0124] 5'- ATGTACGATTATTTTCAATTTACGCAGCAAT -3'

[0125] SEQ ID NO: 12

[0126] 5'- ATACTGAACACTGGAGGGGCAGG -3'

[0127] SEQ ID NO: 13

[0128] 5'- ATGGCGAGATACGATTATTTTCAATTTACGC -3' SEQ ID NO: 14

[0129] 5'- ATACTGAATATCATCAATGGGGCAGAA -3'

[0130] SEQ ID NO: 15

[0131] 5'- ATGTACGATTATTTTCAATTTACGCAGCAAT -3' SEQ ID NO: 16

[0132] 5'- ATACTTAACATCGGCCGGGCAG -3'

[0133] SEQ ID NO: 17

[0134] 5'- ATGTACGATTATTTTCAATTTACGCAGCAAT -3' SEQ ID NO: 18

[0135] 5'- ATACTGAATACTATTGTTTGGGCAAAAATG -3'.

Claims

1. PbrRALF6 Genes or related to the above PbrRALF6 The application of gene-related biomaterials in at least one of the following (a1)-(a6): (a1) Application in promoting pollen tube growth in pear flowers; (a2) Application in the preparation of products that promote the growth of pear pollen tubes; (a3) Application in improving ROS levels at the tips of pear pollen tubes; (a4) Application in the preparation of products that improve the ROS level at the tip of pear pollen tubes; (a5) Application in improving the efficiency of pear in vitro pollination; (a6) Application in the preparation of products that improve the efficiency of pear in vitro pollination; The aforementioned PbrRALF6 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The above and PbrRALF6 Gene-related biological materials are at least one of the following (c1)-(c6): (c1) PbrRALF6 Proteins encoded by genes; (c2) contains the above PbrRALF6 Gene expression cassettes; (c3) contains the above PbrRALF6 A recombinant vector of the gene, or a recombinant vector containing the expression cassette described in (c2); (c4) contains the above PbrRALF6 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in (c2), or recombinant microorganisms containing the recombinant vector described in (c3); (c5) contains the above PbrRALF6 A transgenic plant cell line containing the gene, or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3); (c6) contains the above PbrRALF6 Magnetic transfection reagent for genes, or magnetic transfection reagent containing the expression cassette described in (c2), or magnetic transfection reagent containing the recombinant vector described in (c3); The application described is overexpression.

2. The application according to claim 1, characterized in that, The PbrRALF6 The protein encoded by the gene is either (d1) or (d2): (d1) A protein with the amino acid sequence shown in SEQ ID NO.2; (d2) A fusion protein to which a protein tag is attached to the N-terminus and / or C-terminus of (d1).

3. The application according to claim 1, characterized in that, The above PbrRALF6 The gene is stably overexpressed in pear pollen tubes, promoting pollen tube growth and / or increasing ROS levels at the pollen tube tip.

4. The application according to claim 3, characterized in that, The pear pollen was treated in vitro using magnetic transfection overexpression technology. PbrRALF6 The gene is stably overexpressed in pear pollen tubes.

5. A method for promoting the growth of pollen tubes in pear blossoms, characterized in that, The as described in claim 1 PbrRALF6 The gene is stably overexpressed in pear pollen tubes, promoting pollen tube growth.

6. A method for increasing the ROS level at the tip of pear pollen tubes, characterized in that, The as described in claim 1 PbrRALF6 The gene was stably overexpressed in pear pollen tubes, increasing the ROS level at the tip of the pollen tubes.

7. A method for improving the efficiency of in vitro pollination of pears, characterized in that, The as described in claim 1 PbrRALF6 The gene is stably overexpressed in pear pollen tubes, promoting pollen tube growth and improving the efficiency of pear in vitro pollination.

8. The method according to any one of claims 5-7, characterized in that, The pear pollen was treated in vitro using magnetic transfection overexpression technology. PbrRALF6 The gene is stably overexpressed in pear pollen tubes.

9. The method according to claim 8, characterized in that, The magnetic transfection overexpression technique specifically includes the following steps: (1) Design primers for PCR amplification PbrRALF6 Genes, will be described PbrRALF6 The gene was inserted into the XbaI and BamHI restriction sites of the LAT52::GFP vector to construct the recombinant plasmid PbrRALF6. - LAT52::GFP; (2) MagTransf® transfection reagent and the recombinant plasmid PbrRALF6-LAT52::GFP were mixed to prepare MagTransf® / PbrRALF6. - LAT52::GFP transfection reagent, using the MagTransf® / PbrRALF6 described above. - Pollen cells were treated with LAT52::GFP transfection reagent.

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