CN117886908A - Pear DEFL27 protein and in-vitro expression method and application thereof - Google Patents

Abstract

The invention discloses a pear DEFL27 protein, an in vitro expression method and application thereof, wherein the amino acid sequence of the pear DEFL27 protein is shown as SEQ ID NO. 1. Designing a primer clone pear DEFL27 protein gene PbrDEFL27, and constructing an escherichia coli recombinant expression plasmid PbrDEFL27-PET30a; transferring the constructed recombinant expression plasmid PbrDEFL27-PET30a into escherichia coli Rosetta (DE 3) to construct a pear DEFL27 recombinant expression strain; carrying out protein induction expression on the recombinant expression strain, and carrying out primary purification on the expressed crude protein by using a nickel column affinity chromatography method; desalting and purifying the initially purified DEFL27 protein by using a protein ultrafiltration tube; and then treating the pear pollen tube with the purified pear DEFL27 protein. The result shows that the protein can promote the elongation of the pollen tube and the enhancement of the active oxygen signal of the tip of the pollen tube.

Description

Pear DEFL27 protein and in-vitro expression method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a pear DEFL27 protein, an in-vitro expression method and application thereof. A pear DEFL27 protein is cloned, expressed and purified from pear flower columns, and pear pollen tubes are treated by the protein, so that the elongation promoting effect of the pear pollen tubes and the enhancement effect of tip active oxygen signals are proved.

Background

Pear is one of three fruits in China, the effective yield of pear depends on successful pollination and fertilization, and the precondition of pollination and fertilization is the normal growth of pollen tubes. Besides external climate and other factors, signal molecules such as active oxygen, polypeptide, hormone and the like in the flower column can influence the growth of pollen tubes. Polypeptide signal molecules, such as rapid alkalization factors, thioflavins, and various cysteine-rich proteins, play an extremely important role in the fertilization of pear pollination. However, the genes encoding the polypeptides are generally short and are not easy to annotate in the genome, so that resistance is brought to subsequent functional research. The pear DEFL27 protein is mainly in plant tissues such as pollen, leaves and the like, and subcellular localization shows that the pear DEFL27 protein is mainly distributed outside cells, and part of pear DEFL27 protein is also distributed between cell membranes and cell walls. Including plant defensins, function during the course of the plant immune response. The DEFL27 proteins identified by the invention are distributed in pear flower columns and participate in the growth and development regulation process of pear pollen tubes. However, the separation and purification difficulty of the purely natural DEFL27 protein is high, and the cost input is increased by utilizing chemical technology for synthesis.

By utilizing the genetic engineering technology, the prokaryotic expression strain can be transformed by constructing a prokaryotic expression vector. And then the expression and purification of the pear DEFL27 protein are completed through the steps of IPTG induced protein expression, column chromatography separation and purification of the protein and the like. The pear DEFL27 protein separated and purified by the method can completely meet the experimental requirements, and after the pear pollen tube is treated by the protein, the pear DEFL27 protein is found to promote the elongation of the pear pollen tube and remarkably enhance the active oxygen signal at the tip of the pollen tube.

Disclosure of Invention

The invention aims to provide a pear DEFL27 protein, and a coding gene and application thereof.

The invention also aims at providing an efficient and energy-saving method for separating and purifying the DEFL27 protein from pears.

The invention also aims to provide a method for promoting the elongation of the pollen tube and improving the pollen germination rate.

The aim of the invention can be achieved by the following technical scheme:

in a first aspect, the invention claims a pear DEFL27 protein, the amino acid sequence of which is shown in SEQ ID NO. 1. The protein is separated from flower columns of Dangshan pear, and can regulate the growth and development process of pear pollen tubes.

ERSVRLFSTAFVFVLLLVATGMMGPMLAEGRTCESQSRKFKGACLSRSNCASVCQT EGFPGGHCRGFRRRCFCTKHC(SEQ ID NO.1)。

In a second aspect, the invention claims a gene PbrDEFL27 encoding the aforementioned pear DEFL27 protein, the nucleotide sequence of the gene PbrDEFL27 being shown in SEQ ID NO. 2.

GAGCGTTCCGTGCGTTTGTTTTCGACTGCCTTCGTCTTTGTTCTTCTTTTGGTGGCTACAGGGATGATGGGACCAATGCTTGCGGAGGGTAGGACCTGTGAGTCTCAGAGCCGCAAGTTCAAGGGAGCCTGCCTGAGTAGAAGCAACTGTGCATCTGTTTGCCAGACTGAGGGCTTTCCTGGAGGCCATTGTCGTGGCTTTCGCCGCAGATGCTTCTGCACTAAACATTGT(SEQ ID NO.2)。

In a third aspect, the invention claims a biological material comprising the above-described gene PbrDEFL27, said biological material being an expression cassette, a recombinant vector, a host bacterium or a transgenic cell line.

Further, the recombinant vector described in the above biological material is a recombinant expression vector.

In a specific embodiment of the present invention, the host bacterium is Rosetta (DE 3), but is not limited thereto.

In a fourth aspect, the present invention claims the above-mentioned pear DEFL27 protein, the above-mentioned gene PbrDEFL27, or the above-mentioned biomaterial for use in (1) or (2) or (3) as follows:

(1) Promoting the elongation of pollen tube and improving the germination rate of pollen;

(2) The active oxygen signal of the tip of the pollen tube is enhanced;

(3) Improving pollen fertility rate.

Further, the above application is: the pear DEFL27 protein is adopted to treat pollen so as to promote the elongation of pollen tubes and improve the germination rate of the pollen. Or the gene PbrDEFL27 is adopted to construct a recombinant expression vector and the transgenic engineering bacteria are adopted to express the pear DEFL27 protein in vitro, or the biological material is adopted to express the pear DEFL27 protein in vitro, and then the pear DEFL27 protein obtained by in vitro expression is utilized to treat pollen so as to promote the elongation of pollen tubes and improve the pollen germination rate.

In a fifth aspect, the invention claims a method for promoting pollen tube elongation to increase pollen germination rate, wherein the pear DEFL27 protein is used for treating pollen to promote pollen tube elongation to increase pollen germination rate.

In a sixth aspect, the invention claims a method for in vitro expression of pear DEFL27 protein, comprising the steps of:

(1) Extracting total RNA of a flower column of Dangshan pear, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by using the cDNA as a template to obtain a gene PbrDEFL27 with a nucleotide sequence shown as SEQ ID NO. 2;

(2) Constructing a prokaryotic expression vector of the pear DEFL27 protein escherichia coli;

(3) Transferring the prokaryotic expression vector constructed in the step (2) into an escherichia coli strain Rosetta (DE 3) by a heat shock method to obtain a pear DEFL27 protein prokaryotic expression strain;

(4) Culturing the expression strain with a liquid culture medium, placing the culture solution on ice for 10-30 minutes after reaching a certain OD value, and adding IPTG to induce protein expression;

(5) Purifying and inducing to obtain pear DEFL27 protein with an amino acid sequence shown as SEQ ID NO. 1;

(6) And (5) expression identification of the recombinant pear DEFL27 protein.

As a preferable technical scheme, the flower column sample of the Dangshan pear in the step (1) is obtained from a lake-maturing experimental base of Nanjing agricultural university, and after the flower column is manually removed, quick-freezing is carried out by using liquid nitrogen, and then the flower column sample is stored in a refrigerator at the temperature of minus 80 ℃.

Further preferably, the nucleotide sequences of the forward primer and the reverse primer used for PCR amplification of the full length of the gene PbrDEFL27 in the step (1) are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

Forward primer: 5'-GATATCGGATCCGAATTCGAGCGTTCCGTGCGTTTG-3' (SEQ ID NO. 3);

reverse primer: 5'-GGTGGTGGTGGTGCTCGAGACAATGTTTAGTGCAGAAGCATCTG-3' (SEQ ID NO. 4).

Further preferably, the reaction system of the PCR amplification in the step (1) is: 2X Phanta Max Buffer. Mu.l, forward and reverse primer 2. Mu.l, cDNA template 1. Mu.l, dNTP Mix (10 mM each) 1. Mu.l, phanta Max Super-Fidelity DNA Polymerase. Mu.l, ddH ₂ O18 μl; the PCR amplification procedure was: the first step is pre-denatured at 95 ℃ for 3 minutes, the second step is pre-denatured at 95 ℃ for 15 seconds, annealed at 60 ℃ for 15 seconds, extended at 72 ℃ for 1 minute, cycled 35 times, and the third step is extended at 72 ℃ for 10 minutes.

Further preferably, in the step (2), the E.coli vector used for constructing the prokaryotic expression vector of the pear DEFL27 protein is PET-30a, and the cleavage sites thereof are EcoRI and XhoI.

As a detailed technical scheme, in the step (2), the gene PbrDEFL27 is connected to two ends of the digestion site of the vector PET-30a to form a prokaryotic expression vector.

Further preferably, the heat shock method in step (3) refers to adding the prokaryotic expression vector plasmid to E.coli strain Rosetta (DE 3), standing on ice for 30 minutes, then standing on ice for 45 seconds in a water bath at 42℃for 2 minutes, and shaking at 37℃for 50 minutes to 1 hour.

It is further preferred that the liquid medium in step (4) is E.coli medium whose main components are peptone, yeast extract and sodium chloride, which are common commercial medium known to those skilled in the art, such as liquid LB medium, which contains 100. Mu.g/ml kanamycin. When the OD value reaches 0.4-0.6, the bacterial liquid is placed on ice for 10-30 minutes, then an inducer is added, the inducer is IPTG with the final concentration of 0.5mmol/L, the induction time is 8-10 hours, and the temperature is 25 ℃.

Further preferably, the detailed steps for purifying the pear DEFL27 protein mentioned in step (5) are: after the completion of the induction of expression, the cells were resuspended in 1 XPBS by centrifugation at 12000rpm at 4℃for 10 minutes using an ultracentrifuge, and the supernatant was collected by decanting, and the suspension was disrupted to a clear state using an ultrasonic disrupter. Then, the mixture was ultracentrifuged at 4℃and 12000rpm for 8 minutes, and the supernatant was filtered and collected with a 0.45 μm aqueous filter membrane, and purified by using a nickel column affinity chromatography method. Purified protein was collected, concentrated and desalted using a 10kDa ultrafiltration tube at 4℃and 7000rpm, and finally the concentrated protein was stored in a-80℃refrigerator.

Wherein the ultrasonic power of the ultrasonic crusher is 240W, the condition is that the ultrasonic crusher is driven for 4 seconds and stopped for 3 seconds, and the total time is 20-30 minutes;

the 1 XPBS was purchased from white shark 10 XPBS (phosphate buffer), diluted with distilled water and used;

the N end of the pear DEFL27 protein is provided with a histone Tag (His-Tag).

Further preferably, the method for identifying the pear DEFL27 protein in the step (6) is SDS-PAGE (polyacrylamide gel electrophoresis).

The specific method for promoting the elongation of the pear pollen tube and improving the pollen germination rate or/and enhancing the active oxygen signal at the tip of the pollen tube by adopting the pear DEFL27 protein refers to the pear pollen tube (such as 'Dangshan pear', but not limited to) treated by adopting the pear DEFL27 protein. The specific implementation process is to add pear pollen into a pollen culture medium containing pear DEFL27 protein for culturing. The absolute pixel values of their lengths were then counted and measured using measurement software, and their effects on the growth and development process of the pear pollen tube were then compared by mapping. In order to reduce measurement errors, empty proteins expressed under equivalent conditions were used as negative controls.

As a preferred technical scheme, the liquid pollen culture medium comprises the following formula: 10g of sucrose per 100ml of culture medium; 20g of polyethylene glycol 4000 (PEG 4000); boric acid 0.01g; calcium nitrate tetrahydrate (Ca (NO) ₃ ) ₂ ·4H ₂ O) 0.03g;2- (N-morpholinium) ethanesulfonic acid(MES) 30mM; adjusting pH to 6.0-6.5 with Tris or NaOH. Solid pollen culture medium 1.2% (g/100 ml) low melting agarose was added to liquid pollen culture medium.

As a preferred embodiment, the dye used for treating pear pollen tube tip active oxygen with pear DEFL27 protein is CM-H2DCFDA (diluted) at a final concentration of 20. Mu. Mol. The specific implementation process is as follows: adding pear pollen into solid pollen culture medium, culturing at 25deg.C and 120rpm for 1 hr, dripping liquid pollen culture medium containing pear DEFL27 protein onto the solid pollen culture medium, suspending pollen on the solid pollen culture medium, continuously culturing for 4 hr, adding CM-H2DCFDA with final concentration of 20 μmol into the sucked culture medium, dyeing for 30 min, separating pollen and culture medium dye by centrifuge at 2000rpm for 1min, sucking supernatant, adding appropriate amount of liquid pollen culture medium, centrifuging again (2000 rpm for 1 min), repeating twice, observing and photographing under green fluorescence microscope, and then counting fluorescence intensity.

The invention has the beneficial effects that:

research results show that the pear DEFL27 protein provided by the invention can promote the growth of pear pollen tubes to improve the pollen germination rate, can greatly reduce labor cost and improve pollination efficiency, and has very important theoretical and practical significance for agricultural production.

Drawings

FIG. 1 is a signal peptide predictive diagram of the sequence of the pear DEFL27 protein.

FIG. 2 is a diagram of a clone gel of the gene PbrDEFL27 of the pear DEFL27 protein; wherein 1 is an amplification product, and M is a 2000bp DNA standard molecular mass diagram.

FIG. 3 is a diagram of SDS-PAGE gel of pear DEFL27 protein expression; wherein, left 1 is the total protein of the induced empty prokaryotic expression strain, left 2 is the total protein of the induced prokaryotic expression strain thallus, and M is a protein standard quality diagram.

FIG. 4 is a graph showing pear DEFL27 protein-treated pear pollen tube statistics; wherein, A is a microscope photograph of the recombinant protein treatment and the non-recombinant protein treatment; b is a histogram of pollen tube length statistics (photo statistics at 2, 4, 6 hours, respectively).

FIG. 5 is a graph of observations and statistics of pear pollen tubes using CM-H2 DCFDA; wherein, A is photographed after 4 hours of cultivation, the left is the control right is the treatment group; b is a statistical line graph of fluorescence intensity.

Detailed Description

Reference will now be made in detail to the detailed embodiments of the present invention, examples of which are illustrated for the purpose of explanation only, and examples are not to be construed as limiting, as persons of skill in the art may adapt the detailed description to the essential features and objects thereof.

Example 1: construction of PbrDEFL27-PET30a recombinant plasmid

The flower column sample of the Dangshan pear is taken from a lake-maturing experimental base of Nanjing agricultural university, the flower column is manually taken down, quick-frozen by using liquid nitrogen, and then the flower column sample is stored in a refrigerator at the temperature of minus 80 ℃. In the experiment, the flower column of the Dangshan pear frozen at the temperature of-80 ℃ is taken out, and the total RNA of the flower column is extracted by using a plant total RNA extraction kit of the Noruzhan; reversing the extracted total RNA of the flower column into cDNA by using a full-gold reverse transcription kit; identifying and removing nucleotide sequences corresponding to the signal peptide of the polypeptide, and designing primers (shown as SEQ ID NO.3 and SEQ ID NO. 4) which comprise a gene sequence and a homology arm sequence, wherein the homology arm sequence comprises a part of vector skeleton and enzyme cleavage site sequences (EcoRI and XhoI); using the obtained cDNA as a template, and carrying out PCR amplification by using high-fidelity enzyme of the Norvezan, wherein the PCR amplification reaction system is as follows: 2X Phanta Max Buffer. Mu.l, forward and reverse primer 2. Mu.l, cDNA template 1. Mu.l, dNTP Mix (10 mM each) 1. Mu.l, phanta Max Super-Fidelity DNA Polymerase. Mu.l, ddH ₂ O18 μl; the PCR amplification procedure was: the first step is pre-denatured at 95 ℃ for 3 minutes, the second step is pre-denatured at 95 ℃ for 15 seconds, annealed at 60 ℃ for 15 seconds, extended at 72 ℃ for 1 minute, cycled 35 times, and the third step is extended at 72 ℃ for 10 minutes. Then, the PCR amplified products were identified by electrophoresis, and the length was judged to be approximately identical to the actual length of the gene, as shown in FIG. 2.

The PCR amplification product is recovered by using an agarose gel recovery kit of Shanghai worker to obtain a gene PbrDEFL27; then using a ClonExpress IIOne Step Cloning Kit homologous recombinase of NorvezanThe gene PbrDEFL27 and the PET-30a vector were ligated (cleavage sites EcoRI and XhoI) as follows: 5 Xbuffer 2. Mu.l, exnaseII 1. Mu.l, genes were added according to their concentrationThe carrier is added according to a conversion formuladdH ₂ O X μl, total line volume 10 μl, was reacted in a PCR apparatus at 37deg.C for 30 min to ligate the recombinant plasmid PbrDEFL27-PET30a; the recombinant plasmid was transferred into E.coli strain DH 5. Alpha. Using the heat shock method (adding the prokaryotic expression vector plasmid to E.coli strain, standing on ice for 30 minutes, then standing on ice for 45 seconds in a water bath at 42℃for 2 minutes, shaking culture at 37℃for 50 minutes to 1 hour, tu Ban, and overnight culture in a incubator at 37 ℃). The following day, monoclonal spots were picked and colonies were identified by PCR screening using novinaian 2 x Rapid Taq Master Mix. Sending the correct strain to Shanghai Biotechnology Co., ltd for Sanger sequencing to verify whether the strain accords with a reference gene sequence, and sending a test result to show that the sequence of a pear DEFL27 protein coding region is shown as SEQ ID NO. 2; the amino acid sequence of the pear DEFL27 protein is shown as SEQ ID NO. 1. The recombinant plasmid was designated as PbrDEFL27-PET30a.

Example 2: expression of pear DEFL27 protein gene in escherichia coli

1. Obtaining prokaryotic expression strain for expressing pear DEFL27 protein

The recombinant plasmid with correct sequence is transferred into escherichia coli strain Rosetta (DE 3) by a heat shock method, the plasmid is added according to concentration conversion, the adding amount is 10-100ng, the resistance of a culture dish is kanamycin (100 mug/ml), and the culture is carried out in a constant temperature incubator at 37 ℃ for overnight culture. The next day, colonies of positive clones with normal sizes on the dishes were selected and PCR verified for successful transfer. Successful strains were grown overnight with shaking in a shaker at 37℃and 220rpm, and glycerol bacteria (sterilized glycerol volume percent concentration 50%, bacteria: glycerol=7:3, v: v) were maintained to give prokaryotic expression strains of pear DEFL27 protein.

2. Inducible expression of pear DEFL27 recombinant protein

Taking out the stored prokaryotic expression strain from the refrigerator at the temperature of-80 ℃ according to the following ratio of 1:50 was added to 20ml of liquid LB medium (100. Mu.g/ml kanamycin resistance), placed at 37℃and shake cultivated at constant temperature of 220rpm for 10 hours, followed by 1:50 volume ratio, adding the bacterial liquid into 200ml liquid kanamycin-resistant LB culture medium, shake culturing at constant temperature of 37 ℃ and 220rpm for 3-4 hours, taking out the bacterial liquid, and immediately placing on ice for 10-20 minutes. Subsequently, IPTG (2 ml of uninduced bacterial liquid can be taken as a negative control before addition) with a final concentration of 0.5mmol/L is added to the bacterial liquid, the bacterial liquid is subjected to shaking induction for 10 hours in a shaking table at 25 ℃ and 220rpm, and 2ml of the induced bacterial liquid is taken. After centrifugation at 1200rpm of 2ml of the bacterial liquid after and before induction, the supernatant was poured, suspended in 1 XPBS, followed by adding a protein loading buffer and boiling in hot water. Subsequently, the loading amount of each well was 10. Mu.l by SDS-PAGE, and running gel was used to verify whether induction was successful.

Example 3: separation, purification, expression and identification of recombinant pear DEFL27 protein

The bacterial liquid which is successfully expressed is centrifuged for 10min at the temperature of 4 ℃ and the speed of 12000rpm to collect bacterial bodies. The cells were suspended in 1 XPBS, and the cells were disrupted by an ultrasonic disruption apparatus at 240W for 4 seconds and at 3 seconds until the bacterial liquid became clear. The disrupted bacterial cells were centrifuged again at 12000rpm at 4℃for 8 minutes, and the supernatant was collected by a syringe and filtered through a 0.45 μm aqueous filter (ice-working). Then, PBS was added to the pre-packed nickel column for washing, and then the supernatant was slowly added to the nickel column for loading, and after washing the nickel column with 20mL of 5mM imidazole buffer (pH=7.9), the nickel column was washed with 20mL of 20mM imidazole buffer (pH=7.9) to remove impurity proteins. Elution of pear DEFL27 protein by adding 10mL of 100mM concentration elution buffer (pH=7.9) followed by filtration and desalting of the protein using 10kDa protein ultrafiltration tube manufactured by Millipore company, at 4℃and 7000rpm for 20 minutes each time, adding pear pollen culture medium instead of PBS system after PBS filtration was completed and continuing centrifugation, collecting the protein after 4-5 times filtration (cut-off of about 1-1.5 mL) was started, and the collected protein was stored to-80 ℃. Finally, SDS-PAGE analysis was performed with a small amount of protein, and the results are shown in FIG. 3, lane 2.

Example 4: application of recombinant pear DEFL27 protein

Preparing a liquid pollen culture medium: 10g of sucrose per 100ml of liquid medium; 20g of polyethylene glycol 4000 (PEG 4000); boric acid 0.01g; calcium nitrate tetrahydrate (Ca (NO) ₃ ) ₂ ·4H ₂ O) 0.03g; 30mM of 2- (N-morpholino) ethanesulfonic acid (MES); adjusting Ph to 6.0-6.5 with Tris or NaOH.

A solid pollen culture medium was prepared, and 1.2% (g/100 ml) of low-melting agarose was added to the liquid pollen culture medium.

The collected and purified pear DEFL27 protein is used for treating the pollen of the Dangshan pear, and the pollen is stored at the temperature of minus 20 ℃ and is required to be put to the temperature of 4 ℃ in advance before being used. The pear DEFL27 protein is added into 1ml of liquid pollen culture medium according to the addition amount of 10nmol/L of final concentration, and after being fully mixed, about 0.1g of pollen is added, and the mixture is cultured at 25 ℃ and 120 rpm. The pollen tube lengths were statistically photographed at 2, 4, and 6 hours, respectively, measured using image pro plus software, and the data were plotted using prism software, as shown in fig. 4, a for the pollen tube length control before and after the addition of the purified protein, and B for the pollen tube length statistical histogram before and after the addition of the purified protein.

In order to evaluate the promotion effect of pear DEFL27 on the growth and development of pollen tubes, the same package of pollen is treated by empty protein expressed in a time period under the same condition, and the effect of the empty protein on the growth and development of the pear pollen tubes is counted by observation and photographing at the same time, and the empty protein is used as a control of an experimental group.

About 0.1g of "Dangshan pear" pollen is added into the solid pollen culture medium, after culturing for 1 hour at 25 ℃ and 120rpm, 200 mu L of liquid pollen culture medium with the final concentration of 10nmol/L of purified pear DEFL27 protein is dripped into the solid pollen culture medium, the pollen on the solid pollen culture medium is suspended, the culture is continued for 4 hours, and after the medium is sucked out, CM-H2DCFDA (diluted use) with the final concentration of 20 mu mol is added into the medium to dye the pollen tube active oxygen. After 30 minutes, centrifuging at normal temperature and 2000rpm for 1min, discarding supernatant, adding liquid pollen culture medium again, centrifuging (2000 rpm,1 min), washing off coloring agent, repeating for 2-3 times, and taking photos under green fluorescence microscope, and counting fluorescence intensity, wherein the result is shown in figure 5.

In order to evaluate the promotion effect of pear DEFL27 on the growth and development of pollen tubes, the same package of pollen is treated by using empty protein expressed in a time period under the same condition, and the same package of pollen is simultaneously dyed, observed and photographed, and the fluorescence intensity of the empty protein is counted.

Claims (9)

1. A pear DEFL27 protein is characterized in that the amino acid sequence of the pear DEFL27 protein is shown as SEQ ID NO. 1.

2. The gene PbrDEFL27 for encoding the pear DEFL27 protein according to claim 1, wherein the nucleotide sequence of the gene PbrDEFL27 is shown as SEQ ID No. 2.

3. A biological material comprising the gene PbrDEFL27 according to claim 2, characterized in that said biological material is an expression cassette, a recombinant vector, a host bacterium or a transgenic cell line.

4. The biomaterial according to claim 3, wherein the recombinant vector is a recombinant expression vector.

5. The pear DEFL27 protein of claim 1, the gene PbrDEFL27 of claim 1, or the biological material of claim 3 or 4 for use in (1) or (2) or (3) as follows:

(1) Promoting the elongation of pollen tube and improving the germination rate of pollen;

(2) The active oxygen signal of the tip of the pollen tube is enhanced;

(3) Improving pollen fertility rate.

6. The use of claim 5, wherein said pear DEFL27 protein is used to treat pollen to promote elongation of pollen tubes to increase pollen germination.

7. A method for promoting pollen tube elongation and increasing pollen germination rate, characterized in that the pear DEFL27 protein of claim 1 is used for treating pollen to promote pollen tube elongation and increase pollen germination rate.

8. An in vitro expression method of pear DEFL27 protein is characterized by comprising the following steps:

(1) Extracting total RNA of a flower column of Dangshan pear, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by using the cDNA as a template to obtain a gene PbrDEFL27 with a nucleotide sequence shown as SEQ ID NO. 2;

(2) Constructing a prokaryotic expression vector of the pear DEFL27 protein escherichia coli;

(3) Transferring the prokaryotic expression vector constructed in the step (2) into an escherichia coli strain Rosetta (DE 3) by a heat shock method to obtain a pear DEFL27 protein prokaryotic expression strain;

(4) Culturing the expression strain with a liquid culture medium, placing the culture solution on ice for 10-30 minutes, and adding IPTG to induce protein expression;

(5) Purifying and inducing to obtain pear DEFL27 protein with an amino acid sequence shown as SEQ ID NO. 1.

9. The method according to claim 8, wherein the forward primer and the reverse primer for PCR amplification of the full length of the gene PbrDEFL27 in step (1) have nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.3, respectively

NO. 4.