Small peptides that promote in vitro growth of rice pollen tubes, their expression methods and applications

Abstract

This invention discloses a small peptide protein that promotes the in vitro growth of rice pollen tubes, its expression method, and its application, belonging to the field of biotechnology. The small peptide protein for promoting the in vitro growth of rice pollen tubes in this invention is THION22, and its amino acid sequence is shown in SEQ ID No. 2. The expression method of this small peptide protein is as follows: the rice THION22 gene is cloned, transformed and expressed in *E. coli* to obtain a recombinant vector, and the obtained recombinant vector is used for the expression and purification of the small peptide protein. This invention applies THION22 small peptide protein to promote the in vitro growth of rice pollen tubes, specifically by collecting rice pollen grains from dehiscent anthers, transferring the pollen grains to a liquid culture medium containing THION22 small peptide protein, and culturing them under humid conditions in the dark. This invention, by applying THION22 small peptide protein in vitro to the liquid culture medium for rice pollen germination, can prolong the pollen tube rupture time, promote pollen tube growth, and maintain the integrity of the pollen tubes, which has important application value for rice hybridization breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a small peptide protein that promotes the in vitro growth of rice pollen tubes, its expression method, and its application. Background Technology

[0002] Angiosperms require double fertilization to form seeds and complete their reproduction. Double fertilization involves a complex and continuous process of interaction between the stamen and pistil. This interaction begins when pollen, dispersed by a medium, lands on the stigma. After undergoing processes such as pollen capture, adhesion, and hydration, the mature stigma recognizes and accepts the pollen grains. Upon successful recognition, the pollen grain germinates a pollen tube, which passes through the stigma, guiding tissue, and ultimately reaches the ovule to release sperm cells, completing double fertilization. The pollen tube plays a crucial role in this complex process; therefore, its growth is equally vital for plant double fertilization.

[0003] Researchers have conducted extensive studies on the molecular mechanisms of female-male interactions during fertilization using model plants such as Arabidopsis thaliana and Sophora flavescens, achieving a series of significant breakthroughs in the field of pollen tube guidance. Studies have shown that signaling molecules secreted by various cells in female tissues and gametophytes interact with receptors in the pollen tube, precisely regulating pollen tube guidance. Secretory signals from the pistil include small peptides, glycoproteins, and hormones. Among these, the signal transduction pathway formed by secreted small peptides and receptor-like protein kinases located on the pollen tube has been found to play a crucial regulatory role in multiple processes of pollen tube guidance. Small peptides are small biomolecules widely found in eukaryotes, generally not exceeding 100 amino acids. Existing research indicates that plant small peptides can participate in the regulation of many important biological processes in the form of signaling molecules, including regulating plant growth, development, reproduction, symbiotic relationships with microorganisms, and physiological processes such as stress responses.

[0004] Rice (Oryza sativa) is an important food crop and a typical angiosperm that requires double fertilization to produce seeds and complete its reproduction. Compared with Arabidopsis thaliana, rice pollen has poor viability and quickly loses its activity after being isolated from the plant. This poses a significant obstacle to research on in vitro pollination and pollen tube growth in rice. Furthermore, during in vitro pollen tube culture, pollen tubes rupture after only 10 minutes of germination, leading to growth stagnation. Therefore, extending the growth time of rice pollen tubes and maintaining their integrity is of significant practical value for rice hybridization breeding.

[0005] THION proteins are plant thioproteins widely found in various plants, such as those in the Poaceae, Loranthaceae, and Brassicaceae families, including Arabidopsis thaliana. THION family proteins generally contain no more than 100 amino acids, and each contains a signal peptide at its N-terminus. Studies have reported that THION proteins are widely involved in plant defense against biological stresses such as diseases and pests. THION22 is a cysteine-rich small peptide protein containing 119 amino acids, including a 28-amino acid signal peptide at its N-terminus (MEVKKVAMVAVCCMFILLFPGQQQQVAA). As a small peptide protein secreted by the pistil, THION22 participates in receptor binding on rice pollen, maintaining pollen tube integrity and promoting pollen tube growth. Summary of the Invention

[0006] To address the above problems, this invention provides a small peptide protein that promotes the in vitro growth of rice pollen tubes, its expression method, and its application. The THION22 small peptide protein is obtained through prokaryotic expression and purification in Escherichia coli. When the THION22 small peptide protein is applied in vitro to rice pollen germination medium, it can promote the growth of rice pollen tubes and maintain the integrity of the rice pollen tubes.

[0007] This invention is achieved through the following technical solution:

[0008] A small peptide protein that promotes the in vitro growth of rice pollen tubes, the small peptide protein being THION22 small peptide protein, the amino acid sequence of which is shown in SEQ ID No. 2.

[0009] The recombinant vector for the small peptide protein that promotes the in vitro growth of rice pollen tubes, as described above, was obtained by cloning the rice THION22 gene and transforming it into Escherichia coli.

[0010] Furthermore, the method for constructing the recombinant vector includes the following steps:

[0011] (1) Using cDNA from mature rice spikelet tissue as a template, the CDS sequence of the THION22 gene was amplified and cloned into the pET28a(+)-6xHis-MBP vector, and then transformed into competent Escherichia coli cells by heat shock.

[0012] (2) Select suitable single colonies for PCR identification and sequencing comparison. After confirming that the results are correct, expand the culture and extract the recombinant plasmid.

[0013] (3) The recombinant plasmid from step (2) was transferred into the prokaryotic expression strain BL21(DE3) of Escherichia coli using heat shock for expression.

[0014] The method for expressing and purifying small peptide proteins in a recombinant vector as described above includes the following steps:

[0015] (1) The constructed Escherichia coli strain containing recombinant plasmid was cultured and then isopropyl-β-D-thiopyranoside (IPTG) was added to induce the expression of THION22 small peptide protein.

[0016] (2) Collect bacterial cells by centrifugation, resuspend them in phosphate buffered saline containing imidazole, break the cells, and then separate soluble proteins from the cell debris under high-speed centrifugation. The target protein is detected by gel electrophoresis.

[0017] (3) Load the 6xHis-MBP labeled protein onto a Ni2+ affinity column and wash it with phosphate-buffered saline containing imidazole. Then, elute the target protein from the column with phosphate-buffered saline containing imidazole. Finally, under centrifugation and concentration conditions, exchange all proteins into Tris buffer and store the resulting THION22 small peptide protein concentrate.

[0018] Further, in step (1), the OD of the *E. coli* strain after culture... 600 The value is 0.6 to 0.7.

[0019] Further, in step (1), the induction of THION22 small peptide protein expression is carried out at a temperature of 15-18℃ and a rotation speed of 170-200rpm for 13-15h.

[0020] The application of the small peptide protein that promotes the in vitro growth of rice pollen tubes, as described above, involves applying the THION22 small peptide protein to promote the in vitro growth of rice pollen tubes.

[0021] Further, the application specifically involves: collecting rice pollen grains from dehisced anthers, transferring the fresh pollen grains to a liquid culture medium containing THION22 small peptide protein, and culturing them in the dark under humid conditions at 28–30°C for 10–40 min; the components and contents of the liquid culture medium are: 1–50 nM THION22 small peptide protein, 15–20% sucrose, 8–12% PEG4000, 2–4 mM Ca(NO3)2·4H2O, 35–40 mg / L boric acid, and 2–4 mg / L vitamin B1.

[0022] Furthermore, the components and contents of the liquid culture medium are as follows: 10 nM THION22 small peptide protein, 20% sucrose, 10% PEG4000, 3 mM Ca(NO3)2·4H2O, 40 mg / L boric acid and 3 mg / L vitamin B1.

[0023] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0024] 1. This invention utilizes THION22 small peptide protein, obtained through prokaryotic expression and purification in *E. coli*. When applied in vitro to rice pollen germination liquid culture medium, this THION22 small peptide protein prolongs pollen tube rupture time, effectively promoting pollen tube growth while maintaining pollen tube integrity. The effect of promoting pollen tube growth and maintaining its integrity is most pronounced when the concentration of THION22 small peptide protein in the culture medium is 10 nM. This has significant application value for rice pollen tube research and rice hybridization breeding.

[0025] 2. In this invention, compared with rice pollen germination liquid culture medium without THION22 peptide protein, when the concentration of THION22 peptide protein in the culture medium is 10 nM, within 40 min, the pollen tube rupture rate can be reduced by at least 44%, the pollen tube length can be extended by at least 173 μm, and the pollen tube growth rate can be increased by at least 8 μm / min. It is evident that in vitro application of THION22 peptide protein can effectively prolong the pollen tube rupture time, promote rice pollen tube growth, and maintain the integrity of rice pollen tube growth. Attached Figure Description

[0026] Figure 1 This is a diagram illustrating the prokaryotic expression and purification process of the THION22 small peptide protein in Example 1.

[0027] Figure 2 This is a microscopic observation of the effect of different concentrations of THION22 small peptide protein on pollen tube growth in Example 2.

[0028] Figure 3 This is a comparison graph showing the effect of different concentrations of THION22 small peptide protein on pollen breakage rate in Example 2.

[0029] Figure 4 This is a comparison diagram of the effects of different concentrations of THION22 small peptide protein on pollen tube growth length in Example 2.

[0030] Figure 5 This is a microscopic observation of the effect of 10 nM THION22 small peptide protein on pollen tube growth in Example 3.

[0031] Figure 6 This is a comparison chart showing how THION22 small peptide protein at a concentration of 10 nM reduced pollen breakage rate in Example 3.

[0032] Figure 7 This is a comparison chart showing how THION22 small peptide protein at a concentration of 10 nM promotes pollen tube growth length in Example 3.

[0033] Figure 8 This is a comparison chart showing the rate at which THION22 small peptide protein at a concentration of 10 nM promotes pollen tube growth in Example 3. Detailed Implementation

[0034] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0035] Example 1: Expression and purification of THION22 small peptide protein in Escherichia coli prokaryotes

[0036] SEQ ID No. 1: Nucleotide sequence of the rice THION22 gene

[0037] ATGGAGGTGAAGAAGGTGGCCATGGTTGCTGTCTGCTGCATGTTCATACTGCTGTTTCCAGGCCAGCAGCAGCAGGCTGGCTGCCATGTCCAGGATCTGCAGATGCTACCACGAATGCTTGCCCAACTGCGGCCTGCGCAATTCTCGCTCCTTCTGCAAGGTGTTCTGCGGCAGCTGCTGC GTCTTCAATCCAGTTCACAATTGCACTAGCACCGATGCGGCGGCCGCGGCGCCAGCGATCGCCGGAGACGACTGCAGAATGATCTGCCTGAACTCCTTCTGCGGCGAGGCAGCTACAGGCTACTCGGGGCGAAACGATGCTGATGCTGCAGCTTGTCTCGATGGCTGCAGCAAAGGATGA

[0038] SEQ ID No. 2: Amino acid sequence of THION22 small peptide protein

[0039] MSRICRCYHECLPNCGLRNSRSFCKVFCGSCCVFNPVHNCTSTDAAAAAPAIAGDDCRMICLNSFCGEAATGYSGRNDADAAACLDGCSKG

[0040] The following describes the process of expressing and purifying THION22 small peptide protein in E. coli prokaryotes (e.g.) Figure 1 As shown):

[0041] Figure 1In this study, a small peptide protein (29aa-91aa) of THION22 was expressed in vitro using prokaryotic expression. The protein fragment was approximately 10kDa in size. After being tagged with a 6xHis-MBP tag protein, the recombinant protein was approximately 56kDa in size. R1 represents the protein before elution, E30 represents the protein after elution with 30mM imidazole, and R2 represents the purified protein.

[0042] I. Construction of the carrier

[0043] (1) Using cDNA from mature rice spikelet tissue as a template, the CDS sequence (29aa-91aa) of the THION22 gene was amplified. The sequence was then cloned into a pET28a(+) vector containing a 6xHis-MBP tag using EcoRI and XbaI restriction sites, and transformed into DH5α competent Escherichia coli cells via heat shock transformation. After culturing at 37℃ for 12–16 h, suitable single colonies were picked, identified by PCR, sequenced, and confirmed to be correct. These colonies were then expanded and recombinant plasmids were extracted.

[0044] (3) The recombinant plasmid in (1) was transferred into the prokaryotic expression Escherichia coli strain BL21(DE3) by heat shock. After culturing in a 37℃ incubator for 12-16 h, suitable single colonies were picked and identified by PCR, sequencing and comparison. After confirming that the colonies were correct, prokaryotic expression was performed.

[0045] II. Protein expression and purification in prokaryotes

[0046] (1) The *E. coli* BL21(DE3) containing the recombinant plasmid constructed above was cultured at 37°C for approximately 200 mL until OD was reached. 600 After the value reached 0.6, 1 mM isopropyl-β-D-thiopyranoside was added and protein expression was induced for 14 h at 16℃ and 180 rpm.

[0047] (2) Escherichia coli BL21 cells containing recombinant plasmids were collected by centrifugation at 5000 rpm for 15 min, resuspended in phosphate-buffered saline (PBS; pH 7.5) containing 10 mM imidazole, and disrupted using a low-temperature ultra-high pressure cell disruptor. Soluble proteins were separated from the cell debris by high-speed centrifugation at 12000 rpm for 1 h, and the target proteins were detected by SDS-PAGE gel electrophoresis.

[0048] (3) Load the 6xHis-MBP labeled protein onto a Ni2+ affinity column and wash with 100 mL of washing buffer (PBS containing 20-100 mM imidazole). Elute the target protein from the column with PBS containing 300 mM imidazole. Use a centrifuge to exchange all proteins into Tris buffer (20 mM Tris-HCl, 150 mM NaCl [pH 7.5]) and concentrate to approximately 14.9 mg / mL for storage.

[0049] Example 2: Effects of different concentrations of THION22 small peptide protein on in vitro growth of rice pollen tubes

[0050] (1) First, prepare the liquid culture medium for in vitro germination of rice pollen tubes: 20% sucrose, 10% PEG4000, 3mM Ca(NO3)2·4H2O, 40mg / L boric acid and 3mg / L vitamin B1.

[0051] (2) The THION22 small peptide protein purified in Example 1 was diluted with liquid culture medium to form a mixture with concentrations of 0 nM, 1 nM, 10 nM, 25 nM and 50 nM.

[0052] (3) In the field or greenhouse, collect rice pollen grains with dehiscent anthers by gently shaking the panicles of flowering wild-type rice ZH11. Immediately transfer these fresh pollen grains to blank and liquid culture media containing different concentrations of THION22 small peptide protein, and culture them in the dark under humid conditions at 30°C.

[0053] (4) After germination for 30 minutes, the germination and growth of pollen were observed and photographed in a bright field of view using a ZEISS488 microscope, and the length of pollen tubes was measured using ImageJ software.

[0054] The above experimental results are as follows Figure 2 , 3 As shown in Figure 4. Figure 2 Microscopic observation of the effects of different concentrations of THION22 small peptide protein on pollen tube growth. Figure 3 A comparative graph showing the effect of different concentrations of THION22 small peptide protein on pollen breakage rate. Figure 4 This is a comparison of the effects of different concentrations of THION22 peptide protein on pollen tube growth length.

[0055] Depend on Figure 3It was found that when the concentration of THION22 peptide protein was 0 and 1 nM, the pollen tube rupture rates were 80% and 68%, respectively, while when the concentrations were 10, 25, and 50 nM, the rupture rates were 27%, 30%, and 35%, respectively. This indicates that the pollen tube rupture rate was lowest at a concentration of 10 nM. Compared to a concentration of 0 nM, a concentration of 10 nM reduced the pollen tube rupture rate by 53%. This demonstrates that applying THION22 peptide protein in vitro to the liquid culture medium for rice pollen germination can reduce the pollen tube rupture rate and maintain pollen tube integrity.

[0056] Depend on Figure 4 It was found that when the concentration of THION22 peptide protein was 0 and 1 nM, the pollen tube length was 160 μm and 192 μm, respectively, while when the concentration was 10, 25, and 50 nM, the pollen tube length was 347 μm, 318 μm, and 307 μm, respectively. This indicates that the pollen tube length was longest at a THION22 peptide protein concentration of 10 nM. Compared to a THION22 peptide protein concentration of 0 nM, a concentration of 10 nM resulted in a pollen tube elongation of 187 μm. This demonstrates that THION22 peptide protein can effectively promote the in vitro growth of rice pollen tubes.

[0057] Example 3: Effect of 10 nM THION22 small peptide protein on in vitro growth of rice pollen tubes

[0058] (1) Prepare a liquid culture medium containing 10 nM THION22 small peptide protein for rice pollen using the same method as in Example 2.

[0059] (2) Drop liquid culture medium onto a concave glass slide, shake the pollen grains of wild-type rice ZH11 that is flowering onto the slide, place it in a humid box that can be protected from light, and incubate it in a 30°C incubator.

[0060] (3) The germination and growth of rice pollen tubes were observed and statistically analyzed at 10 min, 20 min, 30 min and 40 min after germination.

[0061] The above experimental results are as follows Figure 5 , 6 As shown in Figures 7 and 8. Figure 5 Microscopic observation of pollen tube growth promoted by 10 nM THION22 small peptide protein. Figure 6 A comparison of the effects of 10 nM THION22 peptide protein on pollen breakage rate. Figure 7A comparison of pollen tube growth length promoted by THION22 small peptide protein at a concentration of 10 nM. Figure 8 A comparison of the rate at which 10 nM THION22 peptide promotes pollen tube growth.

[0062] Depend on Figure 5 It was found that, through observation of in vitro growth of rice pollen tubes at four different time points (10, 20, 30, and 40 minutes), when 0 nM THION22 protein was applied, the pollen tube rupture rate increased with the extension of time, and the growth rate began to decrease after 10 minutes. However, after applying 10 nM THION22 protein, the length of rice pollen tubes increased and the growth rate also improved at all four time points, with the greatest difference observed at 10 minutes.

[0063] Depend on Figure 6 It was found that when the concentration of THION22 peptide protein was 0 nM, the pollen tube rupture rate increased from 23% to 91% within 10–40 minutes after pollen tube germination. However, when the concentration of THION22 peptide protein was 10 nM, the pollen tube rupture rate increased from 13% to 47%. This indicates that after in vitro application of 10 nM THION22 peptide protein, the pollen tube rupture rate decreased by at least 44% within 40 minutes. This demonstrates that THION22 peptide protein can reduce the pollen tube rupture rate and prolong the pollen tube rupture time.

[0064] Depend on Figure 7 It was found that when the concentration of THION22 peptide protein was 0 nM, the pollen tube length increased from 42 μm to 225 μm within 10–40 min after pollen tube germination. When the concentration of THION22 peptide protein was 10 nM, the pollen tube length increased from 110 μm to 398 μm. This indicates that after applying 10 nM of THION22 peptide protein in vitro, the pollen tube length increased by at least 173 μm within 40 min. This demonstrates that THION22 peptide protein can effectively promote pollen tube growth.

[0065] Depend on Figure 8It was found that when the concentration of THION22 peptide protein was 0 nM, the pollen tube growth rate increased from 4 μm / min to 8 μm / min within 0–10 min and 10–20 min after pollen tube germination, and then began to decline after 20–30 min. However, when the concentration of THION22 peptide protein was 10 nM, the pollen tube growth rate increased from 10 μm / min to 16 μm / min within 0–10 min and 10–20 min after pollen tube germination. This indicates that after in vitro application of 10 nM THION22 peptide protein, the pollen tube growth rate increased by at least 8 μm / min within 20 min. This demonstrates that THION22 peptide protein can effectively promote the growth rate of pollen tubes.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a small peptide protein that promotes the in vitro growth of rice pollen tubes, characterized in that, The small peptide protein was used to promote the in vitro growth of rice pollen tubes. The small peptide protein was THION22 small peptide protein, and its amino acid sequence is shown in SEQ ID No.

2.

2. The application of the small peptide protein for promoting in vitro growth of rice pollen tubes according to claim 1, characterized in that, The specific application involves collecting rice pollen grains from dehisced anthers, transferring the fresh pollen grains to a liquid culture medium containing THION22 small peptide protein, and culturing them in the dark under humid conditions at 28–30°C for 10–40 min. The components and contents of the liquid culture medium are: 1–50 nM THION22 small peptide protein, 15–20% sucrose, 8–12% PEG4000, 2–4 mM Ca(NO3)2·4H2O, 35–40 mg / L boric acid, and 2–4 mg / L vitamin B1.

3. The application of the small peptide protein for promoting in vitro growth of rice pollen tubes according to claim 2, characterized in that, The components and contents of the liquid culture medium are as follows: 10 nM THION22 small peptide protein, 20% sucrose, 10% PEG4000, 3 mM Ca(NO3)2·4H2O, 40 mg / L boric acid and 3 mg / L vitamin B1.

4. The application of the small peptide protein for promoting in vitro growth of rice pollen tubes according to claim 1, characterized in that, The method for expressing and purifying the small peptide protein includes the following steps: (1) The constructed Escherichia coli strain containing recombinant plasmid was cultured and then isopropyl-β-D-thiopyranoside was added to induce the expression of THION22 small peptide protein. (2) Collect cells by centrifugation, resuspend them in phosphate buffered saline containing imidazole, break the cells, and then separate soluble proteins from the cell debris under high-speed centrifugation. The target protein is detected by gel electrophoresis. (3) Load the 6×His-MBP labeled protein onto a Ni2+ affinity column and wash it with phosphate-buffered saline containing imidazole. Then, elute the target protein from the column with phosphate-buffered saline containing imidazole. Then, under centrifugation and concentration conditions, exchange all proteins into Tris buffer and store the resulting THION22 small peptide protein concentrate.

5. The application of the small peptide protein for promoting in vitro growth of rice pollen tubes according to claim 4, characterized in that, In step (1), the OD of the *E. coli* strain after culture... 600 The value is 0.6 to 0.

7.

6. The application of the small peptide protein for promoting in vitro growth of rice pollen tubes according to claim 4, characterized in that, In step (1), the expression of THION22 small peptide protein is induced at a temperature of 15-18℃ and a rotation speed of 170-200 rpm for 13-15 h.

7. The application of a recombinant vector expressing a small peptide protein that promotes the in vitro growth of rice pollen tubes, characterized in that, The rice THION22 gene was cloned to obtain a recombinant vector, which was then transformed into Escherichia coli to express a small peptide protein, which was used to promote the in vitro growth of rice pollen tubes. The nucleotide sequence of the rice THION22 gene is SEQ ID No. 1, and the amino acid sequence of the small peptide protein is SEQ ID No.

2.

8. The application of the recombinant vector expressing the small peptide protein that promotes the in vitro growth of rice pollen tubes according to claim 7, characterized in that, The method for constructing the recombinant vector includes the following steps: (1) Using cDNA from rice spikelet tissue at maturity as a template, the CDS sequence of the THION22 gene was amplified and cloned into the pET28a(+)-6×His vector, and then transformed into competent Escherichia coli cells by heat shock. (2) Select suitable single clones for PCR identification and sequencing comparison. After confirming that the sequence is correct, expand the culture and extract the recombinant plasmid. (3) The recombinant plasmid from step (2) was transferred into the Escherichia coli strain for expression using heat shock.

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