Application of Rice THION22 Small Peptide Protein in Breaking Reproductive Isolation between Indica and Japonica Rice Subspecies

Abstract

The present invention discloses an application of a rice THION22 small peptide protein in breaking the reproductive isolation between indica and japonica subspecies of rice, belonging to the field of biotechnology. The present invention breaks the pre-zygotic reproductive isolation of the indica and japonica subspecies of rice by applying the rice THION22 small peptide protein in vitro during the indica-japonica hybridization process. The application includes the rice THION22 small peptide protein promoting the growth of japonica rice pollen tubes in the pistil of the indica rice variety, and the rice THION22 small peptide promoting the acquisition of indica-japonica hybrid F1 hybrid seeds. The present invention effectively promotes the hybridization of japonica rice pollen with some indica rice varieties, promotes the growth of japonica rice pollen tubes, produces more F1 hybrid seeds, improves the efficiency of indica-japonica hybridization breeding, and has important application value for rice hybridization breeding and heterosis practice.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an application of a rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies. Background Art

[0002] Reproductive isolation is the primary mechanism for the formation of new species and for limiting gene flow between related species. It primarily refers to the inability of species to mate, or the production of infertile offspring when mating occurs. It plays a decisive role in the formation of new species. Reproductive isolation is divided into prezygotic reproductive isolation (an early-acting barrier) and postzygotic reproductive isolation (a late-acting barrier). In flowering plants, prezygotic reproductive isolation occurs through the interaction between male gamete pollen and female reproductive organs; postzygotic reproductive isolation occurs when the zygote formed after fertilization is unable to form fertile offspring normally. Currently, research on reproductive isolation mainly focuses on postzygotic reproductive isolation, with less research on prezygotic reproductive isolation.

[0003] Rice (Oryza sativa) is a major food crop encompassing over 20 wild species and two cultivated species (Asian rice and African rice). Asian cultivated rice consists of two subspecies (japonica and indica). The extensive genetic diversity of rice can generate heterosis, also known as hybrid vigor, in which hybrid rice exhibits superior traits compared to the parental inbred lines. However, reproductive isolation is prevalent between different rice species and subspecies, significantly hindering the ability to harness heterosis to produce superior offspring during breeding. The significant prezygotic reproductive isolation observed in indica-japonica hybrids makes F1 seeds from these hybrids difficult to obtain. Currently, relatively little research has examined prezygotic reproductive isolation within the indica-japonica subspecies of rice, a key factor hindering the utilization of heterosis to produce superior rice varieties.

[0004] THION22 is a cysteine-rich protein with a full-length amino acid sequence of 119 amino acids, including a 28-amino acid N-terminal signal peptide and 14 cysteines within the 91-amino acid C-terminus. THION22, a small peptide secreted by the pistil, binds to receptors on pollen, maintaining pollen tube integrity and promoting its growth. The THION22 peptide, cloned from japonica rice, effectively promotes pollen tube growth both in vitro and in vivo. Summary of the Invention

[0005] To address the above problems, the present invention provides an application of rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies. By applying rice THION22 small peptide protein in vitro during the indica-japonica hybridization process, the growth of japonica rice pollen tubes in indica rice pistils can be promoted, thereby obtaining more F1 generation hybrid seeds and improving the efficiency of indica-japonica hybridization breeding.

[0006] The present invention is achieved through the following technical solutions:

[0007] A rice THION22 small peptide protein is used to break the reproductive isolation of rice indica and japonica subspecies. The rice THION22 small peptide protein is applied in vitro during the indica and japonica hybridization process to break the pre-zygotic reproductive isolation of rice indica and japonica subspecies.

[0008] Furthermore, the rice THION22 peptide protein promotes the growth of pollen tubes in japonica rice in the pistil of indica rice varieties, specifically by:

[0009] (1) When the rice is in its flowering stage, the indica rice variety is used as the female parent, and the rice is manually emasculated and bagged;

[0010] (2) Collect pollen from flowering japonica rice varieties and quickly transfer the pollen grains to the stigma of indica rice varieties;

[0011] (3) After pollination, rice THION22 peptide protein was applied to the base of the pistil of indica rice.

[0012] Furthermore, the concentration of the rice THION22 small peptide protein is 8-12 nM, and the volume is 8-12 uL.

[0013] Furthermore, the rice THION22 peptide promotes the acquisition of indica-japonica hybrid F1 generation hybrid seeds, and the specific operation is as follows:

[0014] (1) When the rice is in its flowering stage, the indica rice variety is used as the female parent, and the rice is manually emasculated and bagged;

[0015] (2) Cut off the flowering ears of japonica rice varieties and place them upside down on the pistils of indica rice varieties for pollination;

[0016] (3) After pollination, the entire indica rice panicle is evenly sprayed with rice THION22 peptide protein and then bagged.

[0017] Furthermore, the concentration of the rice THION22 small peptide protein is 8-12 nM, and the volume is 2-3 mL.

[0018] Furthermore, the amino acid sequence of the rice THION22 small peptide protein is shown as SEQ ID No.2.

[0019] Furthermore, the rice THION22 small peptide protein is obtained by prokaryotic expression and purification in Escherichia coli.

[0020] Furthermore, the male parent in the indica-japonica rice hybridization process is the japonica rice variety ZH11, and the female parent is the indica rice variety IR26, MH63, MH86 or YZX.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0022] 1. The present invention applies rice THION22 peptide protein in vitro during the hybridization process of indica and japonica rice, which can effectively promote the hybridization of japonica rice pollen with some indica rice varieties, promote the growth of japonica rice pollen tubes, produce more F1 hybrid seeds, and improve the efficiency of indica and japonica hybrid breeding. It has important application value for rice hybrid breeding and heterosis practice.

[0023] 2. During indica-japonica rice hybridization, the present invention, by applying 10 nM of rice THION22 peptide protein in vitro, enabled japonica rice pollen to germinate on the stigma of indica rice pistils, significantly increased the rate of pollen tube production, and increased the rate of japonica rice pollen tubes reaching ovules by over 30%, compared to when no THION22 peptide protein was applied. Furthermore, the invention promoted the production of F1 hybrids, increasing the seed set rate of F1 generation by over 27%, significantly improving the efficiency of indica-japonica hybridization breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a microscopic observation of the rice THION22 peptide protein promoting the growth of japonica rice ZH11 pollen tubes in Example 1.

[0025] Figure 2 This is a statistical graph showing the rate of pollen tube growth reaching ovules in japonica rice ZH11 promoted by rice THION22 small peptide protein in Example 1.

[0026] Figure 3 This is an observation diagram of the rice THION22 small peptide protein promoting the acquisition of indica-japonica hybrid F1 generation hybrid seeds in Example 2.

[0027] Figure 4 This is a statistical graph showing the effect of the rice THION22 peptide protein on promoting the seed setting rate of the F1 generation of indica-japonica hybrids in Example 2. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below through examples. These examples are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0029] SEQ ID No. 1: Nucleotide sequence of rice THION22 gene

[0030] ATGGAGGTGAAGAAGGTGGCCATGGTTGCTGTCTGCTGCATGTTCATACTGCTGTTTCCAGGCCAGCAGCAGCAGGCTGGCTGCCATGTCCAGGATCTGCAGATGCTACCACGAATGCTTGCCCAACTGCGGCCTGCGCAATTCTCGCTCCTTCTGCAAGGTGTTCTGCGGCAGCTGCTGC GTCTTCAATCCAGTTCACAATTGCACTAGCACCGATGCGGCGGCCGCGGCGCCAGCGATCGCCGGAGACGACTGCAGAATGATCTGCCTGAACTCCTTCTGCGGCGAGGCAGCTACAGGCTACTCGGGGCGAAACGATGCTGATGCTGCAGCTTGTCTCGATGGCTGCAGCAAAGGATGA

[0031] SEQ ID No. 2: Amino acid sequence of rice THION22 small peptide protein

[0032] MSRICRCYHECLPNCGLRNSRSFCKVFCGSCCVFNPVHNCTSTDAAAAAPAIAGDDCRMICLNSFCGEAATGYSGRNDADAAACLDGCSKG

[0033] The rice THION22 small peptide protein of the present invention is obtained through prokaryotic expression and purification in Escherichia coli.

[0034] Example 1 Rice THION22 small peptide protein promotes the growth of japonica rice ZH11 pollen tubes in the pistil of indica rice varieties

[0035] (1) Indica rice varieties International Rice 26 (IR26), Minghui 63 (MH63), Minghui 86 (MH86), and Yuzhenxiang (YZX) were grown together with japonica rice variety ZH11 in a rice smart greenhouse. The conditions were set as follows: temperature 26-28°C, air humidity 50%, light 14 h, dark 10 h;

[0036] (2) When the rice plant reaches the flowering stage, the indica rice varieties IR26, MH63, MH86, and YZX are used as female parents and manually emasculated using scissors, tweezers, and other tools. Three suitable ears of each variety are selected and emasculated and bagged in the afternoon of the previous day.

[0037] (3) At noon the next day, pollen from the flowering japonica rice variety ZH11 was collected with the smooth surface of weighing paper, and then the pollen grains were quickly applied to the stigma of each indica rice variety using a brush. Since the activity of rice pollen will decrease quickly after being separated from the body, the time from pollen grains falling off to landing on the stigma should be controlled within 5 minutes, so a large number of repeated pollination operations are required;

[0038] (4) 5 min after pollination, a volume of 10 μL of rice THION22 peptide protein at a concentration of 10 nM was applied to the base of the pistil of the indica rice variety using a syringe;

[0039] (5) 2 hours after pollination, the pistils of the pollinated indica rice varieties were removed and fixed with FAA fixative (50% ethanol: glacial acetic acid: formaldehyde = 89:6:5) for 24 hours;

[0040] (6) After fixation, the pistil was dehydrated using 70%, 50%, and 30% alcohol, respectively, for 10 min each time.

[0041] (7) After dehydration, the pistil was immersed in distilled water for 10 min and then softened by heating with 10 M NaOH solution in a 56°C water bath for 5–8 min.

[0042] (8) After softening, the pistil was rinsed several times with distilled water and then stained with 0.1% aniline blue for 12 h. Finally, the attachment and germination of pollen on the stigma of the pistil and the elongation of the pollen tube were observed under a laser confocal microscope (Leica SP2) and photographed.

[0043] In the above experiment, the blank control group was used without applying rice THION22 peptide protein (ie, the concentration of rice THION22 peptide protein was 0 nM). Figure 1 and Figure 2 shown. Figure 1 This is a microscopic observation of the effect of rice THION22 small peptide protein on the pollen tube growth of japonica rice ZH11. Figure 2 This is a statistical graph showing the rate of pollen tube growth reaching ovules in japonica rice ZH11.

[0044] Depend on Figure 1It can be seen that when the concentration of rice THION22 peptide protein was 0nM, after pollen from the japonica rice variety ZH11 was applied to the stigmas of the pistils of four indica rice varieties, only 5-20% of the pollen tubes were able to grow normally to the base of the ovule, and about 80% of the pollen tubes did not germinate or ruptured and stalled in the style. However, when the rice THION22 peptide protein was applied at 10nM, the pollen of the japonica rice variety ZH11 was able to germinate on the stigmas of the pistils of the indica rice varieties, and the proportion of pollen tubes produced increased by 30-40%. This shows that during the indica-japonica hybridization process, the in vitro application of rice THION22 peptide protein can promote the growth of japonica rice pollen tubes in the pistils of indica rice varieties.

[0045] Depend on Figure 2 When the concentration of rice THION22 peptide protein was 0 nM, the rates of pollen tubes reaching ovules in japonica rice ZH11 were 15%, 20%, 10%, and 5%, respectively, for indica rice varieties MH63, MH86, IR26, and YZX. However, when 10 nM of rice THION22 peptide protein was applied, the rates of pollen tubes reaching ovules in japonica rice ZH11 were 45%, 54%, 60%, and 40%, respectively. Compared to the two, the 10 nM application of rice THION22 peptide protein increased the rate of pollen tubes reaching ovules in japonica rice ZH11 by over 30%. This suggests that in vitro application of rice THION22 peptide protein can effectively increase the rate of pollen tubes reaching ovules in japonica rice during indica-japonica hybridization.

[0046] Example 2 Rice THION22 peptide promotes the acquisition of indica-japonica hybrid F1 generation hybrid seeds

[0047] (1) The experimental materials and rice planting conditions used were the same as those in Example 1, and both were planted in a rice smart greenhouse;

[0048] (2) The combination of indica and japonica varieties was consistent with that in Example 1. The male parent was the japonica rice variety ZH11, and the female parents were the indica rice varieties IR26, MH63, MH86, and YZX. Two male sterile lines, Gufeng A (GFA) and Jinlong 3A (JL3A), were added for comparison.

[0049] (3) The emasculation method was manual emasculation. Six ears of each indica rice variety were selected for emasculation in the afternoon of the previous day and bagged.

[0050] (4) At noon the next day, gently cut off the flowering ears of the japonica rice variety ZH11, then invert it on the pistil of the indica rice variety and quickly rotate the ears for pollination. Repeat pollination three times for each female parent.

[0051] (5) 5 minutes after pollination, the entire indica rice panicle was evenly sprayed with 2-3 mL of rice THION22 peptide protein at a concentration of 10 nM, and then bagged;

[0052] (6) After the F1 generation hybrid seeds grow and mature, observe and count them.

[0053] In the above experiment, the blank control group was not sprayed with rice THION22 peptide protein (ie, the concentration of rice THION22 peptide protein was 0 nM). Figure 3 and Figure 4 shown. Figure 3 This is an observation diagram of how rice THION22 peptide protein promotes the production of F1 hybrid seeds of indica and japonica hybrids. Figure 4 The statistical results show that rice THION22 small peptide protein promotes the seed setting rate of F1 generation of indica-japonica hybrid.

[0054] Depend on Figure 3 It can be seen that when the concentration of rice THION22 peptide protein is 0nM, almost no F1 generation seeds are obtained during the indica-japonica hybridization process. However, when the rice THION22 peptide protein is sprayed at 10nM, a large number of F1 generation seeds can be obtained during the indica-japonica hybridization process. At the same time, when the male sterile lines GFA and JL3A are used as the female parents, the rice THION22 peptide protein can also promote the production of F1 generation hybrids.

[0055] Depend on Figure 4 When the concentration of rice THION22 peptide protein was 0 nM, the F1 seed set rates in hybrids using the japonica rice variety ZH11 as the male parent and the indica rice varieties MH63, MH86, IR26, YZX, and the male-independent lines GFA and JL3A as the female parents were 12%, 22%, 7%, 5%, 11%, and 8%, respectively. When sprayed with 10 nM rice THION22 peptide protein, the F1 seed set rates were 41%, 49%, 40%, 41%, 54%, and 40%, respectively. Spraying with 10 nM rice THION22 peptide protein increased the F1 seed set rate by over 27%. This suggests that rice THION22 peptide protein can effectively promote the production of F1 seeds in indica-japonica hybrids, increase the number of F1 hybrid seeds, and improve the efficiency of indica-japonica hybrid breeding.

[0056] 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 in the scope of protection of the present invention.

Claims

1. An application of a rice THION22 peptide protein in breaking reproductive isolation between indica and japonica subspecies of rice, characterized in that: In vitro application of THION22 peptide protein breaks the prezygotic reproductive isolation of the indica and japonica subspecies of rice during hybridization; The amino acid sequence of the rice THION22 small peptide protein is shown in SEQ ID No.

2.

2. The use of the rice THION22 small peptide protein according to claim 1 in breaking the reproductive isolation of indica and japonica rice subspecies, characterized in that The rice THION22 small peptide protein promotes the growth of pollen tubes in japonica rice in the pistil of indica rice varieties.

3. The use of the rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies according to claim 2, characterized in that: The application is specifically: (1) When the rice is in its flowering stage, the indica rice varieties are used as the female parent, and the rice is manually detasseled and bagged; (2) Collect pollen from flowering japonica rice varieties and quickly transfer the pollen grains to the stigma of indica rice varieties; (3) After pollination, rice THION22 peptide protein was applied to the base of the pistil of indica rice.

4. The use of the rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies according to claim 3, characterized in that: The concentration of the THION22 small peptide protein is 8-12 nM, and the volume is 8-12 uL.

5. The use of the rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies according to claim 1, characterized in that: The rice THION22 peptide promotes the acquisition of indica-japonica hybrid F1 generation hybrid seeds.

6. The use of the rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies according to claim 5, characterized in that The application is specifically: (1) When the rice is in its flowering stage, the indica rice varieties are used as the female parent, and the rice is manually detasseled and bagged; (2) Cut off the flowering ears of japonica rice varieties and place them upside down on the pistils of indica rice varieties for pollination; (3) After pollination, the entire indica rice panicle is evenly sprayed with rice THION22 peptide protein and then bagged.

7. The use of the rice THION22 small peptide protein according to claim 6 in breaking the reproductive isolation of rice indica and japonica subspecies, characterized in that The concentration of the rice THION22 small peptide protein is 8-12 nM, and the volume is 2-3 mL.

8. The use of the rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies according to claim 1, characterized in that: The rice THION22 small peptide protein is obtained through prokaryotic expression and purification in Escherichia coli.

9. The use of the rice THION22 small peptide protein in breaking the reproductive isolation of indica and japonica rice subspecies according to claim 1, characterized in that: The male parent in the indica-japonica hybridization process is the japonica rice variety Zhonghua No. 11, and the female parent is the indica rice variety International No. 26, Minghui 63, Minghui 86 or Yuzhenxiang.

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