Method for breaking through plant reproductive isolation or distant hybridization incompatibility

Abstract

The present invention relates to ARP2 of plant stigma cells; the invention relates to application of Ca < 2 + > transport capacity damage of homologous genes 1 and 1 in breaking through reproduction isolation of plants. According to the method for breaking through the plant reproductive isolation, a foundation is laid for further research on the relationship of small peptides for regulating and controlling reproductive isolation calcium signals through co-receptors, and a simple and effective target is provided for gene modification for breaking through the reproductive isolation.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a method for breaking through plant reproductive isolation or distant hybridization incompatibility. Background Art

[0002] Interspecific hybridization refers to the hybridization between two different species, genera, or even more distantly related species. This hybridization can break species restrictions, increase genetic variation, and integrate the biological characteristics of the two species. This is an important way to create genetic variation, fertile lines, and excellent traits in new lines and new populations. Many crops, such as rice, sunflower, rapeseed, and sorghum, use interspecific hybridization technology in the breeding process. The breeding of ornamental flowers such as chrysanthemums also uses existing varieties and wild species. However, due to the distant relationship, the reproductive organs, tissues, cells, and gametes of the two parents are not compatible, and it is common for the hybridization to fail to pollinate normally, fertilize, or develop normally. In agricultural production and breeding research, interspecific hybridization incompatibility can be overcome by selecting appropriate parents and paying attention to reciprocal crosses, repeated pollination during the bud stage, treatment with chemicals (such as sodium salicylate), and somatic cell fusion.

[0003] Successful interspecific hybridization in plants depends on pollination and fertilization, which occurs in a series of processes from pollen grain germination to pollen tube growth, from double fertilization to zygote and endosperm development. In natural environments, plants have evolved corresponding systems to prevent heterologous pollen from fertilizing. Heterologous pollen has a low degree of adhesion on the stigma, and pollen grains are difficult to germinate successfully on the stigma. Even if some pollen grains can germinate successfully, a large amount of callus produced in the stigmatic papilla cells will hinder the growth and development of pollen tubes, causing abnormal phenomena such as bending and bifurcation of pollen tubes. A large amount of callus will hinder the extension of pollen tubes, resulting in failure to fertilize. After interspecific hybridization pollination, even if fertilization can be completed normally, embryo abortion will occur in many species and seeds cannot be formed. After interspecific hybridization pollination, a large amount of callus will accumulate in the stigmatic papilla cells. This is a specific reaction of the stigma to reject heterologous pollen during interspecific hybridization, and its reaction intensity is closely related to the relationship between the hybrid parents.

[0004] Previously, scientists have confirmed that interspecific hybrid incompatibility in plants is mainly due to the inability to complete pollination, but its specific molecular mechanism is still unclear. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention proposes a Ca- 2+ Application of impaired transport capacity in breaking reproductive isolation in plants.

[0006] In some embodiments, the reproductive isolation is caused by interspecific hybrid incompatibility.

[0007] In some embodiments, the interspecies hybrid incompatibility is stigma-specific interspecies incompatibility.

[0008] In some embodiments, the plant is a cruciferous plant.

[0009] In some embodiments, the cruciferous plant is Arabidopsis thaliana, broccoli, rapeseed, cabbage, Chinese cabbage, shepherd's purse, camelina, salt mustard, rocket, radish, mustard white, or thrush.

[0010] In some embodiments, the amino acid sequence of ARP2;1 is: the sequence shown in SEQ ID NO.1; or a sequence having at least 80% homology with SEQ ID NO.1.

[0011] The present application also relates to a method for allowing a plant to break through reproductive isolation, comprising: causing the Ca of ARP2;1 of the plant stigma cell to 2+ Impaired transport capacity.

[0012] In some embodiments, wherein Ca 2+ The impaired transport capacity is caused by substitution, insertion or deletion mutation of ARP2;1 itself in plant stigma cells, or by the inhibition of ARP2;1 activity by ARP2;1 inhibitors.

[0013] In some embodiments, the ARP2;1 inhibitor is GdCl3.

[0014] In some embodiments, the ARP2;1 inhibitor comprises FER.

[0015] In some embodiments, the amino acid sequence of FER is as shown in SEQ ID NO.2; or a sequence having at least 80% homology to SEQ ID NO.2.

[0016] In some embodiments, the FER-containing inhibitor is a FER-ANJ complex, a FER-CVY1 complex, a FER-HERK1 complex, or a FER-CVY1-HERK1 complex.

[0017] In some embodiments, the amino acid sequence of CVY1 is as shown in SEQ ID NO.3; or a sequence having at least 80% homology to SEQ ID NO.3; the amino acid sequence of HERK1 is as shown in SEQ ID NO.4; or a sequence having at least 80% homology to SEQ ID NO.4.

[0018] The method for breaking through plant reproductive isolation proposed in this application not only lays the foundation for further studying the relationship between small peptides and calcium signals regulating reproductive isolation through co-receptors, but also provides a simple and effective target for genetic modification to break through reproductive isolation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein:

[0020] Figure 1 1 is an expression pattern diagram of AtARP2 in Arabidopsis thaliana detected by semi-quantitative PCR method according to one embodiment of the present invention; wherein root represents root, stem represents stem, flower represents flower, silique represents silique, cauline leaf represents cauline leaf, and rosette leaf represents rosette leaf;

[0021] Figure 2 is a subcellular localization map of AtARP2 according to one embodiment of the present invention;

[0022] Figure 3 The invention relates to the identification of T-DNA insertion mutants of AtARP2;1 according to one embodiment of the present invention; wherein, Figure 3 A is a schematic diagram of the T-DNA insertion site of AtARP2;1 according to one embodiment of the present invention; Figure 3 B is the identification result of atarp2;1 mutant according to one embodiment of the present invention;

[0023] Figure 4 The germination and growth of shepherd's purse pollen on the stigma of Col-0 type Arabidopsis and its atarp2;1 mutant according to an embodiment of the present invention; wherein the pollen tube growth experiment was conducted in the stigma half body of Arabidopsis wild type and atarp2;1 mutant, the horizontal stigma represents the stigma, and the vertical pollen represents the pollen; the results of Arabidopsis pollen observation under a stereo microscope 6 hours after pollination, and the results of shepherd's purse pollen observation under a stereo microscope 2 days after pollination, bar = 500 μm;

[0024] Figure 5 The germination and growth of shepherd's purse pollen on the stigma of Arabidopsis ecotypes Col-0 and Cvi-0 according to an embodiment of the present invention; wherein in the two Arabidopsis ecotypes Col-0 and Cvi-0, a pollen tube growth experiment was conducted on the stigma semi-in vivo, the horizontal stigma represents the stigma, and the vertical pollen represents the pollen; the results of Arabidopsis pollen were observed under a stereo microscope 6 hours after pollination, and the results of shepherd's purse pollen were observed under a stereo microscope 2 days after pollination, bar = 500 μm;

[0025] Figure 6The pollen tube of A. aconitifolia germinates and extends on the stigma of Arabidopsis mutant atarp2;1 and ecotype Cvi-0 according to an embodiment of the present invention; wherein the horizontal stigma represents the stigma, and the vertical pollen represents the pollen; the aniline blue staining experimental results of the stigma of Arabidopsis wild type Col-0, atarp2;1 mutant and wild type Cvi-0 6 hours after Arabidopsis pollen pollination and 2 days after A. aconitifolia pollen pollination, bar = 200 μm;

[0026] Figure 7 is a sequence alignment of AtARP2;1 of different ecotypes according to one embodiment of the present invention; wherein, Figure 7 A is the protein sequence of 9 different AtARP21 molecules according to one embodiment of the present invention; Figure 7 B is the distribution of different types of AtARP2;1 molecules in Arabidopsis ecotypes according to an embodiment of the present invention;

[0027] Figure 8 This is the protein polymer structure prediction of AtARP2; 1A and AtARP2; 1B by AlphaFold according to an embodiment of the present invention; wherein the horizontal direction represents mono-nonamerization: monomer: monomer, dimer: dimer, trimer: trimer, tetramer: tetramer, pentamer: pentamer, hexamer: hexamer, heptamer: heptamer, octamer: octamer, nanomer: nonamer; the vertical direction is AtARP2; 1A and AtARP2; 1B.

[0028] Fig. 9 This is an enlarged view of the octamer of AtARP2;1B protein according to an embodiment of the present invention; wherein the green circled portion in the middle is the valine at position 2;

[0029] Fig.10 is a comparison of the calcium channel activities of AtARP2; 1A and AtARP2; 1B according to an embodiment of the present invention; wherein, Fig.10 A is a whole-cell current diagram of a negative control (injection of distilled water), a positive control (AtCNGC14), and an African clawed frog oocyte expressing AtARP2; 1A, AtARP2; 1B in a perfusion solution containing 20 mM CaCl2 according to an embodiment of the present invention; Fig.10 B is the inward current magnitude of the negative control (injection of distilled water), the positive control, and the Xenopus oocytes expressing AtARP2; 1A and AtARP2; 1B in the perfusion solution containing 20 mM CaCl2 recorded at a clamping voltage of -140 mV according to one embodiment of the present invention (n=5);

[0030] Fig.11 The present invention is a yeast complementation experiment according to an embodiment of the present invention to verify the difference in calcium channel activity between AtARP2;1A and AtARP2;1B; a filter paper containing 50 μg of α factor is placed on a plate coated with a wild-type yeast strain and cch1 / mid1 yeast mutants respectively transformed with an empty vector (negative control), MtCNGC15 (positive control), and AtARP2;1A and AtARP2;1B, and the yeast growth condition is observed after culturing at 28°C for 48 hours.

[0031] Fig.12 This is a comparative analysis of the calcium channel activities of AtARP2; 1A and AtARP2; 1B by calcium fluorescence imaging of HEK293T cells according to one embodiment of the present invention; the cell calcium fluorescence imaging experiment measures the changes in calcium ion fluorescence in the negative control (empty vector) and HEK293T cells expressing AtCNGC18 (positive control), AtARP2; 1A, and AtARP2; 1B, and the arrow indicates the time of applying calcium ions;

[0032] Fig.13 This is an activity analysis of AtARP2;1 transporting sodium and potassium monovalent cations according to one embodiment of the present invention; wherein Fig.13 A is a whole-cell current diagram of a negative control (injection of distilled water), a positive control (the potassium ion positive control is AtKAT1, and the sodium ion positive control is AtHKT1) and an African clawed frog oocyte expressing AtARP2;1 in a perfusion solution containing 20 mM KCl and 20 mM NaCl, respectively, according to an embodiment of the present invention; Fig.13 B is the statistics of the inward current of negative control (injection of distilled water), positive control and Xenopus oocytes expressing AtARP2;1 in perfusion solution containing 20mM KCl or 20mM NaCl recorded at a clamping voltage of -140mV according to one embodiment of the present invention (n=5);

[0033] Fig.14 This is an activity analysis of AtARP2;1 transporting magnesium and calcium divalent cations according to one embodiment of the present invention; wherein, Fig.14 A is a whole-cell current diagram of a negative control (injection of distilled water), a positive control (magnesium ion positive control is OsHKT2;4, calcium ion positive control is AtCNGC14) and an African clawed frog oocyte expressing AtARP2;1 in a perfusion solution containing 20mM MgCl2 and 20mM CaCl2, respectively, according to an embodiment of the present invention; Fig.14B is the magnitude of the inward current of the negative control (injection of distilled water), the positive control and the Xenopus oocyte expressing AtARP2;1 in the perfusion solution containing 20mM MgCl2 or 20mM CaCl2 recorded at a clamping voltage of -140mV according to one embodiment of the present invention (n=5);

[0034] Fig.15 is a relationship between AtARP2;1 channel activity and calcium ion concentration according to an embodiment of the present invention; wherein, Fig.15 A is a schematic diagram of a two-electrode voltage clamp recording procedure according to an embodiment of the present invention: the holding voltage is 0 mV, the clamping voltage is applied for 0.8 s (the clamping voltage is decreased from 40 mV to -140 mV by 20 mV each time), and then restored to the holding voltage of 0 mV for 1.2 s; whole-cell current graphs of a negative control (injected with distilled water) in a perfusion solution containing 30 mM CaCl2 and whole-cell current graphs of an African clawed frog oocyte expressing AtARP2;1 in perfusion solutions containing 0 mM, 5 mM, 10 mM and 30 mM CaCl2, respectively; Fig.15 B is a current-voltage relationship curve of an African clawed frog oocyte expressing AtARP2;1 in a perfusion solution containing 0mM, 5mM, 10mM and 30mM CaCl2 according to an embodiment of the present invention (n=3);

[0035] Fig.16 This is a pharmacological analysis of the calcium permeability activity of AtARP2;1 according to an embodiment of the present invention; wherein, Fig.16 A is a whole-cell current diagram of a negative control (injected with distilled water) and an African clawed frog oocyte expressing AtARP2;1 in a perfusion solution containing 20 mM CaCl2, 20 mM CaCl2 and a chloride channel inhibitor DIDS, and 20 mM CaCl2 and a calcium channel inhibitor GdCl3, respectively, according to an embodiment of the present invention; Fig.16 B is the magnitude of the inward current of the negative control (injected with distilled water) and the Xenopus oocyte expressing AtARP2;1 recorded at a clamping voltage of -140 mV according to one embodiment of the present invention in the perfusion solution containing 20 mM CaCl2, 20 mM CaCl2 and DIDS, and 20 mM CaCl2 and GdCl3, respectively (n=5);

[0036] Fig.17 is the regulation of the calcium ion transport activity of AtARP2;1A according to one embodiment of the present invention; wherein, Fig.17A is a schematic diagram of a two-electrode voltage clamp recording procedure according to an embodiment of the present invention: the holding voltage is 0 mV, the clamping voltage is applied for 0.8 s (the clamping voltage is decreased from 40 mV to -140 mV by 20 mV each time), and then restored to the holding voltage of 0 mV for 1.2 s; whole-cell current graphs of negative control (injected with distilled water), positive control (expressing AtCNGC14), Xenopus oocytes expressing AtARP2;1, and AtARP2;1 co-expressed with AtANJ, AtFER, AtCVY1, AtHERK1, etc., respectively, in a perfusion solution containing 20 mM CaCl2; Fig.17 B is the inward current magnitudes of negative control (injection of distilled water), positive control, Xenopus oocytes expressing AtARP2;1, and AtARP2;1 co-expressed with AtANJ, AtFER, AtCVY1, AtHERK1, etc., recorded at a clamping voltage of -140 mV according to one embodiment of the present invention in the perfusion solution containing 20 mM CaCl2 (n=5);

[0037] Fig.18 This is an analysis of the calcium ion permeable channel activity of rapeseed BnARP2;1 according to one embodiment of the present invention; Fig.18 A is a schematic diagram of a two-electrode voltage clamp recording procedure according to an embodiment of the present invention: the holding voltage is 0 mV, the clamping voltage is applied for 0.8 s (the clamping voltage is decreased from 40 mV to -140 mV by 20 mV each time), and then restored to the holding voltage of 0 mV for 1.2 s; whole-cell current graphs of negative control (injected with distilled water), Xenopus oocytes expressing BnARP2;1, and BnARP2;1 co-expressed with BnFER in perfusion solution containing 20 mM CaCl2; Fig.18 B is the inward current magnitudes of negative control (injection of distilled water), positive control, Xenopus oocytes expressing ARP2;1, and BnARP2;1 and BnFER co-expressed in the perfusion solution containing 20 mM CaCl2, respectively, recorded at a clamping voltage of -140 mV according to one embodiment of the present invention (n=5);

[0038] Fig.19The present invention is a phylogenetic analysis of the ARP2;1 molecule of the cruciferous plant according to an embodiment of the present invention; wherein the following abbreviations represent species: Aa: Arabis alpina, alpine thaliana; Al: Arabidopsis lyrata, lyre-leaf Arabidopsis; As: Arabidopsis suecica, Swedish Arabidopsis; At: Arabidopsis thaliana, Arabidopsis; Bc: Brassicacretica, pagoda flower; Bn: Brassica napus, rapeseed; Bo: Brassica oleracea, cabbage; Br: Brassicarapa, cabbage; Cr: Capsella rubella, light red shepherd's purse; Cs: Camelina sativa, camelina; Es: Eutremasalsugineum, salt mustard; Ev: Eruca vesicaria, rocket; Rs: Raphanus sativus, radish; Sa: Sinapis alba, white mustard; Ta: Thlaspi arvense, thlaspi arvense. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In the following detailed description, reference may be made to the various specification drawings that are part of the present application and are used to illustrate specific embodiments of the present application. In the accompanying drawings, similar reference numerals describe substantially similar components in different figures. The various specific embodiments of the present application are described below in sufficient detail so that a person of ordinary skill in the art with relevant knowledge and skills in the art can implement the technical solutions of the present application. It should be understood that other embodiments may also be used or structural, logical or electrical changes may be made to the embodiments of the present application.

[0041] Previous studies have shown that the main reason for interspecific hybrid incompatibility is that pollen hydration cannot proceed normally. In the process of pollen recognizing the stigma, a variety of signal molecules: calcium ions, ROS, small peptides, etc. can regulate the hydration reaction of pollen. For example, in self-incompatible species, pollen secretes small peptides that bind to receptor-like kinases (RLKs) on the stigma, resulting in a burst of ROS that inhibits the hydration reaction, thereby blocking the growth and extension of pollen tubes.

[0042] However, these important research advances are mostly limited to the study of self-incompatible species. There are still huge gaps in the research on interspecific hybrid incompatibility between self-compatible species and hybrid incompatibility between different subspecies of the same species.

[0043] Although people have preliminarily discovered that ARP2;1 in Cruciferae is involved in the reaction of pollen incompatibility between different species, there has been no report on the biochemical function of ARP2;1 and the specific mechanism of regulating reproductive isolation. In addition, there are still many gaps in the analysis of the biochemical functions of most members of the ARPs family that have important physiological functions and contain transmembrane domains. Since these transmembrane proteins are similar to the partial structure of the temperature-sensing molecule TRP (Transient Response Potential) family protein that won the 2021 Prize in Physiology or Medicine, this application uses plant electrophysiology as the basic method to functionally screen members of the ARPs family containing more than 3 transmembrane structures. Under the existing screening conditions, the only member ARP2;1 that can record electrophysiological activity was found. The confirmation of this biochemical function will provide a clear direction for the study of the physiological functions of this protein family, and also provide theoretical guidance and genetic resources for plant reproductive isolation and crop hybrid breeding.

[0044] The terms used in this article have the following meanings:

[0045] The "reproductive isolation" mentioned in this article is also called "interspecific barrier" in this article. It refers to the isolation mechanism that prevents closely related plant groups from mating under natural conditions, or even if they can mate, they cannot produce offspring or cannot produce fertile offspring. For example, time isolation caused by non-overlapping flowering periods, spatial isolation caused by long distances between species, mismatched reproductive organs caused by mismatched morphology and position of stamens and pistils, and physiological isolation.

[0046] Reproductive isolation is the basis of species differentiation and maintenance. Physiological reproductive isolation of plants is caused by the phenomenon of interspecific hybrid incompatibility. Research on the mechanism of overcoming interspecific hybrid incompatibility can not only provide a theoretical basis for species maintenance and differentiation, but also provide targets for molecular breeding and genetic modification for interspecific hybrid breeding and increasing crop yields.

[0047] Flowering plants can be divided into two types: self-compatible (SC) plants and self-incompatible (SI) plants. Most self-pollinating plants are SC species, whose pollen can complete pollination on their own stigma. SI species are outcrossing, that is, the pollen and stigma come from different plants. SI is a genetic control system for hermaphroditic plants to recognize and reject their own pollen, the so-called intraspecific barrier, which exists in about half of the flowering plant species. Many hermaphroditic plants use this mechanism as a self / non-self pollen recognition system to avoid inbreeding depression by rejecting their own pollen on the surface of the stigma, thereby maintaining genetic diversity within the population. In addition to self-incompatibility, reproductive isolation also exists between different subspecies of some plants.

[0048] The "self-compatible (SC) plants" mentioned herein refer to plants whose pollen can combine with their own stigma and complete the fertilization process to form normal seeds and offspring. There are many types of self-compatible plants, and common wheat, barley, oats, rice, soybeans, peas, peanuts, sesame, mung beans, potatoes, flax, tobacco, etc. all have self-compatibility. Among the cruciferous plants, Arabidopsis thaliana and Brassica napus are both typical self-compatible plants.

[0049] The "self-incompatible (SI) plant" mentioned herein refers to a plant that has complete flowers and can form normal male and female gametes, but cannot bear fruit when self-pollinated. When such plants self-pollinate, pollen falls on their own stigma and cannot germinate or pass through the stigma normally, resulting in failure to complete fertilization, which manifests as the phenomenon of self-pollination not bearing fruit. This phenomenon is widely present in nature, such as rye, sunflower, beet, sweet potato, tea tree and other plants all have self-incompatibility.

[0050] Hybridization as used herein refers to the sexual mating of plant parents with different genetic characteristics to produce offspring with rich genetic diversity. Hybridization can be divided into intra-species hybridization, that is, hybridization between different ecotypes of the same variety; inter-species hybridization, that is, hybridization between different varieties or strains within a species; and inter-specific hybridization, that is, hybridization between different species within the same genus, which may encounter sexual incompatibility barriers.

[0051] The "interspecific hybridization" mentioned in this article refers to the mating between individuals of taxonomic units above species, including hybridization between plants of different species and genera, and even between species of higher taxonomic levels. Breaking through the barriers to interspecific hybridization can expand the gene pool of cultivated crops and even transfer excellent disease-resistant and stress-resistant genes in wild plants to cultivated varieties, creating new germplasm resources.

[0052] The "interspecific hybrid incompatibility" mentioned in this article refers to the pollination and fertilization barriers caused by morphological, physiological or genetic differences when plants of different species or genera are hybridized. Specifically, it manifests as pollen failure to germinate, pollen tube growth stagnation, male and female gametes not combining, zygotic embryos not developing, etc.

[0053] Interspecific barriers restrict the reproduction between species. Current research on interspecific hybridization is usually based on the SI×SC rule: interspecific pollen is unidirectionally incompatible (UI) on SI stigmas, but unidirectionally compatible on SC pistils. That is, the stigma of SC plants is compatible with pollen from both SI and SC species, while the stigma of SI plants is incompatible with pollen from both SI and SC species. The above mechanism can explain the self-incompatibility phenomenon of plants and the incompatibility phenomenon of interspecific hybridization when the mother parent is a self-incompatible species.

[0054] The "stigma-specific interspecific incompatibility" mentioned in this article refers to the phenomenon of hybrid incompatibility caused by the stigma recognition mechanism between plants of different species. Existing studies are almost all based on species with SI as the maternal parent, and there are few studies on the mechanism of hybrid incompatibility between species with SC as the maternal parent. In the Cruciferae family, there is a stigma-specific interspecific incompatibility factor ARP2;1 / SPRI1, which is a membrane protein specifically expressed in the stigma. The stigma of the arp2;1 / spri1 mutant of Arabidopsis thaliana can accept pollen from M. littorea, and its pollen tubes can germinate and extend normally. The interspecific incompatibility mediated by ARP2;1 / SPRI1 is not related to SI. However, its specific molecular mechanism is still unclear.

[0055] Pollen hydration is a key step in determining pollen germination. Pollen incompatibility is mostly due to the inhibition of its hydration, which leads to the inability to fertilize. The stigma is the source of water for pollen hydration. After the compatible pollen is pollinated on the stigma, the pollen surface triggers a series of reactions in the stigma cells, transferring water to the dry pollen grains, where the pollen then absorbs water and germinates. The pollen hydration process is rapid and is affected by the environmental humidity. Pollen hydration and germination experiments of Arabidopsis pollen on various surfaces such as glass coverslips, leaf blades, sepals, immature stigmas and mature stigmas showed that at a relative humidity of 65%, pollen grains can only be hydrated on the stigma. However, at a humidity of 100%, pollen can be hydrated on any surface, but the pollen will not germinate and the pollen tube cannot extend. However, the signaling mechanism inside the pollen grain that controls pollen hydration on the stigma in compatible pollination remains poorly understood.

[0056] The calcium ion concentration in the stigma papillary cells affects pollen hydration, and higher calcium ion concentrations inhibit pollen hydration. However, the role of calcium ions in the pollen-stigma process is still unclear. During the three stages of pollen hydration, germination, and pollen tube extension, the intracytoplasmic calcium ion level of papillary cells near the pollen increases slightly.

[0057] As an important second messenger, calcium ions play a key regulatory role in the process of self-incompatibility. However, the role of calcium ions in the process of interspecific hybridization has been rarely studied. During the three stages of pollen hydration, germination and pollen tube extension, the calcium ion level in the cytoplasm of papillary cells near the pollen increases slightly. At present, preliminary studies have been conducted on calcium ion signals in the mechanism of self-incompatibility. Arabidopsis stigma expressing SRK protein of the self-incompatible species Brassica rapa (B.rapa) was pollinated with SP11 / SCR pollen of B.rapa, which can cause a rapid increase in calcium ion concentration in the cytoplasm of stigma papillary cells, reaching a peak 5 minutes after pollination. However, the calcium ion concentration of papillary cells pollinated with wild-type Arabidopsis pollen did not change significantly. Iwano et al. believed that glutamate receptor-like channels (GLRs) are channels that mediate calcium ion absorption in the papillary cells of cruciferous plants in the phenomenon of self-incompatibility. In addition, the increase in calcium concentration during pollination in homozygous Arabidopsis mutants of GLR3.7 was smaller than that in wild-type plants. However, the mechanism of calcium concentration changes during pollination of self-compatible species and during interspecific pollination is still unclear.

[0058] One of the characteristics of the "Cruciferae" mentioned in this article is that their flowers have four petals (an even number) arranged in a cross shape, hence the name. The Cruciferae family is divided into many genera, such as Brassica, Capsella, Lepidium, Raphanus, Capsella, Arabidopsis, etc. The Cruciferae family has important economic value. For example, many Cruciferae plants have important economic value. Among them, Brassica and Raphanus sativus are the main vegetables and oil crops, or used as spicy condiments, such as rapeseed, cabbage, radish, Chinese cabbage, shepherd's purse, mustard, cabbage, cauliflower, watercress, apetalum, turnip, and iris, which are important vegetables, wild vegetables and feed, etc.; some species are important medicinal plants, such as Isatis root, radish seed, shepherd's purse flower, Brassica, Kochia scoparia, and mustard, etc., some species of extracts can be used to treat skin diseases, respiratory diseases, cardiovascular diseases, etc.; some are ornamental plants, such as violets, osmanthus fragrans, and February orchid; some can be used as dyes and feed, such as woad.

[0059] The Latin name of "Arabidopsis" mentioned in this article is Arabidopsis thaliana. The prefix "at" or "At" in front of the gene name and protein name indicates that the gene or protein is derived from Arabidopsis. For example, AtARP2;1, AtFER, AtCVY1, AtHERK1, AtANJ refer to ARP2;1, FER, CVY1, HERK1, ANJ derived from Arabidopsis; AtCNGC14, AtCNGC18 refer to CNGC14 and CNGC18 derived from Arabidopsis, which are proteins that have been proven to have calcium ion transport activity; AtKAT1 refers to KAT1 derived from Arabidopsis, which is a protein that has been proven to have potassium ion transport activity; AtHKT1 refers to HKT1 derived from Arabidopsis, which is a protein that has been proven to have sodium ion transport activity. The amino acid sequences of the above genes are shown in Table 1:

[0060] Table 1

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Arabidopsis is an annual, thin herbaceous plant of the Cruciferae family and the genus Arabidopsis. It is often used as a model plant for scientific research. It is 20-35 cm tall and covered with simple and branched hairs, including stems, flowers, siliques, cauline leaves, rosette leaves, and roots. The stem is unbranched or branched from the middle and upper parts; the basal leaves are rosette-shaped, also known as rosette leaves, obovate or spoon-shaped, 1-5 cm long, 3-15 mm wide, obtuse or slightly acute at the top, gradually narrowed at the base into a petiole, with a few inconspicuous teeth on the edge, and 2-3 forked hairs on both sides; the cauline leaves are sessile, lanceolate, linear, oblong or elliptical, 5-15 (-50) mm long, 1-2 (-10) mm wide. The inflorescence is a loose raceme, which can be extended to 20 cm when fruiting; the sepals are oblong ovate, about 1.5 mm long, with obtuse apex and sac-shaped base of the outer ring, glabrous or with a few simple hairs on the outside; the petals are white, oblong, 2-3 mm long, obtuse at the apex, and linear at the base. The siliques are 10-14 mm long and less than 1 mm wide, with obtuse or obtuse ends of the fruit petals, with 1 midrib and sparse reticulate veins, mostly orange-yellow or lavender; the fruit pedicels are extended, 3-6 mm long. There is one row of seeds in each room, and the seeds are ovate, small, and reddish brown.

[0067] The "Col-0" and "Cvi-0" mentioned in this article both refer to ecotypes of Arabidopsis thaliana. Arabidopsis thaliana has a wide geographical distribution range and can adapt to a variety of environments. For example, in the Americas, Europe, Asia, Africa and other places, Arabidopsis thaliana has formed ecotypes that adapt to their respective environments. These ecotypes differ in morphology, physiology and genetics to adapt to different climate and soil conditions.

[0068] The "M. littorea" mentioned in this article refers to Malcolmia littorea, which is a plant of the genus Malcolmia in the family Cruciferae, belonging to the phylum Angiosperms, phylum Magnoliaceae, subclass Rosa, superorder Cruciferae, order Brassicaceae.

[0069] The term "ARP" as used herein refers to ankyrin repeat-containing proteins (ARPs). ARPs mediate various physiological processes in plants by interacting with other proteins, and their main structural feature is the presence of ankyrin repeat motifs. ARPs mainly mediate protein-protein interactions in various cellular activities, and their functions include intercellular signal transduction, maintaining cytoskeleton integrity, transcription and cell cycle regulation, immune response, developmental regulation, and material transport, etc., playing an important role in the life process of cells.

[0070] The "ARP2;1" mentioned in this article is also called "SPRI1", which is a member of ARP and has a domain that is highly homologous to the ankyrin repeat structure. SPRI1 is widely present in cruciferous plants, and scholars speculate that it may be involved in the induction effect of cruciferous plants.

[0071] The "homologous genes" mentioned in this article refer to genes with high homology in different plants and with similar functions. These genes may have evolved vertically from the same ancestor and their functions are usually conserved. ARP2;1 is widely present in cruciferous plants and is specifically expressed in flowers. ARP2;1 in various cruciferous plants, such as AaARP2;1 in Arabidopsis alpina, AlARP2 in Arabidopsis lyreleaf, 1, AsARP2 in Arabidopsis swedensis, 1, AtARP2 in Arabidopsis thaliana, 1, BcARP2 in cauliflower, 1, BnARP2 in rapeseed, 1, BoARP2 in cabbage, 1, BrARP2 in Chinese cabbage, 1, CrARP2 in Capsella rubra, 1, CsARP2 in Camelina flax, 1, EsARP2 in Halophora, 1, EvARP2 in Arugula, 1, RsARP2 in Radish, 1, SaARP2 in Mustard, 1, TaARP2 in Capsella oleracea, 1, ThARP2 in Cleome, 1, etc. are the homologous genes mentioned in this application. This application lists several homologous genes, and their species and accession numbers are shown in Table 2:

[0072] Table 2 ARP2;1 protein information

[0073]

[0074] The "homology" described in this article refers to the degree of similarity between sequences. The higher the homology, the higher the degree of similarity of the sequences. There are various methods for detecting gene homology, such as sequence segmentation and alignment, which divides the genome to be compared into non-overlapping segments of fixed length, such as 1000 bases per segment, to improve the accuracy and efficiency of the alignment, and then uses sequence alignment tools (BLAST, MUMmer, etc.) to align the segments and find the best matching position; calculate similarity, calculate the nucleotide or amino acid similarity of the aligned segments, and the ratio of the number of matching bases or amino acids to the total alignment length is the similarity percentage; average similarity evaluation, average the similarity values ​​of all segments to obtain the average nucleotide identity (ANI) between the two genomes. If the ANI is close to 80%, it indicates that the gene homology is high; software-assisted analysis,

[0075] Use the NCBI database to search for gene base sequences, and use BLAST and other rapid comparison software to screen highly similar homologous sequences. Through the above steps, the homology between genes can be scientifically evaluated.

[0076] Genes with a homology greater than 80% are generally considered to be homologous genes. For example, homology greater than 82%, homology greater than 84%, homology greater than 86%, homology greater than 88%, homology greater than 90%, homology greater than 92%, homology greater than 94%, homology greater than 96%, homology greater than 98%, homology is 100%, etc.

[0077] The "T-DNA" mentioned in this article refers to T-DNA insertion mutation, which refers to the random insertion of T-DNA (transfer DNA) into the plant genome, causing the structure of the inserted gene to change, thereby generating a mutation. This mutation is a heritable variation phenomenon on the genomic DNA molecule, which is specifically manifested as a change in the base pair composition or arrangement order of the gene structure.

[0078] The "SALK_047439" and "SALK_085995" mentioned in this article are both Arabidopsis atarp2;1 mutants, which are two types of T-DNA insertions in Col-0 Arabidopsis. The T-DNA insertion position of SALK_047439 is located in the promoter, and the T-DNA insertion position of SALK_085995 is located in the middle and late part of the second exon.

[0079] The “MtCNGC15” mentioned herein refers to CNGC15 derived from Medicago truncatula of the Leguminosae family, which is a protein confirmed to have calcium ion transport activity, and its amino acid sequence is shown in Table 3.

[0080] The “OsHKT2;4” mentioned herein refers to HKT2;4 derived from rice, which is a protein that has been confirmed to have magnesium ion transport activity, and its amino acid sequence is shown in Table 3.

[0081] The "BnARP2;1" and "BnFER" mentioned herein refer to ARP2;1 and FER derived from rapeseed.

[0082] Table 3

[0083]

[0084]

[0085] The present invention provides a Ca-binding protein for the ARP2;1 homologous gene of plant stigma cells. 2+ Application of impaired transport capacity in breaking through plant reproductive isolation. In some embodiments, reproductive isolation is caused by interspecific hybridization incompatibility. Further, in some embodiments, interspecific hybridization incompatibility is stigma-specific interspecific incompatibility.

[0086] In some embodiments, the plant is a crucifer. Further, in some embodiments, the crucifer is Arabidopsis, cauliflower, rape, cabbage, Chinese cabbage, shepherd's purse, camelina, salt mustard, rocket, radish, mustard white, or iris. Those skilled in the art should understand that there are many species of crucifers, and this application only lists some of them to illustrate that the crucifers can use the method of this application to break through the incompatibility of interspecific hybridization, and no longer list the crucifers one by one.

[0087] Those skilled in the art should understand that the ARP2;1 homologous bases mentioned herein are genes with a homology of not less than 80% in different plants and can be used to regulate reproductive isolation of plants, and such genes have calcium ion transport function. For example, in some embodiments, in Col-0 ecotype Arabidopsis, the amino acid sequence of ARP2;1 is as shown in SEQ ID NO.1; in rapeseed, the amino acid sequence of ARP2;1 is as shown in SEQ ID NO.13.

[0088] The present application also relates to a method for allowing a plant to break through reproductive isolation, comprising: causing the Ca of ARP2;1 of the plant stigma cell to 2+ Impaired transport capacity.

[0089] In some embodiments, wherein Ca 2+ The impaired transport capacity is caused by substitution, insertion or deletion mutation of ARP2;1 itself in the plant stigma cells. For example, there are three mutations in the ARP2;1 protein sequence of Cvi-0 type Arabidopsis and Col-0 type Arabidopsis, involving substitution and deletion: there are two amino acid residue mutations (E2V, N92H), and due to the mutation at the stop codon, the length of AtARP2;1B is 12 amino acid residues longer than AtARP2;1A. The ARP2;1 of Col-0 type Arabidopsis has calcium ion transport activity while the Cvi-0 type Arabidopsis does not. Further, the present application relates to two Col-0 type Arabidopsis ARP2;1 gene T-DNA inserted seeds SALK_047439 and SALK_085995, and the ARP2;1 calcium ion activity of the two is affected to different degrees.

[0090] The mutations caused by substitution, insertion or deletion of ARP2;1 itself listed in this application are only examples to illustrate that the calcium ion transport capacity of ARP2;1 is impaired, and are not intended to be limiting of the manner in which the calcium ion transport capacity of ARP2;1 is impaired, or the mutations caused by substitution, insertion or deletion of ARP2;1 itself.

[0091] In some embodiments, wherein Ca 2+The impaired transport capacity is caused by the inhibition of the activity of ARP2; 1 by an ARP2; 1 inhibitor. In some embodiments, the inhibitor can be an inorganic salt inhibitor, such as GdCl3; or an organic inhibitor.

[0092] In some embodiments, the ARP2;1 inhibitor includes FER. In some embodiments, the amino acid sequence of FER is shown in SEQ ID NO.2; or a sequence having at least 80% homology with SEQ ID NO.2. In some embodiments, FER and ARP2;1 are from the same plant species.

[0093] In some embodiments, the inhibitor is a FER-ANJ complex, a FER-CVY1 complex, a FER-HERK1 complex, or a FER-CVY1-HERK1 complex. In some embodiments, the amino acid sequence of CVY1 is shown in SEQ ID NO.3; or a sequence having at least 80% homology to SEQ ID NO.3; the amino acid sequence of HERK1 is shown in SEQ ID NO.4; or a sequence having at least 80% homology to SEQ ID NO.4. In some embodiments, CVY1 and HERK1 are from the same species of plants as ARP2;1.

[0094] Among them, FER, CVY1 and HERK1 are receptor-like kinases. Reports have shown that receptor-like kinases play an important role in the regulation of ROS during the pollination of cruciferous plants. Studies have shown that the FER-CVY1 / ANJ / HERK1-mediated signaling pathway can form interspecies isolation and protect the stigma from the influence of heterospecific pollen. The application of artificially synthesized pollen-derived RALF26 to the stigma can break this isolation.

[0095] The technical solution of the present application will be described below through specific embodiments. Those skilled in the art should understand that the following embodiments are only for illustrating the technical solution of the present application, but not for limiting the protection scope of the present application.

[0096] Example 1: Pollen of Capsella pursica germinates and grows normally on the stigma of atarp2;1 mutant

[0097] 1. Analysis of the expression pattern of AtARP2;1

[0098] The inventors detected the expression level of AtARP2;1 in roots, stems, flowers, siliques, cauline leaves and rosette leaves of wild-type Col-0 Arabidopsis thaliana by semi-quantitative PCR experiments and found that AtARP2;1 was only expressed in flowers (e.g. Figure 1 shown).

[0099] 2. Subcellular localization of AtARP2;1 protein

[0100] The inventors cloned the CDS sequence of AtARP2;1 into the pE3449-GFP vector, fused it with the GFP gene driven by the cauliflower mosaic virus (CaMV) 35S promoter, and transformed it into Arabidopsis protoplasts using polyethylene glycol mediation to make it transiently expressed in the protoplasts. Protoplasts transformed with an empty vector were used as controls. The GFP signal of 35S:AtARP2;1-GFP can only be observed on the cytoplasmic membrane, overlapping with the red fluorescence emitted by the cytoplasmic membrane-specific dye FM4-64, while the control GFP green fluorescence signal appears in multiple subcellular structures such as the cytoplasm and plasma membrane. These results indicate that AtARP2;1 is localized to the cytoplasmic membrane (e.g. Figure 2 shown).

[0101] 3. Identification of atarp2;1 mutant

[0102] In order to study the function of AtARP2;1 in plants, the inventors purchased atarp2;1 mutants atarp2;1-1 (SALK_047439) and atarp2;1-2 (SALK_085995) from ABRC, where the insertion position of SALK_047439 is located in the promoter, and the insertion position of SALK_085995 is located in the middle and late part of the second exon (e.g. Figure 3 As shown in A). According to the identification at the genomic and transcriptional levels, SALK_047439 is a homozygous gene function loss mutant, and SALK_085995 is a homozygous mutant at the genomic level, while at the transcriptional level, its AtARP2;1 gene still has a small amount of transcription, which is a knockdown mutant (as shown in Figure 3 B).

[0103] 4. Shepherd's purse pollen germinates and grows normally on the stigma of the atarp2;1 mutant

[0104] In this example, the T-DNA inserted strain SALK_085995 was used as the atarp2;1 mutant strain. The inventors respectively inoculated the pollen of Shepherd's purse on the wild-type stigma of Arabidopsis Col-0, the wild-type stigma of Arabidopsis Cvi-0 and the stigma of the atarp2;1 mutant. The pollen of Shepherd's purse could not germinate normally on the wild-type Col-0 stigma, but could germinate on the stigma of the atarp2;1 mutant.

[0105] The results of the semi-in vivo pollen tube growth experiment showed that when Arabidopsis thaliana wild-type and atarp2;1 mutant stigmas were inoculated with Arabidopsis thaliana pollen, pollen tubes grew normally. However, when inoculated with Capsella purseus pollen, only a very small number of growing pollen tubes were observed on the wild-type stigmas, while a large number of pollen tubes were clearly observed extending into the culture medium on the atarp2;1 mutant stigmas (e.g. Figure 4 When Arabidopsis thaliana pollen was applied to the stigmas of Col-0 and Cvi-0, different ecotypes of Arabidopsis thaliana, the pollen tubes grew normally. However, when Capsella pursica pollen was applied, no extended pollen tubes were observed on the Col-0 stigma, while a large number of pollen tubes were clearly observed extending into the culture medium on the Cvi-0 stigma (as shown in Figure 2 ). Figure 5 shown).

[0106] The results of aniline blue staining after pollination were consistent with the results of the semi-in vivo experiment. The results of aniline blue staining after pollination showed that only a few pollen grains of Capsella purse germinated on the stigma of Arabidopsis Col-0, a few pollen grains of Capsella purse germinated on the stigma of Arabidopsis Col-0, and a large number of pollen grains germinated on the stigma of the atarp2;1 mutant (such as Figure 6 Combined with the previous experimental results, Cvi-0 and atarp2;1 phenotypes are consistent (as shown in Figure 4 , Figure 5 as well as Figure 6 It can be inferred that AtARP2;1 is involved in the process of preventing the extension of heterospecific pollen tubes in Arabidopsis, and its functional loss can promote the germination of heterospecific pollen during interspecific hybridization, causing pollen tubes to grow on incompatible stigmas.

[0107] Example 2 Sequence feature differences between AtARP2;1A and AtARP2;1B from different ecotypes of Arabidopsis thaliana

[0108] In order to explore the differences in AtARP2;1 molecules in different ecotypes of Arabidopsis thaliana, the inventors analyzed the AtARP2;1 protein sequences of 242 different Arabidopsis thaliana ecotypes. Among them, there are two AtARP2;1 molecules AtARP2;1A and AtARP2;1B containing four transmembrane structures, and there are 7 other AtARP2;1 molecules AtARP2;1C, AtARP2;1D, AtARP2;1E, AtARP2;1F, AtARP2;1G, AtARP2;1H and AtARP2;1I. The AtARP2;1C-AtARP2;1I molecules all have premature transcription termination caused by single base mutations, and their transmembrane structures are also destroyed.

[0109] Furthermore, AtARP2;1A was present in most ecotypes of Arabidopsis (such as Col-0, Ler-0, Ws-0, etc.), with a total of 207, accounting for 85.54% of the total; while AtARP2;1B was relatively rare, existing in 12 ecotypes such as Can-0 and Cvi-0, accounting for 4.96% of the total; other AtARP2;1 with premature transcription termination was only distributed in 23 ecotypes, accounting for 9.50% (such as Figure 7 B).

[0110] Sequence analysis revealed that AtARP2;1A and AtARP2;1B contain 221 and 233 amino acid residues, respectively. Compared with AtARP2;1A, AtARP2;1B has two amino acid residue mutations (E2V, N92H), and due to a mutation at the stop codon, the length of AtARP2;1B is 12 more amino acid residues than AtARP2;1A (e.g. Figure 7 A).

[0111] Example 3 Differences in protein structure between AtARP2;1A derived from Col-0 and AtARP2;1B derived from Cvi-0

[0112] The inventors used Alphafold3 to predict the protein polymer structure of AtARP2; 1A and AtARP2; 1B. After analyzing the prediction results, it was found that the extracellular perspective perpendicular to the cell membrane clearly showed that the monomers of the two proteins, as well as the dimers, trimers and tetramers, could not form a complete pore structure, and had no possibility of forming a calcium ion channel. At the monomer level, since AtARP2; 1B has 12 more amino acids at the C-terminus than AtARP2; 1A, it forms a curved structure similar to a β-turn in the tertiary structure. However, no obvious structural differences were found between the two proteins in the dimers, trimers and tetramers (such as Figure 8 shown).

[0113] The inventors have analyzed the structure of polymers with more subunits. The results show that in the structural predictions of pentamers and above, a clear pore structure can be seen, which meets the basic conditions for the formation of a channel. Therefore, it can be inferred that AtARP2;1 needs at least 5 subunits to form a homologous pentamer (such as Figure 8 shown).

[0114] In the pentamer, hexamer, heptamer and nonamer structures, AtARP2;1A and AtARP2;1B both formed unobstructed pore structures, and theoretically both could transport calcium ions. However, there were obvious differences in the octamer structures of the two. AtARP2;1A had a normal pore structure, while the intracellular end of the pore structure of AtARP2;1B was blocked (e.g. Fig. 9 Therefore, the inventors predicted that AtARP2;1B may not be able to transport calcium ions.

[0115] The inventors also predicted the structures of AtARP2;1 polymers with more subunits (10-19 subunits), and the predicted results were all abnormal structures. This may be because the Alphafold prediction process cannot take into account the effect of the phospholipid bilayer of the cytoplasmic membrane on the protein structure. The confidence of these structures is also low. Therefore, the inventors believe that these results are not of reference value, and it is more likely that the AtARP2;1 molecule has 5-8 subunits.

[0116] Example 4 AtARP2;1A mediates ion transport, while AtARP2;1B does not

[0117] 1. Differences in calcium channel activity between AtARP2;1A and AtARP2;1B

[0118] In this example, homologous genes AtARP2;1A and AtARP2;1B in Col-0 Arabidopsis and Cvi-0 Arabidopsis were selected, and the ion transport activities of the two were recorded when CaCl2 was used as the perfusion fluid. The inventors used a two-electrode voltage clamp to record the ion transport activities of AtARP2;1A and AtARP2;1B, and found that AtARP2;1A had ion transport activity, while AtARP2;1B did not (e.g. Fig.10 shown).

[0119] 2. Functional verification of AtARP2;1A and AtARP2;1B complementary yeast calcium transporter mutants

[0120] MID1 is a transmembrane protein in yeast that participates in calcium ion absorption at low calcium concentrations, and CCH1 is a transmembrane protein that is highly homologous to voltage-gated calcium channels in animals. Both have calcium ion transport activity and participate in the calcium ion absorption process of yeast cells. α factor is the partner pheromone of yeast cells, which can stimulate calcium ion absorption and accumulation in yeast cells. Mutants lacking the MID1 or CCH1 gene have greatly increased sensitivity to α factor, and cch1 / mid1 double mutants will die within a few hours at low calcium concentrations. Using this characteristic of the cch1 / mid1 double mutant, expressing exogenous proteins in vivo can verify whether they have calcium ion channel activity.

[0121] In the yeast complementation experiment, α factor diffused from the cellulose disk into the culture medium, preventing the growth of yeast that cannot absorb calcium ions and forming an "inhibition zone" structure without yeast colonies around it. Yeast that can absorb calcium ions can grow near the cellulose disk.

[0122] Yeast complementation experiments showed that no colonies grew around the cellulose disk of the cch1 / mid1 strain transformed with the empty vector (EV); the expression of the positive control MtCNGC15 or AtARP2;1A in the cch1 / mid1 double mutant enabled the colonies to grow around the cellulose disk, which was consistent with the growth of the wild-type yeast strain; and on the plates coated with the cch1 / mid1 yeast mutant expressing AtARP2;1B, an obvious inhibition zone appeared around the cellulose disk, which was consistent with the negative control (e.g. Fig.11 This result indicates that AtARP2;1A can transport calcium ions into the cell, while AtARP2;1B has no calcium ion transport activity.

[0123] 3. Single-cell fluorescence imaging experiments to verify the calcium transport activity of AtARP2;1A and AtARP2;1B

[0124] Through HEK293T single-cell fluorescence imaging experiments, it can be found that after calcium ion stimulation, the fluorescence intensity of cells expressing AtARP2; 1A increased significantly, which is consistent with the trend of positive control cells expressing AtCNGC18, while the fluorescence intensity of cells expressing AtARP2; 1B did not change significantly, which is similar to the negative control (EV) transformed with an empty vector (such as Fig.12 This result also verifies the conclusion drawn by the present application through the two-electrode voltage clamp system and yeast complementation experiment, that AtARP2; 1A has calcium ion transport activity, while AtARP2; 1B cannot transport calcium ions.

[0125] Previous structural analysis results showed that the intracellular end of the pore structure of AtARP2;1B was blocked. The cause of this structure may be that the C-terminus of the AtARP2;1B molecule is relatively long, and the folded structure it forms makes the protein sterically hindered, forming a special structure that can interact with the N-terminus of AtARP2;1B, thereby changing the spatial configuration of the free N-terminus of the protein and folding toward the center of the pore. The V2 of each subunit approaches each other. Due to the strong hydrophobicity of valine, the V2 of these subunits forms a hydrophobic ring structure by virtue of the hydrophobic interaction between molecules, causing pore blockage (such as Fig. 9 The enlarged image of the AtARP2;1B octamer shown in the figure shows that the area circled in light green is the valine at position 2 of each subunit, which is unable to transport calcium ions. One of the three mutations of AtARP2;1A and AtARP2;1B is E2V, which can also explain the difference in calcium channel activity between AtARP2;1A and AtARP2;1B from a structural perspective. The prediction results of the spatial structure of the octamer can preliminarily determine that the reason why AtARP2;1B cannot transport calcium ions is that the valine interactions between the free N-termini of its subunits cause the channel to be blocked.

[0126] Since AtARP2;1A and AtARP2;1B exist in two different ecotypes, Col-0 and Cvi-0, respectively, combined with previous experimental results, it can be determined that the calcium channel activity of AtARP2;1A is a necessary condition for Arabidopsis to prevent the extension of heterospecific pollen tubes.

[0127] Example 5 AtARP2;1A specifically mediates calcium influx

[0128] As we all know, calcium ions are second messengers in organisms. External stimuli are received by corresponding receptors, activating calcium ion channels to produce specific calcium ion signals (encoding); calcium ions then bind to specific proteins (calcium ion receptors), regulating effector proteins and causing changes in cell activity (decoding); finally, calcium ions are expelled from the cytoplasm to restore the resting state. In the pollination process of Arabidopsis, calcium ions also transmit corresponding signals as second messengers.

[0129] In this example, AtARP2;1 is derived from Arabidopsis thaliana of the Col-0 ecotype. Hereinafter, unless otherwise specified, AtARP2;1 refers to AtARP2;1A.

[0130] 1.AtARP2;1A does not transport monovalent cations

[0131] In the perfusion solution containing 20mM KCl, oocytes expressing the positive control Arabidopsis potassium channel AtKAT1 have significant potassium ion internalization activity, while the current recorded from Xenopus oocytes expressing AtARP2;1 is not significantly different from the negative control injected with ddH2O, which shows that AtARP2;1 has no potassium ion transport activity. Similarly, in the perfusion solution containing 20mMNaCl, frog eggs expressing the positive control typical Arabidopsis sodium ion transporter AtHKT1 can also record significant sodium ion currents, while the current signal recorded from AtARP2;1 is not significantly different from the negative control, and AtARP2;1 has no sodium ion transport activity (such as Fig.13 The above experimental results show that AtARP2;1 does not have monovalent cation transport activity.

[0132] 2. AtARP2; 1A specifically mediates calcium influx

[0133] In the perfusion solution containing 20mM MgCl2, the African clawed frog oocytes expressing the positive control OsHKT2;4 had significant magnesium ion transport activity, while the current recorded by the cells expressing AtARP2;1 was 0.198±0.084μA, slightly higher than the current recorded by the water-injected frog eggs. However, there was no significant difference between AtARP2;1 and the negative control in statistics, which shows that AtARP2;1 has no magnesium ion transport activity. In the perfusion solution containing 20mM CaCl2, the positive control AtCNGC 14 There is obvious calcium ion transport activity. The African clawed frog oocytes expressing AtARP2;1 also recorded an inward current with obvious tail current, which is the characteristic of the typical voltage-dependent calcium ion channel current. When the clamping voltage drops below -60mV, the AtARP2;1 channel is opened (such as Fig.14 The above experimental results show that AtARP2;1 specifically transports calcium ions and is a calcium ion channel.

[0134] 3. The channel activity of AtARP2;1A is calcium ion concentration dependent

[0135] In order to further study the characteristics of AtARP2;1 channels, the inventors used a two-electrode voltage clamp system to analyze the response of AtARP2;1 channels to Ca 2+ To investigate the dependence of calcium ion concentration, the inventors adjusted the concentration of calcium ions in the perfusate and used perfusates containing 0mM, 5mM, 10mM and 30mM CaCl2 to record the electrophysiological activity of Xenopus oocytes expressing AtARP2;1. The experiment found that as the calcium ion concentration in the perfusate increased, the recorded current also showed an increasing trend. When the CaCl2 concentration increased from 5mM to 30mM, the inward current increased from 0.389μA to 3.225μA, and the relationship between the two was almost proportional. It can be seen that the channel activity of AtARP2;1 is linearly related to the calcium ion concentration and has calcium ion concentration dependence (such as Fig.15 shown).

[0136] 4. Pharmacological properties of AtARP2;1A

[0137] Since CaCC channels exist in Xenopus oocytes, AtARP2;1-mediated calcium ion influx may activate endogenous CaCC channels in Xenopus oocytes and lead to chloride ion efflux. Both calcium ion influx and chloride ion efflux may produce strong inward currents. Therefore, the currents recorded previously were mixed currents of CaCC-mediated chloride ion efflux currents and AtARP2;1-mediated calcium ion influx currents. Therefore, the inventors studied the pharmacological properties of AtARP2;1 and added 500 μM 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt (DIDS) to the perfusate. DIDS is an inhibitor of anion transport that can inhibit the chloride ion transport activity of endogenous CaCC in African clawed frog oocytes. Two-electrode voltage clamp recording showed that a small current of only 0.360±0.226 μA could still be recorded in the perfusate containing DIDS, and the characteristic current of CaCC completely disappeared. This current is the pure calcium ion current mediated by ARP2;1. When 50 μM of the typical calcium channel inhibitor GdCl3 was added to the perfusion fluid, the inward current mediated by AtARP2;1 almost disappeared completely, and was only 0.180±0.033 μA, which was not significantly different from the negative control injected with ddH2O, indicating that the calcium influx mediated by AtARP2;1 can be blocked by the calcium channel inhibitor GdCl3, and its transport activity can be basically completely inhibited (e.g. Fig.16 As shown). It can be determined that AtARP2;1 is a typical calcium ion channel.

[0138] Example 6 AtFER-AtANJ kinase complex completely inhibits the channel activity of AtARP2;1A

[0139] Previously, several papers reported that during the pollination of cruciferous plants, the PCP-Bs peptide on the pollen binds to the receptor kinase complex ANJ-FER on the plasma membrane of the papillary cells, thereby inhibiting the activity of RBOHD in the papillary cells and reducing the level of ROS in the stigma. The FER-CVY1 / ANJ / HERK1-mediated signaling pathway can form interspecies isolation and protect the stigma from the influence of heterospecific pollen. The application of artificially synthesized RALF26 derived from pollen to the stigma can break this isolation.

[0140] It can be seen that receptor-like kinases (RLKs) play an important role in ROS regulation during the pollination of cruciferous plants. Existing studies have shown that RLKs are widely involved in calcium ion signaling in plants. It can be inferred that AtRLKs are also involved in the calcium ion signal encoding during the pollination process of Arabidopsis.

[0141] The inventors screened and obtained four receptor-like kinases located on the stigma, namely AtFER, AtANJ, AtCVY1 and AtHERK1, to verify whether they can regulate the calcium channel activity of AtARP2;1.

[0142] The inventors co-expressed AtARP2;1A with AtFER, AtANJ, AtCVY1, and AtHERK1 in Xenopus oocytes, and used a two-electrode voltage clamp to detect its calcium ion transport activity. The results showed that the co-expression of AtANJ, AtCVY1, and AtHERK1 with AtARP2;1A did not significantly change the calcium ion channel activity of AtARP2;1A; while the co-expression of AtFER with AtARP2;1A partially inhibited its calcium ion transport activity; when AtFER, AtANJ, and AtARP2;1A were co-expressed, the calcium ion transport activity of AtARP2;1A was basically completely inhibited; when AtARP2;1A was co-expressed with AtFER and AtCVY1 or AtHERK1, its calcium ion transport activity was not significantly different from that of AtARP2;1A and AtFER (e.g. Fig.17 shown).

[0143] It can be seen that the calcium ion transport activity of AtARP2;1A is regulated by AtFER / AtANJ, and this pattern is highly consistent with the ROS signaling mechanism during the pollination of Cruciferae. It is inferred that the AtARP2;1-AtFER-AtANJ complex is involved in the pollen recognition process of Arabidopsis, recognizing small peptides in pollen and transmitting calcium ion signals to downstream proteins.

[0144] Example 7

[0145] Since ARP2;1 is widely present in cruciferous plants, in order to verify the functional universality of ARP2;1 and related regulatory molecules, the inventors cloned the ARP2;1 and FER genes of rapeseed, expressed or co-expressed them in African clawed frog oocytes, and detected the electrophysiological activities respectively. The experimental results showed that BnARP2;1 of rapeseed also has obvious calcium ion transport activity, and its homologous BnFER can also inhibit BnARP2;1 channel activity (such as Fig.18 As shown in Figure 2 ). This shows that the hybrid incompatibility mechanism regulated by the ARP2;1-ANJ / FER complex is widely present in Cruciferae, and the hybrid incompatibility rule found in the model plant Arabidopsis is universal in Cruciferae.

[0146] As we all know, the cruciferous plants are extremely rich in species, including oil crops such as rapeseed, cash crops such as cabbage, Chinese cabbage, shepherd's purse, and even ornamental plants such as violets and kale. These plants have great economic value (such as Fig.19 The experimental evidence provided in this application not only provides a theoretical basis for achieving interspecific hybridization breeding of cruciferous crops, but also provides target gene resources that can be genetically manipulated, which has potential wide application value.

[0147] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

1. Ca2+ homolog of ARP2;1 in plant stigma cells 2+ Application of impaired transport capacity in breaking reproductive isolation in plants.

2. The use according to claim 1, wherein the reproductive isolation is caused by interspecific hybrid incompatibility.

3. The use according to claim 2, wherein the interspecific hybridization incompatibility is stigma-specific interspecific incompatibility.

4. The use according to claim 1, wherein the plant is a cruciferous plant.

5. The use according to claim 4, wherein the cruciferous plant is Arabidopsis thaliana, cauliflower, rapeseed, cabbage, Chinese cabbage, shepherd's purse, camelina, salt mustard, rocket, radish, white mustard, or iris.

6. The use according to claim 1, wherein the amino acid sequence of ARP2;1 is: the sequence shown in SEQ ID NO.1; or a sequence having at least 80% homology with SEQ ID NO.

1.

7. A method for enabling a plant to break through reproductive isolation, comprising: Ca2+ in ARP2;1 of the plant stigma cells 2+ Impaired transport capacity.

8. The method according to claim 7, wherein Ca 2+ The impaired transport capacity is caused by substitution, insertion or deletion mutation of ARP2;1 itself in plant stigma cells, or by the inhibition of ARP2;1 activity by ARP2;1 inhibitors.

9. The method of claim 8, wherein the ARP2;1 inhibitor is GdCl3.

10. The method of claim 8, wherein the ARP2;1 inhibitor comprises FER.

11. The method according to claim 10, wherein the amino acid sequence of FER is as shown in SEQ ID NO. 2; or a sequence having at least 80% homology with SEQ ID NO.

2. 12 . The method according to claim 8 , wherein the FER-containing inhibitor is a FER-ANJ complex, a FER-CVY1 complex, a FER-HERK1 complex or a FER-CVY1-HERK1 complex.

13. The method according to claim 12, wherein the amino acid sequence of CVY1 is as shown in SEQ ID NO.3; or a sequence having at least 80% homology with SEQ ID NO.3; the amino acid sequence of HERK1 is as shown in SEQ ID NO.4; or a sequence having at least 80% homology with SEQ ID NO.4.

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