Application of citrus S-adenosylmethionine synthetase in improving self-incompatibility

Abstract

The invention provides application of citrus S-adenosylmethionine synthetase in improving self-incompatibility, and belongs to the technical field of biological genetic engineering, and the gene sequence of the citrus S-adenosylmethionine synthetase is shown as SEQ ID: 1. Experiments prove that citrus S-adenosylmethionine synthetase directly interacts with S-RNase to regulate and control a methionine metabolic pathway so as to influence the growth of a pollen tube; the invention discloses a dual regulation and control effect of the citrus S-adenosylmethionine synthetase in a self-incompatible reaction, and provides a new target for improving the self-compatibility of crops. According to the method, the citrus S-adenosylmethionine synthetase is added into pollen culture or methionine is sprayed onto styles, so that the growth of pollen tubes is improved, the self-incompatibility of the citrus is further improved, a new strategy is provided for improving the self-incompatibility of crops, and the problems of low fruit setting rate, high artificial pollination cost and the like caused by the self-incompatibility are hopefully solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological genetic engineering, and more specifically relates to the application of citrus S-adenosylmethionine synthetase in improving self-incompatibility. Background Art

[0002] Citrus is the world's largest fruit, and my country is the world's largest producer and consumer of citrus. However, some grapefruit and lemon varieties have low natural fruit set rates due to their self-incompatibility and weak parthenocarpic ability. In production, artificial assisted pollination is often required to increase yield, resulting in increased production costs and seriously affecting their commercial value.

[0003] Plant self-incompatibility (SI) is a key mechanism for maintaining genetic diversity in flowering plants, achieving reproductive isolation through pollen-stigma recognition. Citrus, a typical species exhibiting gametophytic self-incompatibility (GSI), relies on S-RNase-mediated pollen tube RNA degradation for its SI response. However, existing research has primarily focused on the recognition and degradation of S-RNase, while understanding of downstream signaling pathways remains insufficient. It is urgent to address the regulatory mechanisms of non-S factors in the downstream SI pathways of citrus, thereby overcoming the existing reliance on single-gene regulation by S-RNase.

[0004] S-adenosylmethionine synthase (SAMS) is the rate-limiting enzyme in methionine metabolism. It catalyzes the production of S-adenosylmethionine (SAM), a precursor for methylation reactions and the synthesis of polyamines and ethylene. It plays a central role in plant growth, development, and stress responses. Although studies have reported that SAMS is involved in plant stress tolerance and the regulation of floral organ development, its role in self-incompatibility has not been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of citrus S-adenosylmethionine synthase in improving self-incompatibility, improving citrus self-incompatibility by improving pollen tube growth, providing a new strategy for improving crop self-compatibility, and is expected to solve problems such as low fruit set rate and high artificial pollination cost caused by self-incompatibility.

[0006] To achieve the above object, the present invention provides the use of citrus S-adenosylmethionine synthetase in improving self-incompatibility. The gene sequence of the citrus S-adenosylmethionine synthetase is shown in SEQ ID: 1.

[0007] Furthermore, the coding gene sequence of the citrus S-adenosylmethionine synthetase is shown in SEQ ID: 2.

[0008] Furthermore, pollen tube growth was improved by adding citrus S-adenosylmethionine synthetase to the pollen culture.

[0009] Furthermore, by spraying methionine onto the style, the expression inhibition of citrus S-adenosylmethionine synthase was improved, thereby improving the growth of pollen tubes.

[0010] Furthermore, the spraying concentration of the methionine is 0.9-1.1 mM.

[0011] Compared with the prior art, the present invention has the following technical effects:

[0012] This study experimentally demonstrates that citrus S-adenosylmethionine synthase (CgSAMS4) regulates the methionine metabolic pathway, influencing pollen tube growth, by directly interacting with S-RNase. This study also reveals the dual regulatory role of citrus S-adenosylmethionine synthase in self-incompatibility, providing a new target for improving crop self-compatibility. By adding citrus S-adenosylmethionine synthase to pollen culture media or spraying methionine onto the style, the study improves pollen tube growth, thereby improving citrus self-incompatibility. This provides a new strategy for improving crop self-compatibility and is expected to address issues such as low fruit set and high artificial pollination costs caused by self-incompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 This is a diagram showing the effect of the antisense oligonucleotide provided in Example 2 of the present invention on the growth of pollen tubes by inhibiting the expression of CgSAMS4;

[0015] Figure 2 This is a statistical graph of pollen tube growth length after the antisense oligonucleotide provided in Example 2 of the present invention inhibited the expression of CgSAMS4;

[0016] Figure 3 This is a graph showing the dual luciferase complementation imaging analysis of CgSAMS4 and S-RNase provided in Example 5 of the present invention;

[0017] Figure 4 Pull-down interaction analysis diagram of CgSAMS4 and S-RNase provided in Example 5 of the present invention;

[0018] Figure 5This is a diagram showing the effects of different pollination methods on SAMS activity provided in Example 4 of the present invention;

[0019] Figure 6 This is a graph showing the methionine content in styles during different pollinations provided in Example 4 of the present invention;

[0020] Figure 7 This is an observation diagram of aniline blue staining of a style treated with exogenous methionine provided in Example 4 of the present invention;

[0021] Figure 8 This is a diagram showing the effects of different protein treatments on incompatible pollen tube growth provided in Example 3 of the present invention;

[0022] Figure 9 This is a statistical graph of pollen tube growth length after treatment with different proteins provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The singular forms "a", "an", "the" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0025] Example 1 Antisense Oligonucleotide Primer Design

[0026] In the embodiment of the present invention, antisense oligonucleotide primers are designed for citrus S-adenosylmethionine synthetase, so as to inhibit the expression of the gene encoding citrus S-adenosylmethionine synthetase through antisense oligonucleotide silencing, thereby inhibiting the growth of pollen tubes.

[0027] The gene sequence of the citrus S-adenosylmethionine synthetase (CgSAMS4) of the embodiment of the present invention is shown in SEQ ID: 1, and the sequence of the gene encoding the citrus S-adenosylmethionine synthetase is shown in SEQ ID: 2. The protein encoded by the encoding gene, citrus S-adenosylmethionine synthetase, contains 393 amino acid residues and participates in the catalytic reaction of converting methionine to S-adenosylmethionine (SAM).

[0028] In this embodiment of the present invention, the mfold website (http: / / www.unafold.org / mfold / applications / rna-folding-form.php) was used to predict the secondary structure of the target gene CgSAMS4 mRNA. The configuration with the lowest free energy was selected as a reference. The sequence of the bubble region was used as the target. Based on the principle of base complementary pairing, an antisense oligonucleotide with a length of 20 bp was designed that was completely complementary to the target sequence. Simultaneously, the Sfold website (http: / / sfold.wadsworth.org / cgibin / soligo.pl) was used to screen for available antisense oligonucleotides. Candidate AS-ODN and S-ODN were synthesized by adding thiolation modifications between the four bases at each end. The primer pair information is shown in Table 1, including the primer with the gene sequence shown in SEQ ID: 3 and the primer with the gene sequence shown in SEQ ID: 4. After each primer was dissolved in pollen culture medium to a 100 μM stock solution, pollen tubes were cultured in a ratio of pollen culture medium to stock solution of 3:1.

[0029] Table 1 Antisense oligonucleotide primer pair information

[0030]

[0031] Example 2 In vitro pollen culture

[0032] Crystal pomelo pollen (S genotype is S5S6) was used as the experimental material, and a 1.5 mL centrifuge tube cap was used as a pollen tube culture vessel. The pollen tube was cultured with 100 μL of the pollen medium of Example 1: mother liquor = 3:1, wherein the pollen medium composition is: 10% sucrose, 13.3% PEG-4000, 0.02% MgSO4·7H2O, 0.01% KNO3, 0.03% Ca(NO3)2·4H2O, 0.01% H3BO3, and the pollen was evenly sprinkled on the surface of the liquid culture medium; a clean centrifuge tube box was taken, and an appropriate amount of distilled water was added to the bottom of the box. The centrifuge tube cap was then placed in the box, the centrifuge tube box lid was closed, and the box was placed in a dark incubator at 28°C for 9 hours; the pollen tube was transferred to a glass slide using a 1 ml pipette tip with the tip cut off, covered with a coverslip, and observed and photographed with an inverted microscope. The results are as follows: Figure 1 As shown; using a digital screen (GM185, Gaoman), the pollen tube length was measured and the pollen germination rate was calculated using Image J software. The results are shown in Figure 2 shown.

[0033] Figure 1 、 Figure 2In the figure, CK represents the control group, which was not treated, i.e., no mother solution was added to the liquid culture medium; S-CgSAMS4 represents the liquid culture medium to which a mother solution containing a primer of SEQ ID: 3 was added; and AS-CgSAMS4 represents the liquid culture medium to which a mother solution containing a primer of SEQ ID: 4 was added. Figure 2 Yes Figure 1 Quantitative processing, from Figure 1 、 Figure 2 It can be seen that the average length of pollen tubes in the control group was 579.468±14.3μm, the average length of pollen tubes in the S-CgSAMS4 treatment group was 533.749±24.14μm, and the average length of pollen tubes in the AS-CgSAMS4 treatment group was 372.749±7.29μm, indicating that antisense oligonucleotides inhibiting the expression of CgSAMS4 will affect pollen tube growth and hinder pollen tube growth, thereby proving the importance of citrus S-adenosylmethionine synthase in the reproductive process of citrus.

[0034] Example 3 Effect of CgSAMS4 protein treatment on incompatible pollen tube growth

[0035] Crystal pomelo pollen (S genotype: S5S6) was used as the experimental material. Purified S1-RNase and S2-RNase proteins were added to the same pollen culture medium. S5-RNase and S6-RNase were also added to the same pollen culture medium. The S1-RNase + S2-RNase group was designated as the compatibility treatment group, and the S5-RNase + S6-RNase group was designated as the incompatibility treatment group. A control group without S-RNase was also established, as was a combination of the incompatibility treatment group with recombinant CgSAMS4 protein (Incompatibility + CgSAMS4). Each protein was added to a final concentration of 100 ng / μL. The culture medium was sprinkled with Crystal pollen and incubated at 28°C in a humidified dark environment for 12 hours.

[0036] Observe and take pictures with an inverted microscope. Figure 8 As shown, Figure 8 The scale is 200 μm. A digital screen (GM185, Gaoman) was used to measure the pollen tube length and calculate the pollen germination rate using Image J software. The results are shown in the figure. Figure 9 shown.

[0037] from Figure 8 、 Figure 9As shown in the study, under normal conditions, the average pollen tube length of crystal pomelo was 582.1±36.32 μm. When recombinant S1-RNase and S2-RNase proteins were added to the culture medium, the average pollen tube length increased to 563.3±25.19 μm, showing no significant difference from the control group. However, after the addition of S5-RNase and S6-RNase proteins to the culture medium, the average length was only 332.2±8.83 μm, significantly shorter than the previous two groups. When recombinant CgSAMS4 protein was added to the incompatibility treatment group, the average pollen tube length increased to 468.5±11.95 μm. Although still shorter than the control and affinity treatment groups, it was significantly longer than the incompatibility treatment group. This suggests that CgSAMS4 protein has a certain promoting effect on the growth of incompatibility pollen tubes. Specifically, by adding citrus S-adenosylmethionine synthase CgSAMS4 to the pollen culture medium, pollen tube growth can be improved, thereby alleviating self-incompatibility.

[0038] Example 4 Determination of S-adenosylmethionine synthetase activity and S-adenosylmethionine content

[0039] Proteins were extracted from the styles of unpollinated, cross-pollinated, and self-incompatible pollination plants 4 days after pollination. The S-adenosylmethionine synthase activity and methionine content were determined using the plant S-adenosylmethionine synthase (SAMS) ELISA kit and plant methionine ELISA kit from Shanghai Yuanmu Biotechnology Co., Ltd. according to their product instructions. The results are shown in Figure 2. Figure 5 shown.

[0040] Figure 5 In the table, control represents no pollination, cross-pollination represents cross-compatible pollination, and self-pollination represents self-incompatible pollination. Figure 5 It can be seen that the activity of SAMS in the style after hybrid compatibility pollination is significantly higher than that in the style after self-incompatibility pollination and unpollinated (Control). At the same time, the activity of SAMS in the style after self-incompatibility pollination is significantly higher than that in the style after unpollination. The results show that in hybrid compatibility pollination, the activity of SAMS is significantly higher than that in self-incompatibility pollination, and S-RNase plays a toxic role in the self-incompatibility reaction. Therefore, it can be roughly deduced that S-RNase can inhibit the activity of SAMS. That is, in citrus self-incompatibility, the activity of citrus S-adenosylmethionine synthetase (SAMS) is inhibited, and SAMS is the rate-limiting enzyme that affects the synthesis of S-adenosylmethionine (S-adenosylmethionine) from methionine (also called methionine), and this synthesis pathway is the only pathway in the plant body, so the activity of SAMS is inhibited, the efficiency of this synthesis pathway will be reduced, and methionine will accumulate. Combined with Figure 6 The methionine concentration in the styles of flowers pollinated with self-incompatibility was significantly higher than that in unpollinated flowers and those pollinated with cross-compatibility. There was no significant difference between the methionine concentrations in styles pollinated with cross-compatibility and those pollinated with unpollinated flowers. This suggests that self-incompatibility may specifically induce abnormal accumulation of methionine in the styles, resulting in higher methionine concentrations in styles pollinated with self-incompatibility.

[0041] Example 5 Verification of the interaction between S-adenosine monoamine synthetase and S-RNase

[0042] 1. Tobacco luciferase-protein interaction analysis

[0043] The full-length CDS of S-RNase, with the stop codon removed, was ligated into the JW771 vector; the full-length CDS of CgSAMS4, with the stop codon removed, was ligated into the JW772 vector. The constructed gene vector combinations were then transformed into Agrobacterium tumefaciens strain GV3101. Together with the Agrobacterium containing the helper plasmid pSOUP-p19, the cells were shaken at 28°C and 220 rpm for 16-20 hours. The cells were harvested and resuspended in infection buffer (10mM MES, 0.1mM AS, 10mM MgCl2) to an OD600 of 0.6-0.7. The cells were then mixed in a 1:1:2 ratio of JW771:JW772:pSOUP-p19, inverted to mix thoroughly, and allowed to stand at 28°C for 2 hours. The mixture was injected into the underside of 4-5-week-old tobacco leaves using a 1mL syringe. After 3 days, the leaves were cut and 200 μL of 0.4 mg / mL D-luciferin sodium salt was applied to the back of the leaves. The leaves were then placed in a NightShade LB 985 live imaging instrument and kept in the dark for 5 minutes. The photos were taken and the results were as follows: Figure 3 shown.

[0044] exist Figure 3 In the figure, S1-S10 represent different S-RNases, namely S1-RNase~S 10 -RNase, from Figure 3 As seen in CgSAMS4 and nLUC, S1~S 10 No fluorescence was detected in the three negative control groups: the injection of S-RNase with cLUC, and the injection of nLUC with cLUC, indicating that CgSAMS4 can interact with most S-RNases within plant cells. S-RNase is central to citrus self-incompatibility, and its interaction with S-RNase suggests that this gene may be involved in the self-incompatibility reaction.

[0045] 2. Pull-down

[0046] The segment of the CgSAMS4 stop codon was removed. At the same time, the signal peptide of S-RNase was analyzed and its sequence was determined with the help of SignalP 5.0 signal peptide analysis platform. Afterwards, the CgSAMS4 segment and S-RNase segment after the above treatment were constructed into the pET-32a(+) and pMAL-c2X prokaryotic expression vectors. The fusion proteins His-CgSAMS4 and MBP-S-RNase were obtained by IPTG induction. In the pull-down experiment, Prey protein was the His-CgSAMS4 recombinant protein, Bait protein was the MBP tag protein and MBP-S-RNase fusion protein. The His antibody was used to detect the CgSAMS4 recombinant protein containing the His tag, and the MBP antibody was used to detect the protein containing the MBP tag. The test results are as follows. Figure 4 shown.

[0047] from Figure 4 As shown in the results, the protein encoded by CgSAMS4 interacts with S-RNase in vitro. Figure 3 , both of which together demonstrate that the protein encoded by CgSAMS4 interacts with S-RNase, thus indicating that S-adenosine monoamine synthetase (SAMS) is involved in the self-incompatibility reaction.

[0048] Example 6 Exogenous methionine treatment and observation

[0049] 1. Exogenous methionine spraying and pollination

[0050] Spray the stylar portion of the flowers with methionine at concentrations of 0.1mM, 0.5mM, 1mM, and 5mM. Spray with double-distilled water served as a control. Place flowers in a sulfuric acid paper bag and seal with a paperclip to prevent contamination by pollen. After 4 hours, unbag the flowers and perform cross-compatible and self-incompatible pollination, ensuring visible pollen abundance. After pollination, re-bag the flowers. Four days after spraying, remove all flowers from the bag and remove the stylars.

[0051] 2. Processing and preservation of style:

[0052] Four days after pollination, remove the pollination bag and collect the pollinated styles that have not fallen off in a ziplock bag. Immediately remove the petals and receptacle from the flower, retaining the ovary, style, and stigma. Place the styles in a fixative (prepared immediately for use: anhydrous ethanol: glacial acetic acid = 3:1). Place the styles in the fixative for 24 hours and then wash them 2-3 times with 90% ethanol solution to fix the styles.

[0053] 3. Preparation of aniline blue solution:

[0054] First, prepare a 0.1 mol / L K3PO4 solution, then add 0.1% aniline blue powder, shake thoroughly to mix, and transfer to a brown bottle for storage. After the aniline blue solution is prepared, wash the style sample stored in 70% ethanol with clean water once, then add 4 mol / L NaOH solution, seal, and place in a 65°C water bath for 60 minutes. After the water bath, the style color changes from yellow-white to orange-red. At this time, pour out the NaOH solution in the centrifuge tube and fill it with clean water. Soak for 30 minutes, then pour out and change the water. Repeat the water change step 3-4 times until the style color turns yellow. After the style is cleaned, add the aniline blue solution and soak and dye for 12 hours.

[0055] 4. Fluorescence microscopy observation

[0056] Before observation begins, place a glass slide on a flat surface and add 2-3 drops of 60% glycerol. Wash the style of the flower to be observed in clean water. Use a scalpel to split the style in half lengthwise. Place one half of the style on a glass slide and press it with a coverslip. Then, place the slide under a microscope and observe it at 5x magnification. Observe three styles of each variety. Pollen tubes that germinate to the lower part of the style are considered compatible; those that do not are considered incompatible.

[0057] Observation results such as Figure 7 As shown, in Figure 7 In the figure, AE represent the pollen tube growth after hybrid-compatible pollination treated with methionine; FJ represent the pollen tube growth after self-incompatibility pollination treated with methionine; A and F represent double-distilled water treatment; B and G represent 0.1 mM methionine treatment; C and H represent 0.5 mM methionine treatment; D and I represent 1 mM methionine treatment; E and J represent 5 mM methionine treatment; the scale bar in the figure is 1 mm.

[0058] from Figure 7 As can be seen in the hybrid affinity treatment group, the pollen tubes of the control group, 0.1mM, 0.5mM, and 1mM concentration treatment groups all grew normally in the style ( Figure 7 AD), while in the style treated with 5 mM concentration, although pollen germinated on the stigma, the pollen tube did not grow into the style ( Figure 7 E). This indicates that high concentrations of methionine affect pollen tube growth and can effectively inhibit pollen tube growth in hybrid compatibility pollination.

[0059] At the same time, it was observed that in the self-incompatibility treatment, no pollen tube extension was observed in the style of the control group, 0.1mM, 0.5mM, and 5mM concentration treatments, which is consistent with the performance of the self-incompatibility trait, but pollen tube extension was observed in the style of the 1mM concentration treatment ( Figure 7 I) It shows that in self-incompatibility, appropriate concentration of methionine can promote pollen tube growth.

[0060] In summary, it can be concluded that spraying the style with exogenous methionine at a concentration of 5 mM will inhibit the growth of pollen tubes, while 1 mM will promote the growth of pollen tubes. This reflects that methionine has a dual nature in the growth of pollen tubes: "low concentrations promote pollen tube growth, and high concentrations inhibit growth."

[0061] The examples of the present invention confirm that in the self-incompatibility reaction, S-RNase inhibits the activity of CgSAMS4, resulting in the inability to convert methionine into S-adenosylmethionine. Excessive methionine inhibits the activity and interferes with pollen tube development, thereby proving that CgSAMS4 is involved in the self-incompatibility reaction. Therefore, the expression level of CgSAMS4 can be changed by genetic means, or by adding citrus S-adenosylmethionine synthetase to the pollen culture, or by exogenous spraying of methionine to break the self-incompatibility reaction and promote pollen tube growth, thereby improving citrus self-incompatibility and reducing the cost of artificial pollination.

[0062] The present invention provides an application of citrus S-adenosylmethionine synthase (SAMS) and its encoding gene (CgSAMS4) in regulating plant self-incompatibility. By analyzing its function in the methionine metabolic pathway, it provides a new strategy for improving crop self-compatibility, which is expected to address problems such as low fruit set and high artificial pollination costs caused by self-incompatibility. The present invention reveals that CgSAMS4 regulates self-incompatibility through methionine metabolism, providing a new target for molecular breeding, reducing artificial pollination costs, and increasing citrus yields.

[0063] S1-RNase~S of the embodiment of the present invention 10 -RNase were obtained by prokaryotic expression and purification of the coding genes shown in SEQ ID: 5-14, S1-RNase ~ S 10 The protein sequences of -Rnase are shown in SEQ ID: 15-24.

[0064] The above embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of citrus S-adenosylmethionine synthetase in improving self-incompatibility, characterized in that: The gene sequence of the citrus S-adenosylmethionine synthetase is shown in SEQ ID:

1.

2. The use of citrus S-adenosylmethionine synthetase in improving self-incompatibility according to claim 1, characterized in that: The coding gene sequence of the citrus S-adenosylmethionine synthetase is shown in SEQ ID:

2.

3. The use of the citrus S-adenosylmethionine synthetase in improving self-incompatibility according to claim 1, wherein: Pollen tube growth was improved by adding citrus S-adenosylmethionine synthetase to the pollen culture medium.

4. The use of citrus S-adenosylmethionine synthetase in improving self-incompatibility according to claim 1, characterized in that: By spraying methionine onto the style, the expression inhibition of citrus S-adenosylmethionine synthase was improved, thereby improving the growth of pollen tubes.

5. The use of citrus S-adenosylmethionine synthetase in improving self-incompatibility according to claim 4, characterized in that: The spraying concentration of the methionine is 0.9-1.1 mM.

Images