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

Abstract

This invention provides the application of citrus S-adenosylmethionine synthase in improving self-incompatibility, belonging to the field of biogenetic engineering technology. The gene sequence of citrus S-adenosylmethionine synthase is shown in SEQ ID:1. This invention experimentally demonstrates that citrus S-adenosylmethionine synthase affects pollen tube growth by directly interacting with S-RNase and regulating the methionine metabolic pathway. This invention reveals the dual regulatory role of citrus S-adenosylmethionine synthase in self-incompatibility responses, providing a new target for improving crop self-compatibility. This invention improves pollen tube growth by adding citrus S-adenosylmethionine synthase to pollen culture or spraying methionine onto the style, thereby improving citrus self-incompatibility. This provides a new strategy for improving crop self-compatibility and is expected to solve problems such as low fruit set rate and high artificial pollination costs caused by self-incompatibility.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and more specifically, relates to the application of citrus S-adenosylmethionine synthase in improving self-incompatibility. Background Technology

[0002] Citrus fruits are the world's most popular fruit, and my country is the world's largest producer and consumer of citrus fruits. However, some grapefruit and lemon varieties have low natural fruit set rates due to their self-incompatibility and weak parthenocarpy. In production, artificial pollination is often required to increase yield, which increases production costs and seriously affects commercial value.

[0003] Self-incompatibility (SI) is a crucial mechanism for maintaining genetic diversity in flowering plants, achieving reproductive isolation through pollen-stigma recognition. Citrus, a typical gametophytic self-incompatibility (GSI) species, relies on S-RNase-mediated pollen tube RNA degradation for its SI response. However, current research primarily focuses on the recognition and degradation processes of S-RNases, with insufficient elucidation of downstream signaling pathways. There is an urgent need to elucidate the regulatory mechanisms of non-S factors in downstream pathways of citrus SI, thereby overcoming the current reliance on single-gene regulation by S-RNases.

[0004] S-Adenosylmethionine synthase (SAMS), as the rate-limiting enzyme in methionine metabolism, catalyzes the production of S-adenosylmethionine (SAM), a precursor in methylation reactions and the synthesis of polyamines and ethylene, playing a central role in plant growth, development, and stress responses. Although studies have reported SAMS's involvement in plant stress resistance and floral organ development regulation, its function in self-incompatibility responses has not yet been reported. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides the application of citrus S-adenosylmethionine synthase in improving self-incompatibility, the gene sequence of which is shown in SEQ ID:1.

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

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

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

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

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

[0012] This invention experimentally demonstrates that citrus S-adenosylmethionine synthase (CgSAMS4) affects pollen tube growth by directly interacting with S-RNase and regulating the methionine metabolic pathway. This invention 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 the pollen culture medium or spraying methionine onto the style, this invention improves pollen tube growth, thereby improving citrus self-compatibility. This provides a new strategy for improving crop self-compatibility and is expected to solve problems such as low fruit set rate and high artificial pollination costs caused by self-incompatibility. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a diagram illustrating the effect of antisense oligonucleotide inhibition of CgSAMS4 expression on pollen tube growth, as provided in Example 2 of this invention.

[0015] Figure 2 This is a statistical diagram of pollen tube growth length after antisense oligonucleotides inhibit CgSAMS4 expression, provided in Example 2 of the present invention.

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

[0017] Figure 4 This is a pull-down interaction analysis diagram of CgSAMS4 and S-RNase provided in Embodiment 5 of the present invention;

[0018] Figure 5This is a graph showing the effect 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 the style of flowers during different pollination processes, as provided in Example 4 of the present invention.

[0020] Figure 7 This is an observation image of aniline blue staining on the style of a flower treated with exogenous methionine, provided in Example 4 of the present invention.

[0021] Figure 8 This is a diagram illustrating the effect of different protein treatments on incompatible pollen tube growth, as provided in Example 3 of the present invention.

[0022] Figure 9 This is a statistical chart of pollen tube growth length after different protein treatments provided in Example 3 of the present invention. Detailed Implementation

[0023] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0024] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0025] Example 1: Antisense Oligonucleotide Primer Design

[0026] In this invention, antisense oligonucleotide primers were designed for citrus S-adenosylmethionine synthase to suppress the expression of the gene encoding citrus S-adenosylmethionine synthase by silencing the antisense oligonucleotides, thereby inhibiting pollen tube growth.

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

[0028] This invention utilizes the mfold website (http: / / www.unafold.org / mfold / applications / rna-folding-form.php) to predict the secondary structure of the target gene CgSAMS4 mRNA. The configuration with the lowest free energy is selected as a reference, and the vesicular region sequence is used as the target. Based on the base pairing principle, antisense oligonucleotides with a length of 20 bp that are perfectly complementary to the target sequence are designed. Simultaneously, the Sfold website (http: / / sfold.wadsworth.org / cgibin / soligo.pl) is used to screen for usable antisense oligonucleotides. Thioylation modifications are added to the two ends of candidate AS-ODN and S-ODN four bases before synthesis. Primer pair information is shown in Table 1, including primers with gene sequences as shown in SEQ ID:3 and SEQ ID:4. Both primers are dissolved in pollen medium to prepare 100 μM stock solutions, and pollen tubes are cultured at a pollen medium:stock solution ratio of 3:1.

[0029] Table 1 Information on antisense oligonucleotide primer pairs

[0030]

[0031] Example 2 In vitro pollen culture

[0032] Using crystal pomelo pollen (S genotype S5S6) as experimental material, a 1.5 mL centrifuge tube cap was used as a culture vessel for pollen tubes. The pollen tubes were cultured using 100 μL of the pollen culture medium from Example 1 (stock solution = 3:1). The pollen culture medium consisted of 10% sucrose, 13.3% PEG-4000, 0.02% MgSO4·7H2O, 0.01% KNO3, 0.03% Ca(NO3)2·4H2O, and 0.01% H3BO3. 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. The centrifuge tube cap was then placed inside the box, and the box was closed. The box was placed in a dark incubator at 28°C for 9 hours. The pollen tubes were then transferred to a glass slide using a 1 mL pipette tip with the tip cut off. A coverslip was placed on top, and the tubes were observed and photographed using an inverted microscope. The results are as follows: Figure 1 As shown; using a digital display (GM185, Gaoman), the pollen tube length was measured and the pollen germination rate was statistically analyzed using ImageJ software. The results are as follows. Figure 2 As shown.

[0033] Figure 1 , Figure 2In the table, CK represents the control group, which is not treated in any way, i.e., no stock solution is added to the liquid culture medium; S-CgSAMS4 represents the liquid culture medium with stock solution containing primers of SEQ ID:3; AS-CgSAMS4 represents the liquid culture medium with stock solution containing primers of SEQ ID:4. Figure 2 Yes Figure 1 Quantization processing, from Figure 1 , Figure 2 The study observed that the average pollen tube length in the control group was 579.468±14.3 μm, in the S-CgSAMS4 treatment group it was 533.749±24.14 μm, and in the AS-CgSAMS4 treatment group it was 372.749±7.29 μm. This indicates that the inhibition of CgSAMS4 expression by antisense oligonucleotides affects pollen tube growth, thus hindering pollen tube growth and demonstrating the importance of citrus S-adenosylmethionine synthase in citrus reproduction.

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

[0035] Using crystal pomelo pollen (S genotype S5S6) as experimental material, purified S1-RNase and S2-RNase proteins were added to the same pollen culture medium, as were S5-RNase and S6-RNase proteins. The S1-RNase + S2-RNase group was the compatibility treatment group, and the S5-RNase + S6-RNase group was the incompatibility treatment group. A control group (without S-RNase) and an incompatibility treatment group (with recombinant CgSAMS4 protein added) were also included. The final concentration of each protein was 100 ng / μL. The mixture was sprinkled with crystal pomelo pollen and incubated in a humid, dark environment at 28°C for 12 hours.

[0036] Observe and photograph using an inverted microscope; the results are as follows: Figure 8 As shown, Figure 8 The scale bar length was 200 μm; a digital display (GM185, Gaoman) was used, and ImageJ software was employed to measure pollen tube length and statistically analyze pollen germination rate. The results are as follows: Figure 9 As shown.

[0037] from Figure 8 , Figure 9The study observed that under normal conditions, the average pollen tube length of the 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 decreased to 563.3 ± 25.19 μm, showing no significant difference compared to the control group. However, when S5-RNase and S6-RNase proteins were added, the average length decreased to 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, still shorter than the control and compatibility treatment groups but significantly longer than the incompatibility treatment group. This indicates that CgSAMS4 protein has a certain promoting effect on the growth of incompatible pollen tubes; that is, by adding citrus S-adenosylmethionine synthase CgSAMS4 to the pollen culture medium, pollen tube growth can be improved, thereby improving self-incompatibility.

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

[0039] Proteins were extracted from the styles of unpollinated, hybrid-compatible, and self-incompatible pollinated plants 4 days later. The S-adenosylmethionine synthase (SAMS) ELISA kit and the plant methionine ELISA kit from Shanghai Yuanmu Biotechnology Co., Ltd. were used to determine the S-adenosylmethionine synthase activity and methionine content according to their product instructions. The results are as follows: Figure 5 As shown.

[0040] Figure 5 In this context, "control" represents unpollinated, "cross-pollination" represents hybridization-compatible pollination, and "self-pollination" represents self-incompatible pollination. From... Figure 5 The study observed that the activity of SAMS in the styles after hybrid compatibility pollination was significantly higher than that in the styles of self-incompatible pollination and unpollinated (Control) styles. Furthermore, the SAMS activity in the styles of self-incompatible pollination was significantly higher than that in the unpollinated styles. These results indicate that SAMS activity is significantly higher in hybrid compatibility pollination than in self-incompatible pollination. Since S-RNase exerts toxicity in the self-incompatibility reaction, it can be roughly deduced that S-RNase can inhibit SAMS activity. That is, in citrus self-incompatibility, the activity of citrus S-adenosylmethionine synthase (SAMS) is inhibited. SAMS is the rate-limiting enzyme affecting the synthesis of S-adenosylmethionine (S-adenosylmethionine) from methionine (also called methionine), and this synthetic pathway is the only one in the plant. Therefore, the inhibition of SAMS activity reduces the efficiency of this synthetic pathway, leading to the accumulation of methionine. Figure 6 The methionine concentration in the styles of self-incompatible pollinated flowers was significantly higher than that in unpollinated and hybrid-compatible pollinated flowers, while there was no significant difference in methionine concentration between hybrid-compatible and unpollinated flowers. This suggests that self-incompatible pollination may specifically induce abnormal accumulation of methionine in the styles, i.e., the methionine content is higher in self-incompatible pollinated flowers.

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

[0042] 1. Analysis of the interaction between tobacco luciferase and protein

[0043] The full-length CDS of S-RNase without the stop codon was ligated into the JW771 vector; the full-length CDS of CgSAMS4 without the stop codon was ligated into the JW772 vector. These constructed gene vector combinations were then transformed into Agrobacterium strain GV3101. Agrobacterium strain GV3101, along with Agrobacterium containing the helper plasmid pSOUP-p19, was simultaneously cultured at 28°C and 220 rpm for 16–20 h. The bacterial cells were collected and resuspended in an infection buffer (10 mM MES, 0.1 mM AS, 10 mM MgCl2) to an OD600 of 0.6–0.7. The bacterial solution was then mixed at a ratio of JW771:JW772:pSOUP-p19 of 1:1:2, inverted, and incubated at 28°C for 2 h. The mixed bacterial solution was then injected into the underside of tobacco leaves that were 4–5 weeks old using a 1 mL syringe. Three days later, the leaves were cut off, and 200 μL of 0.4 mg / mL D-fluorescein sodium salt was applied to the underside of the leaves. The leaves were then placed in a NightShade LB 985 in vivo imaging system and treated in darkness for 5 minutes. Photos were taken and observed. The results are as follows. Figure 3 As shown.

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

[0045] 2. Pull-down

[0046] The section removing the stop codon of CgSAMS4 was obtained. Meanwhile, the signal peptide of S-RNase was analyzed using the SignalP 5.0 signal peptide analysis platform to determine its sequence. Subsequently, the processed CgSAMS4 section and S-RNase section were constructed into the prokaryotic expression vectors pET-32a(+) and pMAL-c2X. The fusion proteins His-CgSAMS4 and MBP-S-RNase were obtained by IPTG induction. In the Pull-down experiment, the Prey protein was the His-CgSAMS4 recombinant protein, and the Bait proteins were the MBP-tagged protein and the MBP-S-RNase fusion protein. The His-CgSAMS4 recombinant protein containing the His tag was detected using the His antibody, and the protein containing the MBP tag was detected using the MBP antibody. The detection results are as Figure 4 shown.

[0047] As seen Figure 4 from it, in vitro of plants, the protein encoded by CgSAMS4 interacts with S-RNase. Combining Figure 3 , the two jointly prove that the protein encoded by CgSAMS4 interacts with S-RNase. Therefore, it can be shown that S-adenosylmethionine synthetase (SAMS) is involved in the self-incompatibility reaction.

[0048] Example 6 Exogenous Methionine Treatment and Observation

[0049] 1. Spraying of exogenous methionine and pollination

[0050] The style parts were sprayed with methionine at concentrations of 0.1 mM, 0.5 mM, 1 mM, and 5 mM, and double-distilled water was sprayed as a control. Then, a sulfuric acid paper bag was put on and sealed with a paper clip to prevent contamination by other foreign pollens. After 4 h, the bag was untied, and cross-compatible and self-incompatible pollinations were carried out, ensuring that the pollen amount was visible to the naked eye. After pollination, the bag was resealed. Four days after spraying, all the flowers were unbagged, and the styles were taken.

[0051] 2. Treatment and preservation of styles:

[0052] Four days after pollination of citrus varieties, the pollination bags were removed, and the unpollinated styles that had not fallen were collected into a self-sealing bag. Immediately after collection, the petals and receptacles on the flowers were removed, and the ovary style and stigma were retained and placed in a fixing solution (prepared freshly, absolute ethanol: glacial acetic acid = 3:1). The styles were placed in the fixing solution for 24 h, and the fixed styles were washed 2 - 3 times with a 90% ethanol solution.

[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 column samples stored in 70% ethanol once with water, then add 4 mol / L NaOH solution, seal, and place in a 65℃ water bath for 60 minutes. After the water bath, the column color changes from yellowish-white to orange-red. At this point, pour out the NaOH solution from the centrifuge tube, pour in water, soak for 30 minutes, then pour out the water and change the water. Repeat the water-changing step 3-4 times until the column color turns yellow. After the column is cleaned, soak it in aniline blue solution for 12 hours for staining.

[0055] 4. Observation under a fluorescence microscope

[0056] Before observation, place a glass slide on a flat surface and add 2-3 drops of 60% glycerin. Then, wash the style to be observed in clean water. After washing, use a scalpel to cut the style in half lengthwise. Place one half of the style on the glass slide and press it down with a coverslip. Place the pressed slide under a microscope and observe using a 5x microscope. Observe three styles for each variety. If the pollen tubes germinate to the lower part of the style, the variety is considered compatible; if they do not reach the lower part of the style, the variety is considered incompatible.

[0057] Observation results as follows Figure 7 As shown, in Figure 7 In the figure, AE represents pollen tube growth after methionine-treated hybridization and compatibility pollination; FJ represents pollen tube growth after methionine-treated self-incompatible pollination; 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 long.

[0058] from Figure 7 It was observed that in the hybridization compatibility treatment group, the pollen tubes of the control group, and the 0.1mM, 0.5mM, and 1mM concentration treatment groups all grew normally in the style. Figure 7 AD), while in the styles 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 affinity pollination.

[0059] Meanwhile, it was observed that in the self-incompatibility treatment, no pollen tube extension was observed in the styles of the control group, and the 0.1mM, 0.5mM, and 5mM concentration treatments, consistent with the self-incompatibility trait. However, pollen tube extension was observed in the styles of the 1mM concentration treatment. Figure 7 I). This indicates that, in self-incompatibility, an appropriate concentration of methionine promotes 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 pollen tube growth, while 1 mM will promote pollen tube growth. This demonstrates that methionine has a dual effect on pollen tube growth: "promoting pollen tube growth at low concentrations and inhibiting growth at high concentrations".

[0061] This invention demonstrates that in self-incompatibility, S-RNase inhibits CgSAMS4 activity, preventing methionine from being converted to S-adenosylmethionine. Excessive methionine inhibits pollen tube development, thus proving that CgSAMS4 participates in self-incompatibility. Therefore, the expression level of CgSAMS4 can be altered through transgenic methods, or by adding citrus S-adenosylmethionine synthase to pollen culture, or by exogenously spraying 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] This invention provides an application of citrus S-adenosylmethionine synthase (SAMS) and its encoding gene (CgSAMS4) in regulating plant self-incompatibility. By elucidating its function in the methionine metabolic pathway, this invention offers a new strategy for improving crop self-compatibility, potentially solving problems such as low fruit set and high artificial pollination costs caused by self-incompatibility. This invention reveals that CgSAMS4 regulates self-incompatibility through methionine metabolism, providing a new target for molecular breeding, reducing artificial pollination costs, and increasing citrus yield.

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

[0064] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. The application of citrus S-adenosylmethionine synthase in improving citrus self-incompatibility, characterized in that, The amino acid sequence of the citrus S-adenosylmethionine synthase is shown in SEQ ID:

1.

2. The application of the citrus S-adenosylmethionine synthase as described in claim 1 in improving citrus self-incompatibility, characterized in that, The gene sequence encoding the citrus S-adenosylmethionine synthase is shown in SEQ ID:

2.

3. The application of the citrus S-adenosylmethionine synthase as described in claim 1 in improving citrus self-incompatibility, characterized in that, Pollen tube growth was improved by adding citrus S-adenosylmethionine synthase to the pollen culture medium.

4. The application of methionine in improving self-incompatibility in citrus, characterized in that, Pollen tube growth is improved by spraying methionine onto the style at a concentration of 0.9-1.1 mM.

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