Method for extracting myxococcus flavus dormant mucospore RNA (Ribonucleic Acid)

Abstract

The invention belongs to the field of microbiology and molecular biology, and particularly relates to a method for extracting myxococcus flavus dormant mucospore RNA (Ribonucleic Acid). The method specifically comprises the following steps: taking a certain amount of myxococcus flavus liquid, and inducing sporocarp development by using a common starvation induction method; scraping the formed sporocarp structure, resuspending with sterile water, and destroying the sporocarp structure and mucospore coating by using a certain proportion of organic reagent; centrifuging, taking a water phase, adding an acetate buffer solution with a certain concentration, and precipitating with isopropanol to obtain nucleic acid substances; and removing DNA by using a conventional kit to obtain RNA. Aiming at the characteristic that the mucospore RNA is difficult to extract, the invention develops a simple, convenient and efficient method for extracting the mucospore RNA, and provides an effective way for deeply researching the ecological characteristics of the myxobacteria.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology and molecular biology, and specifically relates to a method for extracting RNA from dormant myxospores of *Myxococcus faecalis*. Background Technology

[0002] Myxobacteria are a group of Gram-negative bacteria with complex multicellular behavior and large genomes. They are known for their unique multicellular gregarious behavior and abundant natural products, and are now classified separately as a phylum called Myxobacteria (Myxobacteria phylum). Myxococcota Unlike the predominantly single-celled lifestyle of bacteria, myxobacteria cells can collaboratively perform multicellular group functions, such as group movement and wolf-pack hunting. As Gram-negative bacteria, myxobacteria can transform into a resilient dormant somatic spore state under certain conditions.

[0003] When nutrients are limited, myxobacterial cells aggregate to form colonies, with intercellular specialization and cooperation to form visible multicellular fruiting bodies. A small portion of these cells differentiate into dormant myxospore bodies or peripheral rod-shaped cells, while most cells lyse during aggregation. Taking the myxobacterial model strain *Myxococcus faecalis* as an example, after starvation induction, the cells initially move and aggregate into mound-like structures, forming clearly defined fruiting bodies approximately 24 hours later, with spherical myxospores beginning to form on their surface.

[0004] The development of multicellular fruiting bodies is a crucial manifestation of the environmental adaptation evolution of myxobacteria. The fruiting body structure and the dormant myxospore body provide myxobacteria with strong resistance, helping strains withstand harsh environments. Under suitable nutritional conditions, myxospores can regerminate into vegetative cells, and the collective germination of spores within the fruiting body structure helps strains rapidly establish population density. Furthermore, the macroscopically visible fruiting body structure also promotes the spread of myxobacteria in the ecological environment. Therefore, the study of myxospores is of great significance for a deeper understanding of the environmental adaptation of myxobacteria.

[0005] Dormant spores possess resistance to acids, alkalis, and hydrolytic enzymes, making them difficult to break down for RNA extraction using conventional bacterial cell RNA extraction reagents. Common spore disruption methods, such as grinding, lead to RNA degradation, resulting in low-quality RNA samples and impacting the accuracy of subsequent assays. Unlike the spore walls of Gram-positive bacteria like Bacillus, which are primarily composed of peptidoglycan, the spore coat of *Myxococcus faecalis* is rich in N-acetylgalactosamine and Nfs protein scaffolds. Therefore, treatment methods for Gram-positive bacterial spores have limited effectiveness against myxococci, and conventional RNA extraction reagents such as Biozol are ineffective at extracting RNA. Due to the characteristics of myxococci, accurate analysis of their gene transcription levels faces challenges, necessitating the development of efficient methods for extracting RNA from myxococci. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention aims to provide a method for extracting RNA from dormant myxospores of *Myxococcus faecalis*.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for extracting RNA from dormant myxospores of *Myxococcus faecalis*, comprising the following steps: (1) Obtain a solution of the fruiting bodies of *Myxococcus faecalis*; (2) Add organic reagent to the fruiting body solution, mix by inverting, and let stand at room temperature. The organic reagent consists of phenol, chloroform and isoamyl alcohol. (3) Centrifuge, remove the supernatant, add sodium acetate buffer to the supernatant and mix well to obtain mixture A, incubate on ice for 5-15 min; (4) Add isopropanol to mixture A and incubate on ice for 5-15 minutes; (5) Centrifuge, discard the supernatant, and wash the nucleic acid precipitate with 75% ethanol; (6) Remove the genomic DNA from the nucleic acid precipitate to obtain the RNA of the dormant myxococcus faecalis.

[0008] In some embodiments of the present invention, step (1) of obtaining the fruiting body solution of *Myxococcus faecalis* includes: starvation-induced sporulation of *Myxococcus faecalis*, scraping off the fruiting bodies, washing with sterile water, and then resuspending the fruiting bodies in sterile water to obtain the solution.

[0009] In some embodiments of the present invention, the starvation-induced sporulation of *Myxococcus faecalis* specifically involves spreading *Myxococcus faecalis* onto a nutrient-free TPM plate to induce fruiting body development.

[0010] In some embodiments of the present invention, *Xanthomonas aeruginosa* with OD600≈1 is prepared to a concentration of 1×10⁻⁶. 9 -1×10 11 A bacterial culture of 100 cells / mL was spread in 200 μL onto a nutrient-free TPM plate to induce fruiting body development.

[0011] In some embodiments of the present invention, the starvation induction time is 0h-120h, preferably 0-72h, more preferably 6-48h, including 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, and 48h.

[0012] In some embodiments of the present invention, the fruiting body is washed with sterile water two or more times.

[0013] In some embodiments of the present invention, in step (2), the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1.

[0014] Adding the above-mentioned specific proportions of phenol, chloroform, and isoamyl alcohol can disrupt the structure of the myxospore coat and release the nucleic acid substances within it.

[0015] In some embodiments of the present invention, in step (2), the volume ratio of the organic solvent to the fruiting body solution is 1:1.

[0016] In some embodiments of the present invention, in step (3), the centrifugation speed is 12,000-14,000 rpm, the centrifugation temperature is 4°C, and the centrifugation time is 4-6 min.

[0017] In some embodiments of the present invention, in step (3), the ice bath time is 5-15 min, preferably 7-12 min, more preferably 9-11 min, including 9 min, 10 min and 11 min within the range.

[0018] In some embodiments of the present invention, in step (3), the volume ratio of the supernatant to the sodium acetate buffer is 1:1.

[0019] In some embodiments of the present invention, the supernatant is the upper aqueous phase.

[0020] After high-speed centrifugation, the solution separates into an upper aqueous phase, an intermediate protein layer, and a lower organic phase. The released nucleic acid material is in the upper aqueous phase. Sodium acetate buffer is added to the upper aqueous phase to promote nucleic acid precipitation.

[0021] In some embodiments of the present invention, in step (3), the concentration of sodium acetate buffer is 0.2-0.6M, including the range of 0.2M, 0.3M, 0.4M, 0.5M, and 0.6M.

[0022] In some embodiments of the present invention, in step (3), the pH of the sodium acetate buffer solution is 4.5-5.5, preferably 4.5-5.2, including the range of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 and 5.2.

[0023] In some embodiments of the present invention, in step (4), the volume ratio of mixture A to isopropanol is 1:1.

[0024] In some embodiments of the present invention, in step (4), the ice bath time is 5-15 min, preferably 7-12 min, more preferably 9-11 min, including 9 min, 10 min and 11 min within the range.

[0025] In some embodiments of the present invention, in step (5), the centrifugation speed is 12,000-14,000 rpm, the centrifugation temperature is 4°C, and the centrifugation time is 4-6 min.

[0026] In some embodiments of the present invention, in step (5), the nucleic acid precipitate is washed with 75% ethanol, centrifuged again for 4-6 min, and the supernatant is completely discarded.

[0027] The process involves high-speed centrifugation to obtain a nucleic acid precipitate. After washing the precipitate, it is dissolved in RNase-free water. The nucleic acid extraction efficiency can be detected by agarose gel electrophoresis. At this point, a mixture of DNA and RNA has been extracted.

[0028] In some embodiments of the present invention, in step (6), the removal of genomic DNA from the nucleic acid precipitate can be done using a conventional kit, and no specific limitation is made here.

[0029] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) This invention uses a specific organic reagent to treat the myxospore coat, thereby destroying the scaffold proteins within it. During the experiment, a large amount of white protein precipitate appeared rapidly after the reagent was added, indicating that the reagent was effective.

[0030] (2) In this invention, sodium acetate buffer is added. This buffer is acidic, which helps the subsequent nucleic acid precipitation effect.

[0031] (3) The nucleic acid extracted by this invention was observed to have three clear ribosomal RNA bands and the uppermost genomic band after agarose gel electrophoresis, indicating that the RNA extraction effect was significant. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Appendix Figure 1 The images show the results of myxospore nucleic acid extracted using the method of the present invention in Examples 1-8 of this invention. The four bands shown in the images, from top to bottom, are the genomic DNA band, the 23S rRNA band, the 16S rRNA band, and the 5S rRNA band.

[0034] Appendix Figure 2The images show the electrophoresis results of nucleic acids extracted in Examples 1-8 of this invention after the removal of genomic DNA. The genomic DNA band has been completely removed, and the remaining rRNA band represents the integrity of the extracted mRNA sample.

[0035] Appendix Figure 3 The figures show the results of extracting myxospore RNA using Biozol, a conventional bacterial cell RNA extraction reagent, in Comparative Examples 1-8 of this invention. As a control group, myxospores were formed after 24 hours of starvation induction and gradually matured. As can be seen in the figure, after starvation induction, it was difficult to extract high-quality RNA samples with Biozol reagent, and almost no RNA samples could be extracted after 24 hours of spore formation. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0039] Biological material: *M. xanthus* DK 1622 was obtained from D. Kaiser, Stanford University. Its genome sequence is: a single circular chromosome of 9,139,763 bp (GenBank accession number CP0001 13); genome annotation information is available in: TIGRAnnotation genelist.

[0040] Example 1 (1) When the yellow myxocytocium cells with OD600≈1 are concentrated 35 times, the cell concentration is approximately 1×10⁻⁶. 10 per mL.

[0041] (2) Take 200 μL of *Myxococcus faecalis* cells from step (1) and add an equal volume of phenol:chloroform:isoamyl alcohol = 25:24:1. Mix thoroughly by inverting the container. The purpose of this step is to disrupt the myxospore coat barrier and intracellular proteins, releasing the nucleic acid substances within.

[0042] (3) Centrifuge at 13,000 rpm and 4 ℃ for 5 min. Take the upper aqueous phase (about 200 μL) and add an equal volume of pre-cooled 0.6 M sodium acetate buffer (pH 5.2) to promote nucleic acid precipitation.

[0043] (4) Add the above solution to pre-cooled isopropanol at a volume ratio of 1:1, mix well, and place on ice for 10 min to allow nucleic acid precipitation.

[0044] (5) Centrifuge at 13,000 rpm and 4 ℃ for 5 min, discard the supernatant. At this time, the nucleic acid precipitate will be at the bottom of the centrifuge tube. Wash the precipitate with 75% ethanol, centrifuge again for 5 min, and discard the supernatant completely.

[0045] (6) Dissolve the precipitate at the bottom of the centrifuge tube in 100 μL of RNase-free water to obtain a nucleic acid solution, and store 80 μL at -80℃ for later use.

[0046] (7) Take 2 μL of nucleic acid solution from step (6) and use the genomic removal kit (HiScript® III RT SuperMix for qPCR (+gDNA wiper), Cat.: R323, Novizan) to remove genomic DNA from the sample to obtain 10 μL of RNA sample.

[0047] Example 2 (1) Take 200 μL of the solution, which has a concentration of approximately 1×10⁻⁶. 10 Yellow myxocytocium cells per mL were spread onto nutrient-free TPM plates (10 mM Tris-HCl, 8 mM MgSO4, 1 mM K2HPO4-KH2PO4, 1.5% Agar; pH 7.6) and starved at 30°C for 6 h to induce fruiting body development. The fruiting bodies were scraped off and washed twice with sterile water.

[0048] (2) Resuspend the fruiting body in 200 μL of sterile water, add an equal volume of phenol:chloroform:isoamyl alcohol = 25:24:1 reagent, and mix by inverting the container. The purpose of this step is to disrupt the myxospore coat barrier and intracellular proteins, releasing the nucleic acid substances within.

[0049] (3) Centrifuge at 13,000 rpm and 4 ℃ for 5 min. Take the upper aqueous phase (about 200 μL) and add an equal volume of pre-cooled 0.6 M sodium acetate buffer (pH 5.2) to promote nucleic acid precipitation.

[0050] (4) Add the above solution to pre-cooled isopropanol at a volume ratio of 1:1, mix well, and place on ice for 10 min to allow nucleic acid precipitation.

[0051] (5) Centrifuge at 13,000 rpm and 4 ℃ for 5 min, discard the supernatant. At this time, the nucleic acid precipitate will be at the bottom of the centrifuge tube. Wash the precipitate with 75% ethanol, centrifuge again for 5 min, and discard the supernatant completely.

[0052] (6) Dissolve the precipitate at the bottom of the centrifuge tube in 50 μL of RNase-free water to obtain a nucleic acid solution, and store 30 μL at -80℃ for later use.

[0053] (7) Take 2 μL of nucleic acid solution from step (6) and use the genomic removal kit (HiScript® III RT SuperMix for qPCR (+gDNA wiper), Cat.: R323, Novizan) to remove genomic DNA from the sample to obtain 10 μL of RNA sample, and store at -80℃ for later use.

[0054] Example 3 The operation steps are the same as in Example 2, except that the starvation induction time for *Myxococcus faecalis* is 12 hours.

[0055] Example 4 The operation steps are the same as in Example 2, except that the starvation induction time for *Myxococcus faecalis* is 24 hours.

[0056] Example 5 The operation steps are the same as in Example 2, except that the starvation induction time for *Myxococcus faecalis* is 36 hours.

[0057] Example 6 The operation steps are the same as in Example 2, except that the starvation induction time for *Myxococcus faecalis* is 48 hours.

[0058] Example 7 The operation steps are the same as in Example 2, except that the starvation induction time for *Myxococcus faecalis* is 72 hours.

[0059] Example 8 The operation steps are the same as in Example 2, except that the starvation induction time for *Myxococcus faecalis* is 120 h.

[0060] Comparative Example 1 A concentration of approximately 1×10⁻⁶ μL is taken as 200 μL. 10 Myxococcus faecalis cells per mL were used to extract myxospore RNA using Biozol (Cat.: BSC51M1), a standard bacterial cell RNA extraction reagent. The experimental method was in accordance with the reagent instructions. The extracted RNA was dissolved in 100 μL of RNase-free water and stored at -80℃ for later use.

[0061] Comparative Example 2 The extraction method was the same as in Comparative Example 1, except that the yellow myxococcus was starved for 6 hours. The starvation induction conditions were the same as in step (1) of Example 2. The extracted RNA was dissolved in 50 μL of RNase-free water and stored at -80℃ for later use.

[0062] Comparative Example 3 The extraction method was the same as in Comparative Example 2, except that *Myxococcus faecalis* was starved for 12 hours.

[0063] Comparative Example 4 The extraction method was the same as in Comparative Example 2, except that *Myxococcus faecalis* was starved for 24 hours.

[0064] Comparative Example 5 The extraction method was the same as in Comparative Example 2, except that *Myxococcus faecalis* was starved for 36 hours.

[0065] Comparative Example 6 The extraction method was the same as in Comparative Example 2, except that *Myxococcus faecalis* was starved for 48 hours.

[0066] Comparative Example 7 The extraction method was the same as in Comparative Example 2, except that *Myxococcus faecalis* was starved for 72 hours.

[0067] Comparative Example 8 The extraction method was the same as in Comparative Example 2, except that *Myxococcus faecalis* was starved for 120 h.

[0068] Take 5 μL of the nucleic acid solution extracted in Examples 1-8 and perform agarose gel electrophoresis. The results are as follows: Figure 1 As shown in the figure, the four bands displayed in the sample, from top to bottom, are the genomic DNA band, the 23S rRNA band, the 16S rRNA band, and the 5S rRNA band.

[0069] 10 μL of RNA extracted from Examples 1-8 and 5 μL of RNA extracted from Comparative Examples 1-8 were respectively subjected to agarose gel electrophoresis. The results are as follows: Figure 2 and Figure 3 As shown, Figure 2 The genomic DNA band was completely removed, leaving only the rRNA band, and the extracted mRNA sample showed high integrity. However, the results of extracting myxospore RNA using the conventional bacterial cell RNA extraction reagent Biozol show that after starvation induction, Biozol reagent struggled to extract high-quality RNA samples, and almost no RNA was extracted after 24 hours of spore formation. Figure 3 ).

[0070] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting RNA from dormant myxospores of *Myxococcus faecalis*, characterized in that, Includes the following steps: (1) Obtain a solution of the fruiting bodies of *Myxococcus faecalis*; (2) Add organic reagent to the fruiting body solution, mix by inverting, and let stand at room temperature. The organic reagent consists of phenol, chloroform and isoamyl alcohol. (3) Centrifuge, remove the supernatant, add sodium acetate buffer to the supernatant and mix well to obtain mixture A, incubate on ice for 5-15 min; (4) Add isopropanol to mixture A and incubate on ice for 5-15 minutes; (5) Centrifuge, discard the supernatant, and wash the nucleic acid precipitate with 75% ethanol; (6) Remove the genomic DNA from the nucleic acid precipitate to obtain the RNA of the dormant myxococcus faecalis.

2. The method as described in claim 1, characterized in that, In step (1), the process of obtaining the fruiting body solution of *Myxococcus faecalis* includes: starvation-induced spore formation of *Myxococcus faecalis*, scraping off the fruiting bodies, washing with sterile water, and then resuspending the fruiting bodies in sterile water to obtain the solution.

3. The method as described in claim 2, characterized in that, The starvation-induced sporulation of *Myxococcus faecalis* specifically involves spreading *Myxococcus faecalis* onto a nutrient-free TPM plate to induce fruiting body development.

4. The method as described in claim 1, characterized in that, In step (2), the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:

1.

5. The method as described in claim 1, characterized in that, In step (2), the volume ratio of the organic solvent to the fruiting body solution is 1:

1.

6. The method as described in claim 1, characterized in that, In step (3), the volume ratio of the supernatant to the sodium acetate buffer is 1:

1.

7. The method as described in claim 1, characterized in that, In step (3), the concentration of sodium acetate buffer is 0.2-0.6M.

8. The method as described in claim 1, characterized in that, In step (3), the pH of the sodium acetate buffer solution is 4.5-5.

5.

9. The method as described in claim 1, characterized in that, In step (4), the volume ratio of mixture A to isopropanol is 1:

1.

10. The method as described in claim 1, characterized in that, In step (5), the centrifugation time is 4-6 min.

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