CN115873843A - Method for extracting total RNA of bacteria-algae symbiotic spheres - Google Patents

Abstract

A method for extracting the total RNA of the symbiotic spheres of bacteria and algae, comprising the following steps: placing the symbiotic spheres of bacteria and algae in a sterile pre-cooled mortar, adding quartz sand and liquid nitrogen 1 to 3 times the mass of the symbiotic spheres of bacteria and algae (using After the bacteria and algae symbiosis balls are all submerged in liquid nitrogen), they are quickly and fully ground to obtain a cell homogenate, and the cell homogenate supernatant is collected after centrifugation for subsequent total RNA extraction; the bacteria and algae symbiosis balls are mold and microalgae symbiosis Cultivate to form. The invention replaces simple liquid nitrogen grinding or quartz sand grinding by adding quartz sand combined with liquid nitrogen to quickly freeze and grind the symbiotic balls of bacteria and algae, so that the demand for symbiotic balls of bacteria and algae during RNA extraction is greatly reduced, and the grinding consumption is significantly shortened. hour. This method is also conducive to grinding the symbiotic balls of bacteria and algae into powder, improving the full contact between the cell homogenate and the RNA isolater extractant after the symbiotic balls of bacteria and algae are ground, thereby reducing the residual amount of polysaccharides and proteins in the outer cell membrane in the total RNA extract, Improve the extraction concentration and quality of total RNA of bacteria and algae symbiosis balls.

Description

A kind of method for extracting the total RNA of bacteria and algae symbiosis

technical field

The invention relates to the field of bioengineering, in particular to a method for extracting total RNA of bacteria and algae symbiosis balls.

Background technique

Microalgae are the most widely distributed microorganisms in nature. They can grow under stressful environmental conditions, such as heavy metal pollution, high salinity, nutrient stress, and extreme temperature. The cell walls of microalgae are rich in extracellular polysaccharides. Polymers (EPS), proteins and nucleic acids, can bind charged metal ions, heavy metals and organic pollutants, and microalgae have good biosorption potential.

There are many methods for harvesting algae from suspension, such as centrifugation, gravity sedimentation, natural pressure filtration, chemical flocculation, electrocoagulation, and vacuum filtration. However, these methods have low efficiency and high energy consumption, are not suitable for large-scale industrial recovery of microalgae, and the small size of microalgae cells (diameter <30um) and adverse characteristics such as electrostatic repulsion between cells significantly limit the large-scale commercialization of microalgae. economic viability and sustainability of chemical production.

Fungi can form spheres to adsorb microalgae in the culture medium to form fungal-microalgae symbiotic hyphae balls, and their hyphae can be used as carrier materials for immobilized microalgae, which is conducive to separating the balls from the medium by filtration and reducing The operating cost of the microalgae harvesting process is reduced, and the mutual beneficial symbiosis between filamentous fungi and microalgae can also increase the biomass of microalgae and reduce the cost of cultivation, and form a fungal-microalgae symbiotic system with microalgae. Therefore, the co-cultivation of algae and filamentous fungi has become a new method to harvest microalgae efficiently.

At present, fungi-microalgae symbiotic mycelium has significant advantages in microalgae harvesting and heavy metal treatment, but there is no detailed report on the analysis of the molecular mechanism at the transcription level. One of the problems is: the layers of filamentous fungi Thickening, the common liquid nitrogen grinding cannot quickly and fully disperse the fungi-microalgae symbiotic mycelium into powder, and the traditional SDS method and CTAB method cannot extract a large amount of RNA, which is easily affected by residual polysaccharides and glycoproteins The purity of total RNA after extraction leads to low extraction efficiency and quality of total RNA from symbiotic mycelia, and directly affects the accuracy of transcriptome sequencing results. However, there is no widely recognized and effective method for the extraction of total RNA from symbiotic mycelium.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a simple, safe, effective, fast and less polluting method for extracting the total RNA of the symbiotic spheres of bacteria and algae.

One of the technical solutions adopted by the present invention to solve its technical problems is:

A method for extracting the total RNA of the symbiotic sphere of bacteria and algae, comprising the following steps:

1) Cell homogenization treatment: place the bacteria and algae symbiosis balls in a sterile pre-cooled mortar, add quartz sand and liquid nitrogen (so that the bacteria and algae symbiosis balls are all submerged in liquid nitrogen) middle), quickly and fully grind to obtain a cell homogenate, collect the supernatant of the cell homogenate after centrifugation, and set aside; the symbiosis ball of bacteria and algae is formed by the symbiotic culture of mold and microalgae;

2) Transfer the cell homogenate supernatant obtained in step 1) into a centrifuge tube, add 1mL RNA isolater for every 5-10× ¹⁰⁶ cells, and let it stand for 5-10 minutes; after the sample is completely melted, continue to pipette until it is lysed liquid transparent;

3) Transfer the lysate to a centrifuge tube, centrifuge at 12000×g, 4°C for 5 minutes, and absorb the supernatant of the lysate; since the bacteria and algae symbiosis balls are ground by quartz sand and liquid nitrogen, the homogenate sample contains more protein and polysaccharides centrifuge to remove the precipitate composed of impurities including fungal cell outer membrane polysaccharides and high-molecular-weight DNA, and collect the supernatant of the lysate to initially enrich the total RNA of the symbiosis sphere.

4) Add 500-700 μl of chloroform/isoamyl alcohol to the supernatant of the lysate in step 3), vortex and mix, and let stand at 4°C for more than 10 minutes; centrifuge at 12000×g, 4°C for 10 minutes to make The aqueous phase and organic phase of RNA are separated efficiently, and RNA forms a gelatinous precipitate on the bottom of the tube or on the side wall of the tube, remove the supernatant,

5) Add at least 1mL 75% ethanol to wash the precipitate after step 4), flick the bottom of the tube to suspend the precipitate, and turn it upside down 8-15 times, let stand at room temperature for 3-5min, then centrifuge at 12000×g 4°C for 5min , discard the supernatant;

6) In a clean environment, dry the precipitate at room temperature for 2-5 minutes, add an appropriate amount of RNase-free ddH ₂ O to dissolve the precipitate, and if necessary, use a pipette to gently blow a few times and mix well. After it is completely dissolved, you can get Take a small amount of total RNA for detection, and store the rest at -85 to -65°C for future use.

The method for extracting total RNA of bacteria and algae symbiotic balls of the present invention uses quartz sand and liquid nitrogen to grind the bacteria and algae symbiotic balls together, which not only increases the damage to the cell walls of fungi and microalgae, but also improves the extraction efficiency of total RNA, and at the same time, greatly It reduces the demand and grinding time for total RNA extraction of symbiotic balls of bacteria and algae raw materials; and it is beneficial to grind the symbiosis balls of bacteria and algae into powder, and improve the full contact of cell homogenate and RNA isolater extractant after the symbiosis balls of bacteria and algae are ground, and then Reduce the residual amount of extracellular membrane polysaccharides and proteins in the total RNA extract, and improve the quality of the total RNA of the symbiosis sphere.

In the present invention, for the first time, RNA isolater is used to separate and extract the RNA of two symbiotic microorganisms in the symbiosis sphere, and the strong cracking ability of the RNA isolater is used to increase the damage to the cell wall and cytoplasm of the fungus, and protect the total RNA of the symbiosis sphere integrity.

The symbiosis ball of bacteria and algae is a symbiosis ball of bacteria and algae formed by the symbiosis culture of mold and any one microalgae of Synechocystis sp., Synechococcus, Chlorella and Cyanothrix.

The symbiosis ball of bacteria and algae is a symbiosis ball of bacteria and algae formed by the symbiotic culture of Aspergillus fumigatus or Penicillium and Synechcystis sp. PCC6803. Preferably, after mixing 100 mL of Synechcystis sp. PCC6803 (concentration of 1 to 2×10 ⁸ /mL) with mycelial balls (100) with a diameter of 3-5 mm after 48 hours of cultivation, place them in a 30°C 150rpm Shaker symbiotic culture.

Aspergillus fumigatus (Aspergillus fumigatus) is a laboratory isolated and screened Aspergillus fumigatus. The screened samples were obtained from heavy metal wastewater samples from Liuxi, Dongping Town, Yangdong District, Yangjiang City, Guangdong Province. The filamentous fungus Aspergillus fumigatus was screened on a plate containing printing and dyeing wastewater. Firstly, the collected wastewater samples (200uL) were applied to solid plates containing printing and dyeing wastewater, and the plates were placed in a constant temperature incubator at 30°C for 5-7 days. Repeat at least 5 times on the PDA plate containing printing and dyeing wastewater, and finally obtain a single filamentous fungus. The DNA of the filamentous fungi was extracted and sent for sequencing, and the sequencing results were compared with the NCBI database through the BLAST gene bank, and the sequencing results of the strains were used to construct a phylogenetic tree (as shown in Figure 1) using MEGA6.06, and the bacterial species were identified They are: Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ NO.1.

Inoculate 4% of Aspergillus fumigatus conidia suspension in 100mL liquid medium (glucose (15g), KH ₂ PO ₄ (1.0g), Na ₂ HPO ₄ 12H ₂ O (2.9g), NH ₄ (SO ₄ ) ₂ (1.0g), MgSO ₄ .7H ₂ O (0.5g), NaCl (0.5g), beef extract (1.0g), distilled water 1000mL), cultured on a constant temperature shaker at 30°C 150rpm/min for 48h. Synechocystis sp. PCC6803, inoculated with 5% microalgae suspension in 100mL BG-11 medium, at 30°C, light-dark ratio 12:12h, light intensity 2000Lux light incubator static culture, shake the bottle manually twice a day , to prevent adherent growth. The fungal mycelium formed after 48 hours of cultivation was separated from the liquid medium by filtration, washed with normal saline for 3 times, and mixed with microalgae in the logarithmic phase (concentration: 1-2×10 ⁸ /mL) The number of balls: the volume of microalgae (ml) = 1: 0.67 ~ 1.5 for symbiotic culture.

Preferably, in step 1), the diameter of the symbiotic balls of bacteria and algae is 0.2-0.3 cm, and the number of symbiosis balls of bacteria and algae ground in a single time is 6-8.

The dosage of the quartz sand is 1-1.5 times of that of the bacteria and algae symbiosis ball, and generally the dosage of the quartz sand for a single grinding is 0.05-0.1 g.

Preferably, in step 4), the chloroform/isoamyl alcohol volume ratio is 24:1.

In step 5), the 75% ethanol is prepared with RNase-free ddH ₂ O.

Preferably, in step 6), the amount of RNase-free ddH ₂ O used is 30 μL.

The beneficial effects of the method for extracting the total RNA of the bacteria and algae symbiosis spheres of the present invention:

The method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present invention is to quickly freeze and grind the bacteria and algae symbiotic spheres by adding quartz sand combined with liquid nitrogen, instead of simple liquid nitrogen grinding or quartz sand grinding, so that the RNA extraction has no effect on the bacteria and algae symbiotic spheres. The demand is greatly reduced and the grinding time is significantly shortened. This method is also conducive to grinding the symbiotic balls of bacteria and algae into powder, improving the full contact between the cell homogenate and the RNA isolater extractant after the symbiotic balls of bacteria and algae are ground, thereby reducing the residual amount of polysaccharides and proteins in the outer cell membrane in the total RNA extract, Improve the extraction concentration and quality of total RNA of bacteria and algae symbiosis balls.

In the present invention, for the first time, RNA isolater is used to separate and extract the RNA of two symbiotic microorganisms in the symbiosis sphere, and the strong cracking ability of the RNA isolater is used to increase the damage to the cell wall and cytoplasm of the fungus, and protect the total RNA of the symbiosis sphere integrity.

The invention avoids the harm of DEPC water to the human body in the traditional extraction method, and has important significance for the research on the molecular mechanism of the transcription level of the fungus and microalgae symbiotic spheres.

The highest concentration of total RNA extracted by this extraction method can reach more than 495.03ng/uL, and the A260/A280 is greater than 1.9. The total RNA extract is not contaminated by DNA and protein, and the quality is high, and the gel electrophoresis band of the total RNA is clear and bright. It shows that the extraction method of the present invention does not degrade the RNA of the bacteria and algae symbiotic spheres.

Description of drawings

Fig. 1—the phylogenetic tree of Aspergillus fumigatus adopted in the extraction method of total RNA of bacteria and algae symbiosis of the present invention;

Fig. 2 - the method for extracting the total RNA of the bacteria and algae symbiosis balls of the present invention and the RNA concentration distribution diagram of the total RNA extraction of the bacteria and algae symbiosis balls treated with cell-free homogenate, quartz sand alone, and liquid nitrogen alone;

Fig. 3 - the method for extracting the total RNA of the symbiosis sphere of bacteria and algae of the present invention and the A ₂₆₀ /A ₂₈₀ ratio analysis chart of the total RNA extraction of the symbiosis sphere of bacteria and algae treated with cell-free homogenate, quartz sand alone, and liquid nitrogen alone;

Fig. 4—the total RNA gel electrophoresis figure obtained in the extraction method of total RNA of bacteria and algae symbiotic spheres of the present invention;

Fig. 5—A comparative analysis diagram of total RNA concentration and A ₂₆₀ /A ₂₈₀ extracted by the method for extracting total RNA of symbiotic balls of bacteria and algae in Examples 1-3.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

The microbial source that the present invention adopts is respectively as follows:

The filamentous fungus is Aspergillus fumigatus isolated and screened in the laboratory. The screening sample comes from the heavy metal wastewater water sample of Liuxi, Dongping Town, Yangdong District, Yangjiang City, Guangdong Province. The filamentous fungus Aspergillus fumigatus was screened on a plate containing printing and dyeing wastewater. Firstly, the collected wastewater samples (200uL) were applied to solid plates containing printing and dyeing wastewater, and the plates were placed in a constant temperature incubator at 30°C for 5-7 days. Repeat at least 5 times on the PDA plate containing printing and dyeing wastewater, and finally obtain a single filamentous fungus. The DNA of the filamentous fungi was extracted and sent for sequencing, and the sequencing results were compared with the NCBI database through the BLAST gene bank, and the sequencing results of the strains were used to construct a phylogenetic tree (as shown in Figure 1) using MEGA6.06, and the bacterial species were identified They are: Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ NO.1.

The microalgae was Synechcystis sp. PCC6803, which was purchased from the Chinese Academy of Sciences Wildlife Germplasm Bank-Freshwater Algae Species Bank.

Comparative Example 1

A method for extracting the total RNA of the symbiotic sphere of bacteria and algae, comprising the following steps:

1) Take 6 bacteria and algae symbiotic balls with a diameter of 0.3cm and put them in a container, then transfer the cell homogenate supernatant obtained in step 1) into a centrifuge tube, add 500uL RNA isolater, and let stand for 10min; wait until the sample is completely melted After that, continue pipetting until the lysate is transparent;

2) Transfer the lysate to a centrifuge tube, centrifuge at 12000×g, 4°C for 5 min, and absorb the supernatant of the lysate;

3) Add 600 μl of chloroform/isoamyl alcohol to the supernatant of the lysate in step 3), vortex and mix, let stand at 4°C for more than 10 minutes; centrifuge at 12000×g, 4°C for 10 minutes, remove the supernatant,

4) Add at least 1 mL of 75% ethanol to wash the precipitate after step 4), flick the bottom of the tube to suspend the precipitate, and turn it upside down 10 times. After standing at room temperature for 3 minutes, centrifuge at 12000×g 4°C for 5 minutes, discard the upper clear;

5) Dry the precipitate at room temperature for 2 minutes in a clean environment, add 30 μL of RNase-free ddH ₂ O to dissolve the precipitate, and if necessary, use a pipette to gently blow and mix for a few times, and the total RNA can be obtained after it is completely dissolved , take a small amount for testing, and store the rest at -80°C for later use.

Comparative example 2

A method for extracting the total RNA of the symbiotic sphere of bacteria and algae, comprising the following steps:

1) Put 6 bacteria and algae symbiosis balls with a diameter of 0.3cm in a sterile mortar, add 0.05g of quartz sand with the weight of the bacteria and algae symbiosis balls, fully grind to obtain a cell homogenate, and collect the supernatant of the cell homogenate after centrifugation , standby; the bacteria and algae symbiosis ball is a bacteria and algae symbiosis ball formed by the symbiotic culture of Aspergillus fumigatus and Synechcystis sp.PCC6803;

2) Transfer the cell homogenate supernatant obtained in step 1) into a centrifuge tube, add 500uL RNA isolater, and let it stand for 10 minutes; after the sample is completely melted, continue blowing until the lysate is transparent;

3) Transfer the lysate to a centrifuge tube, centrifuge at 12000×g, 4°C for 5 min, and absorb the supernatant of the lysate;

4) Add 600 μl of chloroform/isoamyl alcohol to the supernatant of the lysate in step 3), vortex and mix, let stand at 4°C for more than 10 minutes; centrifuge at 12000×g, 4°C for 10 minutes, remove the supernatant,

5) Add at least 1 mL of 75% ethanol to wash the precipitate after centrifugation in step 4), flick the bottom of the tube to suspend the precipitate, and turn it upside down 10 times. After standing at room temperature for 3 minutes, centrifuge at 12000×g 4°C for 5 minutes, discard the upper clear;

6) Dry the precipitate at room temperature for 2 minutes in a clean environment, add 30 μL of RNase-free ddH ₂ O to dissolve the precipitate, and if necessary, use a pipette to gently blow and mix for a few times, and the total RNA can be obtained after it is completely dissolved , take a small amount for testing, and store the rest at -80°C for later use.

Comparative Example 3

Compared with Comparative Example 2, the method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment has the following differences:

The consumption of described quartz sand is 0.1g.

Comparative Example 4

Compared with Comparative Example 2, the method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment has the following differences:

The consumption of described quartz sand is 0.15g.

Comparative Example 5

The method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment comprises the following steps:

The operation of step 1) is: put 6 bacteria and algae symbiotic balls with a diameter of 0.3cm in a sterile pre-cooled mortar, add an appropriate amount of liquid nitrogen (generally add more than 1/2 of the volume of the mortar), and grind Obtain the cell homogenate in 30 s, collect the supernatant of the cell homogenate after centrifugation, and set aside; the symbiosis ball is a symbiosis ball formed by the symbiotic culture of Aspergillus fumigatus and Synechcystis sp.PCC6803;

2) Transfer the cell homogenate supernatant obtained in step 1) into a centrifuge tube, add 500uL RNA isolater, and let it stand for 10 minutes; after the sample is completely melted, continue blowing until the lysate is transparent;

3) Transfer the lysate to a centrifuge tube, centrifuge at 12000×g, 4°C for 5 min, and absorb the supernatant of the lysate;

4) Add 600 μl of chloroform/isoamyl alcohol to the supernatant of the lysate in step 3), vortex and mix, let stand at 4°C for more than 10 minutes; centrifuge at 12000×g, 4°C for 10 minutes, remove the supernatant,

5) Add at least 1 mL of 75% ethanol to wash the precipitate after centrifugation in step 4), flick the bottom of the tube to suspend the precipitate, and turn it upside down 10 times. After standing at room temperature for 3 minutes, centrifuge at 12000×g 4°C for 5 minutes, discard the upper clear;

6) Dry the precipitate at room temperature for 2 minutes in a clean environment, add 30 μL of RNase-free ddH ₂ O to dissolve the precipitate, and if necessary, use a pipette to gently blow and mix for a few times, and the total RNA can be obtained after it is completely dissolved , take a small amount for testing, and store the rest at -80°C for later use.

Comparative Example 6

Compared with Comparative Example 5, the method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment has the following differences:

The time of the liquid nitrogen grinding treatment is 1 min.

Comparative Example 7

Compared with Comparative Example 5, the method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment has the following differences:

The time for the liquid nitrogen grinding treatment is 2 minutes.

Comparative Example 8

Compared with Comparative Example 5, the method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment has the following differences:

The time for the liquid nitrogen grinding treatment is 3 minutes.

Example 1

The method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment comprises the following steps:

1) Put 6 symbiotic spheres with a diameter of 0.3cm in a sterile mortar, add quartz sand twice the mass (about 0.1g) of the symbiotic spheres, grind for 2 minutes to obtain a cell homogenate, and collect the cells after centrifugation The homogenized supernatant is set aside; the symbiosis ball is a symbiosis ball formed by the symbiotic culture of Aspergillus fumigatus and Synechcystis sp.PCC6803;

2) Transfer the cell homogenate supernatant obtained in step 1) into a centrifuge tube, add 500uL RNA isolater, and let it stand for 10 minutes; after the sample is completely melted, continue blowing until the lysate is transparent;

3) Transfer the lysate to a centrifuge tube, centrifuge at 12000×g, 4°C for 5 min, and absorb the supernatant of the lysate;

4) Add 600 μl of chloroform/isoamyl alcohol (volume ratio: 24:1) to the supernatant of the lysate in step 3), vortex and mix, let stand at 4°C for more than 10min; centrifuge at 12000×g, 4°C for 10min, Remove the supernatant,

5) Add at least 1 mL of 75% ethanol (prepared with RNase-free ddH ₂ O) to wash the precipitate after centrifugation in step 4), flick the bottom of the tube to suspend the precipitate, and turn it upside down 10 times. After standing at room temperature for 3 minutes, 12000 Centrifuge at ×g 4°C for 5 min, discard the supernatant;

6) Dry the precipitate at room temperature for 2 minutes in a clean environment, add 30 μL of RNase-free ddH ₂ O to dissolve the precipitate, and if necessary, use a pipette to gently blow and mix for a few times, and the total RNA can be obtained after it is completely dissolved , take a small amount for testing, and store the rest at -80°C for later use.

In step 1), the bacteria and algae symbiosis balls are obtained by the following method: inoculate 4% of Aspergillus fumigatus conidia suspension in 100mL liquid medium (glucose (15g), KH ₂ PO ₄ (1.0g), Na ₂ HPO ₄ 12H ₂ O (2.9g), NH ₄ (SO ₄ ) ₂ (1.0g), MgSO ₄ .7H ₂ O (0.5g), NaCl (0.5g), beef extract (1.0g), distilled water 1000mL) , cultured on a constant temperature shaker at 30°C 150rpm/min for 48h; Synechocystis sp. PCC6803, inoculated with 5% microalgae suspension in 100mL BG-11 medium, at 30°C, the light-dark ratio was 12:12h, and the light intensity was The 2000Lux light incubator was cultured statically, and the flask was manually shaken twice a day to prevent the growth of the adherent wall; the fungal mycelium ball formed after 48 hours of cultivation was filtered and separated from the liquid medium, washed with normal saline for 3 times, and placed in Microalgae in the logarithmic phase (concentration is 1-2× ¹⁰⁸ /mL) according to the number of mycelial balls: volume of microalgae (ml) = 1:1 (that is, add 100 fungal mycelial balls to 100ml of microalgae) For symbiotic cultivation.

The total RNA concentration and A ₂₆₀ /A ₂₈₀ obtained by the micro-spectrophotometer were compared with the extraction methods of Examples 1 to 8 and Example 1 for detection, and the differences in the total RNA concentration and A ₂₆₀ /A ₂₈₀ were shown in Figure 2 and Figure 2 respectively. 3 shown.

It can be seen from Figure 2 that the total RNA concentration of Comparative Examples 1-3 increased first and then decreased with the increase of the addition of quartz sand, while A ₂₆₀ /A ₂₈₀ increased correspondingly with the increase of the addition of quartz sand, indicating that the concentration of quartz sand When the amount of sand added is less than 0.1g, the increase of the added amount is beneficial to the destruction of the cell wall of the symbiosis ball of bacteria and algae, and is also conducive to the extraction of total RNA. When the amount of quartz sand added is greater than 0.1g, continue to increase the amount of quartz sand, It may be due to the loss of some cell tissues attached to the quartz sand after grinding and subsequent separation with the quartz sand. On the contrary, the extraction concentration of total RNA is reduced, but the purity of the RNA extract is promoted.

The total RNA concentration of Comparative Examples 4-7 increases first and then decreases with the prolongation of liquid nitrogen grinding time, while A ₂₆₀ /A ₂₈₀ first increases and then decreases with the prolongation of liquid nitrogen grinding time, indicating that the liquid nitrogen grinding time is less than 2min The shorter the liquid nitrogen grinding time, the easier it is to grind insufficiently. The prolongation of the grinding time is beneficial to the destruction of the cell wall of the symbiosis ball of bacteria and algae, the finer powder of cell tissue grinding, and the extraction of total RNA. If the nitrogen grinding time is longer than 2 minutes, continue to prolong the grinding time, which may cause partial RNA degradation due to the long liquid nitrogen grinding time, and correspondingly reduce the extraction concentration and quality of total RNA.

The total RNA concentration obtained by the extraction method of Example 1 is directly extracted from the cell-free homogenate (comparative example 1), separate quartz sand (0.1g, comparative example 3), separate liquid nitrogen treatment (2min, Comparative Example 7) has significantly improved, relative to the extraction method of cell-free homogenate treatment, the total RNA extraction concentration has increased by 6.5 times; relative to the same mass of quartz sand grinding treatment, the increase in the total RNA concentration is up to more than 5 times, compared with liquid nitrogen The total RNA concentration of the grinding treatment at the same time was increased by as much as 1.9 times; it shows that the simultaneous grinding of quartz sand and liquid nitrogen to treat the symbiotic spheres of bacteria and algae has a significant synergistic effect on the extraction of the total RNA of the symbiotic spheres of the strains, and the extraction method of Example 2 is adopted The A ₂₆₀ /A ₂₈₀ of the total RNA obtained by the method is equivalent to that obtained by liquid nitrogen treatment alone (2 min, comparative example 7), indicating that the extraction method in this example 1 has a good effect on the separation and extraction of total RNA.

The applicant also carried out gel electrophoresis analysis on the total RNA extract obtained by the extraction method of Example 1, and its gel electrophoresis figure is shown in Figure 4. As can be seen from Figure 4, the extracted total RNA bands are clear and bright, indicating that The extraction method has a strong protection for the RNA of the symbiosis ball of bacteria and algae, and the RNA of the symbiosis ball of bacteria and algae itself is not degraded during the extraction process.

Example 2

Compared with Example 1, the method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment has the following differences:

In step 1), the amount of quartz sand used is 1 times (about 0.05g) the mass of the symbiosis spheres added.

Example 3

Compared with Example 1, the method for extracting the total RNA of the bacteria and algae symbiotic spheres of the present embodiment has the following differences:

In step 1), the amount of quartz sand is 3 times (about 0.15g) of the mass of the bacteria and algae symbiosis ball added.

The total RNA concentration and A ₂₆₀ /A ₂₈₀ obtained by the extraction method of Examples 1 to 3 of the micro-spectrophotometer were used for detection, and the differences in the total RNA concentration and A ₂₆₀ /A ₂₈₀ are shown in Figure 5 respectively.

It can be seen from Figure 5 that when liquid nitrogen and quartz sand are simultaneously ground to treat the symbiosis ball of bacteria and algae, when the amount of quartz sand is less than twice that of the symbiosis ball of bacteria and algae, with the increase of the amount of quartz sand added, the total RNA of the symbiosis ball of bacteria and algae The amount of extraction increases accordingly, but the difference of A ₂₆₀ /A ₂₈₀ is small, that is, the change of the amount of quartz sand added has little effect on the purity of RNA isolation, and when the amount of quartz sand is greater than twice that of the bacteria and algae symbiosis balls, Continuing to increase the amount of quartz sand, the total RNA extraction amount of the symbiosis sphere decreased instead.

In the method for extracting the total RNA of the symbiosis balls of the present invention, the microalgae can also be Synechococcus, Chlorella or Cyanothrix, and when the microalgae is treated with cell homogenization, the number of the symbiosis balls can be determined according to the amount used for grinding. Adjust the size of the mortar and other equipment. For example, if the mortar is slightly larger, the number of bacteria and algae symbiosis balls can be adjusted to 8 in a single grinding, or you can choose a bacteria and algae symbiosis ball with a diameter of 0.2cm; thereby improving the bacteria and algae symbiosis balls Total RNA extraction efficiency; changes in the above technical features can be understood and implemented by those skilled in the art through text descriptions, so no additional drawings are provided for illustration.

Claims (10)

1. The extraction method of the total RNA of the bacteria-algae symbiotic sphere is characterized by comprising the following steps: putting the mycorrhizal symbiotic spheres into an aseptic precooling mortar, adding quartz sand and liquid nitrogen (so that the mycorrhizal symbiotic spheres are completely immersed in the liquid nitrogen) which are 1-3 times of the mass of the mycorrhizal symbiotic spheres, quickly and fully grinding to obtain cell homogenate, centrifuging, and collecting cell homogenate supernatant for subsequent total RNA extraction; the fungus-algae symbiotic ball is formed by symbiotic culture of mould and microalgae.

2. The method for extracting total RNA of the bacteria-algae symbiotic sphere according to claim 1, comprising the following steps:

1) Cell homogenization treatment: placing the mycorrhizal symbiotic spheres in an aseptic precooling mortar, adding quartz sand and liquid nitrogen (so that the mycorrhizal symbiotic spheres are completely immersed in the liquid nitrogen) which are 1-3 times of the mass of the mycorrhizal symbiotic spheres, quickly and fully grinding to obtain cell homogenate, centrifuging and collecting cell homogenate supernatant for later use;

2) Transferring the cell homogenate supernatant obtained in the step 1) into a centrifuge tube according to the proportion of 5-10 multiplied by 10 ⁶ Adding 1mL of RNA isolator into each cell, and standing for 5-10 min; after the sample is completely melted, continuously blowing and beating until the lysate is transparent;

3) Transferring the lysate to a centrifuge tube, centrifuging, and absorbing lysate supernatant;

4) Adding 500-700 mul of chloroform/isoamylol into the supernatant of the lysate obtained in the step 3), performing vortex mixing, standing for more than 10min at 4 ℃, centrifuging, removing the supernatant,

5) Washing the centrifuged precipitate in the step 4) by adding at least 1mL of 75% ethanol, turning upside down and uniformly mixing, standing at room temperature for 3-5min, centrifuging, and removing a supernatant;

6) Drying the precipitate in clean environment at room temperature for 2-5min, adding appropriate amount of RNase-free ddH ₂ Dissolving the precipitate with O to obtain total RNA.

3. The method for extracting total RNA of the fungus-algae symbiotic sphere according to claim 1, wherein the fungus-algae symbiotic sphere is formed by symbiotic culture of mould and any one of Synechocystis sp.

4. The method for extracting total RNA from Synechococcus as claimed in claim 3, wherein the Synechococcus is a Synechococcus formed by symbiotic culture of Aspergillus fumigatus or Penicillium and Synechocystis sp.PCC6803.

5. The method for extracting total RNA of the algal-bacterial symbiotic ball as claimed in claim 4, wherein in the step 1), aspergillus fumigatus (Aspergillus fumigatus) is Aspergillus fumigatus obtained by laboratory separation and screening, and the screening sample is derived from a water sample of the Liuxi heavy metal wastewater from east Tokyo Toxico district of Yangtze river, guangdong province, and the nucleotide sequence of the water sample is shown in SEQ NO. 1.

6. The method for extracting total RNA of the bacteria-algae symbiotic balls according to any one of claims 1 to 5, wherein in the step 1), the diameter of the bacteria-algae symbiotic balls is 0.2 to 0.3cm, and the number of the bacteria-algae symbiotic balls ground in a single time is 6 to 8.

7. The method for extracting total RNA from Synechococcus of claim 2, wherein the amount of the silica sand is 1-1.5 times of the amount of the Synechococcus of claim 2, and the amount of the silica sand used in one-time grinding is 0.05-0.1 g.

8. The method for extracting total RNA from Coccomys bacteria and algae according to claim 2, wherein in step 4), the chloroform/isoamyl alcohol volume ratio is 24.

9. The method for extracting total RNA of Synechococcus according to claim 2, wherein step 5) comprises extracting RNase-free ddH with 75% ethanol ₂ And (O) preparation.

10. The method for extracting total RNA of Synechococcus of claim 2, wherein in step 6), RNase-free ddH is added ₂ The amount of O is 30. Mu.L.

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