阅读说明：本技术 一种高纯种超微藻的分离和鉴定方法 (Separation and identification method of high-purity super microalgae ) 是由 雷瑾 刘常清 史小丽 陈开宁 于 2019-12-09 设计创作，主要内容包括：本发明公开了一种高纯种超微藻的分离和鉴定方法,本方法将流式细胞仪以及分子生物学技术集成协同运用,先采用流式细胞仪分选出特定的超微藻类群,而后进行单细胞孔板培养,扩大培养后采用高通量测序技术进行藻类鉴定即可。此法可高效、快速地分离培养出纯种超微藻,为湖泊水体超微藻的分离培养提供了成套技术,从而为我国藻种库的扩种提供了一部分的理论技术支持。(The invention discloses a method for separating and identifying high-purity super microalgae, which integrates and cooperatively uses a flow cytometer and a molecular biology technology, firstly selects a specific super microalgae group by using the flow cytometer, then carries out single cell pore plate culture, and carries out algae identification by using a high-throughput sequencing technology after amplification culture. The method can separate and culture pure super-microalgae efficiently and quickly, and provides a complete set of technology for separating and culturing the super-microalgae in the lake water body, thereby providing a part of theoretical technical support for the expansion of the seed bank of the algae in China.)

1. A method for separating and identifying high-purity super microalgae is characterized by comprising the following steps:

(1) firstly, collecting water samples for multiple times in a target water body, mixing the water samples, and pre-filtering the water samples by using a 300-mesh bolting silk;

(2) then, selecting fluorescence with excitation wavelengths of 488nm and 640nm as chlorophyll fluorescence, sorting the number of target algae cells in a water sample by a flow cytometer at a sample injection flow rate of 200-300 cells/second, setting lateral scattering light in the sorting process, and enclosing an area within 3 mu m by a niele pellet with the diameter of 3.1 mu m to collect an algae sample of the ultramicro algae cells;

(3) then, diluting the algae sample of the sorted target super-microalgae cells by adopting the filtered lake water;

(4) mixing the diluted algae cell dilution solution with 20% GB11 culture solution, subpackaging in a pore plate, and culturing in an incubator;

(5) taking out the algae cell sap discolored in the step (4), transferring the algae cell sap into a sterile bottle containing BG11 culture solution, and carrying out expanded culture for several days; the algae liquid which is successfully expanded and cultured is collected in a collecting tube after supernatant fluid is removed through centrifugation;

(6) extracting DNA from the algae sample collected in the step (5), and identifying the algae species by a molecular biology technology.

2. The method for separating and identifying super microalgae as claimed in claim 1, wherein the method comprises the following steps: the water sample in the step (1) is obtained by collecting three layers of water, namely surface water within 0.5 m below the water surface of the target water body, middle water and lake bottom water within 0.5 m away from the lake bottom, and mixing the three layers of water.

3. The method for separating and identifying super microalgae as claimed in claim 1, wherein the method comprises the following steps: the lake water used for dilution in step (3) was filtered through a 0.2 μm polycarbonate filter.

4. The method for separating and identifying super microalgae as claimed in claim 1, wherein the method comprises the following steps: the culture conditions of the step (4): the daily lighting period is 16 hours of light and 8 hours of darkness, the culture temperature is 25 +/-1 ℃, and the filtered lake water is supplemented to the original volume every 3 days.

5. The method for separating and identifying super microalgae as claimed in claim 1, wherein the method comprises the following steps: extracting DNA from the algae sample in the step (6) by using a DNeasy Blood & Tissue Kit;

amplifying a target alga-like 18S sequence by adopting 18S rRNA universal primers NS1 (5'-GTAGTCATATGCTTGTCTC-3') and 18L (5'-CACCTACGGAAACCTTGTTACGACTT-3'), performing bidirectional sequencing by utilizing a Sanger method, performing forward and reverse splicing on a sample successfully subjected to bidirectional sequencing, extracting a forward sequence from the sample successfully subjected to forward or reverse sequencing, and performing molecular identification; finally, the species level taxonomy information of the target algae sample is judged based on molecular biology means through local blast comparison of Silva (SILVA _132_ SSURef _ tax _ Silva. fasta) database.

Technical Field

The invention belongs to the technical field of algae separation culture in the fields of environmental science and ecology, and particularly relates to a method for quickly separating pure super microalgae.

Background

The ultramicro algae has huge groups and wide habitat, is generally distributed in various large oceans and lake ecosystems, can efficiently obtain and utilize resources to grow and reproduce due to the advantages of small individual and large specific surface area, and therefore has important contribution to primary productivity of lakes, the annual average contribution rate of the ultramicro algae decreases along with the rising of the nutrient salt level of lakes, and the contribution rates of the ultramicro algae in the pacific lake, the Poyang lake and the nested lake reach 65%, 60% and 23% respectively on the relevant research surface. Super little algae is as an initial key link of little food ring, can be ingested by some heterotrophic flagellates and ciliates, and then becomes the indirect food of large-scale plankton, gets into classical food net from this, finally influences the output of aquatic products fishery. Research shows that the super microalgae also has important influence on the carbon metabolism balance of the water body, and has significance in promoting the material circulation and the energy flow of a water ecosystem. And because the ultra-micro algae is small and lacks of remarkable morphological classification standards, the optical microscope and even the electron microscope are difficult to perform species identification, so that few new species of ultra-micro algae are reported, the cognition of people on the ultra-micro algae is greatly reduced, and the huge and non-trivial biological resources are ignored. Therefore, if the method for rapidly separating, culturing and identifying the super-microalgae is provided, the cognition of the super-microalgae can be greatly improved, so that the effect of the super-microalgae in an ecological system can be known, and a foundation is laid for further exploring natural resources in China.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the existing problems and defects, the invention aims to provide a method for separating and identifying high-purity super-microalgae, which integrates and cooperatively uses a flow cytometer and a molecular biology technology, efficiently and quickly separates and cultures the pure super-microalgae, provides a complete set of technology for separating and culturing the super-microalgae in water bodies such as lakes, oceans and the like, and greatly improves the cognition of people on the super-microalgae, thereby understanding the effect of the super-microalgae in an ecological system and laying a foundation for further exploring natural resources in China.

The technical scheme is as follows: in order to realize the purpose, the invention adopts the following technical scheme: a method for separating and identifying high-purity super microalgae comprises the following steps:

(1) firstly, collecting water samples for multiple times in a target water body, mixing the water samples, and pre-filtering the water samples by using a 300-mesh bolting silk;

(2) then, selecting fluorescence with excitation wavelengths of 488nm and 640nm as chlorophyll fluorescence, sorting the number of target algae cells in a water sample by a flow cytometer at a sample injection flow rate of 200-300 cells/second, setting lateral scattering light in the sorting process, and enclosing an area within 3 mu m by niele globules with the size of 3.1 mu m to collect an algae sample of a proper amount of target super-microalgae cells;

(3) then, the algae sample of the sorted target super-microalgae cells is diluted to 1cell 100 mu L by using the filtered lake water^-1；

(4) Mixing the diluted algae cell dilution solution with 20% GB11 culture solution, subpackaging in a pore plate, and culturing in an incubator;

(5) taking out the algae cell sap discolored in the step (4), transferring the algae cell sap into a sterile bottle containing BG11 culture solution, and carrying out expanded culture for several days; the algae liquid successfully cultured in the enlarged scale is collected into an EP collecting tube after supernatant fluid is removed through centrifugation;

(6) extracting DNA from the algae sample collected in the step (5), and identifying the algae species by a molecular biology technology.

Further, the water sample in the step (1) is obtained by collecting three layers of water, namely surface water within 0.5 m below the water surface of the target water body, middle water and lake bottom water within 0.5 m away from the lake bottom, and mixing the three layers of water.

Further, the lake water used for dilution in the step (3) was filtered through a 0.2 μm polycarbonate filter.

Further, the culture conditions of the step (4): the daily lighting period is 16 hours of light and 8 hours of darkness, the culture temperature is 25 +/-1 ℃, and the filtered lake water is supplemented to the original volume every 3 days.

Further, extracting DNA from the algae-like sample in the step (6) by using a DNeasy Blood & Tissue Kit;

amplifying a target alga-like 18S sequence by adopting 18S rRNA universal primers NS1 (5'-GTAGTCATATGCTTGTCTC-3') and 18L (5'-CACCTACGGAAACCTTGTTACGACTT-3'), performing bidirectional sequencing by utilizing a Sanger method, performing forward and reverse splicing on a sample successfully subjected to bidirectional sequencing, extracting a forward sequence from the sample successfully subjected to forward or reverse sequencing, and performing molecular identification; finally, the species level taxonomy information of the target algae sample is judged based on molecular biology means through local blast comparison of Silva (SILVA _132_ SSURef _ tax _ Silva. fasta) database.

Has the advantages that: compared with the prior art, the invention integrates and cooperatively applies the flow cytometry and the molecular biology technology, can efficiently and quickly separate and culture pure super-microalgae, provides a complete set of technology for separating and culturing the super-microalgae in the lake water body, and provides a part of theoretical technical support for the expansion of the algae seed bank in China.

Drawings

FIG. 1 is a schematic flow chart of the method for separating and identifying the high-purity super microalgae according to the present invention;

FIG. 2 is a schematic diagram of a group of microalgae detected by a flow cytometer in an embodiment of the present disclosure;

FIG. 3 is a DNA electrophoresis diagram of a microalgae according to an embodiment of the present invention.

Detailed Description

The present invention is further illustrated by the following figures and specific examples, which are to be understood as illustrative only and not as limiting the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalent modifications thereof which may occur to those skilled in the art upon reading the present specification.

In the embodiment, based on the time-space distribution characteristics of the Poyang lake super microalgae, the point with high abundance of the target algae species (eukaryotic green algae, Macleaya) is selected to collect the mixed water sample. The method for collecting the target mixed water sample comprises the steps of collecting water samples of a target water body on a surface layer (0.5 m below the water surface), a middle layer and a bottom layer (0.5 m away from the lake bottom) by using a machine glass water sampler, uniformly mixing 3 layers of water samples, placing the mixture in a 4 ℃ heat preservation box for temporary storage, and immediately sending the mixture back to a laboratory for next treatment. The lake water was filtered beforehand using a 0.2 μm polycarbonate filter.

As shown in FIGS. 1-3, a flow cytometer is used to sort a water sample to obtain an algal sample containing 2000 eukaryotic algal cells. The specific process comprises the following steps: the flow cytometer channel was flushed with 75% ethanol in advance, then with PBS for half an hour, and used as the sorting sheath fluid. The workbench of the flow cytometer reaches an aseptic state, a sterile BG11 culture medium for receiving the sorted ultramicro algae is prepared, and fluorescence of chlorophyll of fluorescence FL3 and FL5 channels (excitation wavelengths of 488nm and 640 nm) is selected according to the spontaneous chlorophyll fluorescence difference of the target ultramicro algae; then according to the size of the super micro algae cell below 3 μm, setting side scattered light (FSC, according to the 3.1 μm niel globule of BD company, defining the area within 3 μm, pre-filtering the lake water body to be processed with 300 mesh bolting silk, then injecting sample at low speed of 200 cells/sec, according to the defined range (figure 2), collecting the chlorophyll fluorescence containing the fluorescence difference of self-generated chlorophyll according to the target super micro algae, selecting fluorescence FL3 and FL5 channels (excitation wavelength of 488nm and 640 nm), according to the defined range (figure 2), according to the size of the super micro algae cell below 3 μm, setting side scattered light (FSC, according to the 3.1 μm niel globule of BD company, defining the area within 3 μm, pre-filtering the lake water body to be processed with 300 mesh bolting silk, then injecting sample at low speed of 200 cells/sec, according to the defined range (figure 2), collecting a proper amount of target super microalgae cells. Diluting the sorted eukaryotic algae cell solution to 1 cell.100. mu.L by using the filtered lake water^-1A20% BG11 solution was prepared with sterile water, and 100. mu.L of the algal cell dilution solution and 900. mu.L of the culture solution of LBG11 were transferred to a 48-well medium and cultured. A total of seven plates of 48 well medium were inoculated, for a total of 336 wells. Culturing 48-well culture medium in a light incubator with a 16-hour (light): 8 hours (dark) and the incubation temperature was 25 ℃. After the color of the multi-well medium was changed, 100. mu.L of the algal solution was transferred to a Erlenmeyer flask containing 100 mL of BG11 culture solution, and the culture was expanded in an incubator with light. In total, 85 holes are changed in color, and the algae liquid in 49 holes is successfully cultured in an expanding way. The algal solution was collected by centrifugation for several times and discarded into a 1.5ml EP tube using DNeasy Blood&Tissue Kit (Qiagen)DNA was extracted (FIG. 3). The target alga-like 18S sequence was then amplified using 18S rRNA universal primers NS1 (5'-GTAGTCATATGCTTGTCTC-3') and 18L (5'-CACCTACGGAAACCTTGTTACGACTT-3'). And then performing bidirectional sequencing by using a Sanger method, performing forward and reverse splicing on samples successfully subjected to bidirectional sequencing, extracting a forward sequence from the samples successfully subjected to forward or reverse sequencing, performing molecular identification, and judging the species level taxonomy information of the target algae sample by a molecular biology means through local blast comparison of a Silva (SILVA _132_ SSURef _ tax _ silva.fasta) database of the latest version. Wherein, 11 samples fail to be amplified, 10 samples have repeated peaks, and finally 28 algae samples are successfully identified.

FIG. 3 shows a DNA electrophoresis chart of the microalgae sample of the present embodiment, which is markerDL2000 and sample 1 from left to rightMychonastes homosphaera) Sample 2 (no amplified band), sample 3: (Mychonastes homosphaera) Sample 4 blank, sample 5: (Allovahlkampfiasp.strain SO/1P), sample 6 (Tetrastrum staurogeniaeforme) Sample 7: (Desmodesmus intermedius）。