CN110269014B - RNA interference method of urechis unicinctus larvae - Google Patents
RNA interference method of urechis unicinctus larvae Download PDFInfo
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention provides an RNA interference method of urechis unicinctus larvae, and belongs to the technical field of gene interference. The RNA interference method comprises the following steps: (1) taking a culture water body in a normal breeding state of urechis unicinctus larvae, and filtering seawater in the culture water body by using bolting silk to obtain a concentrated water body with the larva density of 15-60/ml; (2) mixing the concentrated water body with target gene double-stranded RNA to enable the final concentration of the target gene double-stranded RNA to be 0.03-0.3 micromole/liter, and standing for 6-15 hours to obtain an interference water body; (3) adding filtered seawater into the interference water body to the culture water body in the normal state in the step (1), and continuing to raise for more than or equal to 2 days. The RNA interference method provided by the invention can effectively interfere the expression of the target gene of the urechis unicinctus larvae, and is beneficial to the research of the function of the urechis unicinctus gene.
Description
Technical Field
The invention relates to the technical field of gene interference, in particular to an RNA interference method of urechis unicinctus larvae.
Background
Many invertebrates undergo one or more larval stages during their early development, where the larvae, through metamorphosis, develop characteristics that are substantially identical to adult morphology, physiology and ecological habits, which involve the network regulation of a series of genes and vary by species or physiological processes. Urechis animals have unique developmental characteristics and evolutionary status, such as: the structure of the trochophore is more complex and has longer duration than the structures of trochophores of other crown animals; the larva of the body segment has larva body segment, the larva body segment is one of important evolutionary characteristics of the urechis animals, and the structure is simpler and the duration is shorter than that of the typical body segment animal, and the like. However, at present, no research platform for the gene function of the urechis unicinctus larvae is available worldwide, so that research progress of people for exploring the characteristic molecular mechanism is greatly limited.
Disclosure of Invention
In view of the technical problems in the background art, the invention aims to provide an RNA interference method for urechis unicinctus larvae, and aims to construct an RNA interference technology for urechis unicinctus larvae and build a platform for the gene function research of the urechis unicinctus larvae.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an RNA interference method of urechis unicinctus larvae, which comprises the following steps:
(1) taking a culture water body in a normal breeding state of urechis unicinctus larvae, and filtering seawater in the culture water body by using bolting silk to obtain a concentrated water body with the larva density of 15-60/ml;
(2) mixing the concentrated water body with target gene double-stranded RNA to enable the final concentration of the target gene double-stranded RNA to be 0.03-0.3 micromole/liter, and standing for 6-15 hours to obtain an interference water body;
(3) adding filtered seawater into the interference water body to the culture water body in the normal state in the step (1), and continuing to raise for more than or equal to 2 days.
Preferably, the urechis unicinctus larvae are trochophores.
Preferably, when the urechis unicinctus larvae are trochophores, the density of the larvae in the concentrated water body in the step (1) is 40-60 larvae per milliliter.
Preferably, when the urechis unicinctus larvae are trochophores, the standing time in the step (2) is 6-12 hours.
Preferably, the urechis unicinctus larvae are somniferous larvae.
Preferably, when the urechis unicinctus larvae are somnal larvae, the density of the larvae in the concentrated water body in the step (1) is 25-60 larvae per milliliter.
Preferably, when the urechis unicinctus larvae are body segment larvae, the standing time in the step (2) is 10-15 hours.
Preferably, the target gene in step (2) includes one or more of Msx, Lox5 and Antp.
Preferably, when the target gene is an Msx gene, the final concentration of the double-stranded RNA of the target gene is 0.1-0.2 micromole/liter; when the target gene is Lox5 gene, the final concentration of the double-stranded RNA of the target gene is 0.05-0.1 micromole/liter; when the target gene is an Antp gene, the final concentration of the double-stranded RNA of the target gene is 0.05-0.1 micromole/liter.
The invention also provides the application of the RNA interference method in the RNA interference of the trochophore; the trochophore preferably comprises a mollusk trochophore and/or a clitellum trochophore.
Preferably, when the period of continuing the breeding in step (3) is >7 days, the operations of step (1), step (2) and step (3) are repeated in order.
Has the advantages that: the invention provides an RNA interference method of urechis unicinctus larvae, which comprises the following steps: (1) taking a culture water body in a normal breeding state of urechis unicinctus larvae, and filtering seawater in the culture water body by using bolting silk to obtain a concentrated water body with the larva density of 15-60/ml; (2) mixing the concentrated water body with target gene double-stranded RNA to enable the final concentration of the target gene double-stranded RNA to be 0.03-0.3 micromole/liter, and standing for 6-15 hours to obtain an interference water body; (3) adding filtered seawater into the interference water body to the culture water body in the normal state in the step (1), and continuing to raise for more than or equal to 2 days. The RNA interference method provided by the invention can effectively interfere the expression of the urechis unicinctus larva target gene; if the continuous feeding time exceeds 7 days, a second RNA interference can be added, and the system is the same as the steps (1) to (3). The RNA interference method provided by the invention is beneficial to the research of urechis unicinctus gene functions.
Drawings
FIG. 1 shows the test results of example 1 of the present invention, specifically the relative expression level of urechis unicinctus larva Msx mRNA in 3 days of RNA interference; all data are mean ± sem (n ═ 3); setting the Msx expression quantity of a blank control group as standard 1; different letters indicate that there was a significant difference in the expression level of the target gene in different groups of larvae (P < 0.05).
FIG. 2 is a morphological diagram of the urechis unicinctus larva nervous system according to embodiment 1 of the present invention, specifically, the urechis unicinctus larva nervous system for detecting Msx mRNA interference for 7 days by fluorescence immunohistochemistry; all pictures are in a larval ventral view, and the heads of the larval are upward; green is a positive signal for 5-HT (5-hydroxytryptamine), indicating a nerve; blue is a DAPI positive signal, indicating the nucleus; white dashed lines indicate the somite groove, red arrows indicate nervous system differences; spg, the superior esophageal ganglion; cc, periesophageal nerve connection; pk is perikaryoid; vnc abdominal nerve cord. A scale: 100 μm.
FIG. 3 shows the results of the experiment described in example 2 of the present invention, specifically the relative expression level of Urechis unicinctus larva Lox5 mRNA in 3 days of RNAi; all data are mean ± sem (n ═ 3); the blank control group Lox5 expression level was set as standard 1; different letters indicate that there was a significant difference in the expression level of the target gene in different groups of larvae (P < 0.05).
FIG. 4 shows the test results of example 3 of the present invention, specifically the relative expression level of the Antp mRNA in urechis unicinctus larva at 3 days of RNAi; all data are mean ± sem (n ═ 3); the expression level of Antp in the blank control group is set as standard 1; different letters indicate that there was a significant difference in the expression level of the target gene in different groups of larvae (P < 0.05).
Detailed Description
The invention provides an RNA interference method of urechis unicinctus larvae, which comprises the following steps:
(1) taking a culture water body in a normal breeding state of urechis unicinctus larvae, and filtering seawater in the culture water body by using bolting silk to obtain a concentrated water body with the larva density of 15-60/ml;
(2) mixing the concentrated water body with target gene double-stranded RNA to enable the final concentration of the target gene double-stranded RNA to be 0.03-0.3 micromole/liter, and standing for 6-15 hours to obtain an interference water body;
(3) adding filtered seawater into the interference water body to the culture water body in the normal state in the step (1), and continuing to raise for more than or equal to 2 days.
The method is characterized in that the breeding water body of the urechis unicinctus larvae in the normal breeding state is taken firstly. In the present invention, the urechis unicinctus larvae comprise trochophore and/or somniform larvae. In the culture water body under the normal breeding state, the density of the trochophore is usually 5-10 larvae per milliliter; the density of the somite larvae is usually 3-5 larvae per milliliter.
The invention uses bolting silk to filter the seawater in the culture water body to obtain the concentrated water body. In the invention, the volume of the filtered seawater is preferably 80-95% (more preferably 90%) of the volume of the aquaculture water body. In the invention, the aperture of the bolting silk is preferably 200-600 meshes, and more preferably 500 meshes. In the invention, the density of the larvae in the concentrated water body is preferably 15-60 larvae per milliliter. The research of the invention determines that: the larva density of 15-60/ml can be used for RNA interference of urechis unicinctus larvae. In the invention, when the urechis unicinctus larvae are trochophores, the larva density of the concentrated water body in the step (1) is preferably 40-60 larvae per milliliter, and more preferably 50 larvae per milliliter; when the urechis unicinctus larvae are somnal larvae, the density of the larvae in the concentrated water body in the step (1) is preferably 25-60 larvae per milliliter, and more preferably 50 larvae per milliliter.
After the concentrated water body is obtained, the concentrated water body is mixed with the target gene double-stranded RNA. In the invention, the final concentration of the mixed target gene double-stranded RNA is preferably 0.03-0.3 micromole/liter. The research of the invention determines that: the double-stranded RNA with the final concentration of 0.03-0.3 micromole/liter can be used for RNA interference of urechis unicinctus larvae, and when the final concentration of the double-stranded RNA is more than 0.3 micromole/liter, the survival rate of urechis unicinctus trochophore and somite larvae is obviously reduced. In the present invention, the target gene preferably includes one or more of Msx, Lox5, and Antp. In the present invention, when the target gene is an Msx gene, the final concentration of the double-stranded RNA of the target gene is preferably 0.1 to 0.2 μmol/l, and more preferably 0.15 μmol/l; when the target gene is Lox5 gene, the final concentration of the double-stranded RNA of the target gene is preferably 0.05-0.1 micromole/liter, and more preferably 0.05 micromole/liter; when the target gene is an Antp gene, the final concentration of the double-stranded RNA of the target gene is preferably 0.05-0.1 micromole/liter, and more preferably 0.05 micromole/liter.
The invention mixes the concentrated water body with the double-stranded RNA of the target gene, and then stands the mixed water body to obtain the interference water body. In the invention, the standing time is preferably 6-15 hours. The research of the invention determines that: the RNA interference time of 6 to 15 hours can be used for RNA interference of urechis unicinctus larvae, the interference time is longer than 12 hours, and particularly, the survival rate of urechis unicinctus trochophore and somite larvae is obviously reduced when the interference time is longer than 15 hours. In the invention, when the urechis unicinctus larvae are trochophores, the standing time in the step (2) is preferably 6-12 hours, and more preferably 12 hours; when the urechis unicinctus larvae are somniferous larvae, the standing time in the step (2) is preferably 10-15 hours, and more preferably 12 hours.
And (3) after obtaining the interference water body, adding filtered seawater into the interference water body to the culture water body in the normal state in the step (1), and continuing to raise. In the present invention, the period of the continuous feeding is preferably 2 days or more, more preferably 3 days or more. When the continuous feeding time is more than or equal to 3 days, the expression of the detected target gene is obviously reduced. If the time for continuing the feeding exceeds 7 days, the present invention preferably further comprises a second RNA interference, which is performed in the same manner as in the above steps (1) to (3). The RNA interference method provided by the invention is beneficial to the research of urechis unicinctus gene functions.
At present, no RNA interference system of urechis unicinctus larvae exists in the industry, the invention realizes the RNA interference of urechis unicinctus trochophore and somite larvae for the first time, and has the universality of RNA interference technology of annelids and mollusk trochophores. The successful realization of the RNA interference technology of the invention is mainly based on: the method is suitable for a high-density system of the urechis unicinctus larvae, the stability of the double-stranded RNA in seawater and the influence of the double-stranded RNA on the survival rate of the urechis unicinctus larvae.
The invention also provides the application of the RNA interference method in the RNA interference of the trochophore; the trochophore comprises a trochophore of a mollusk and/or a trochophore of a annelid. When the period of continuing the rearing in the step (3) is more than 7 days, the present invention preferably repeats the operations of the step (1), the step (2) and the step (3) in this order.
The terms used in the present invention are explained as follows:
(1) the method comprises the following steps of (1) urechis unicinctus: a group of benthic invertebrates living in the ocean is now advocated to be classified as annelids.
(2) Urechis unicinctus: an urechis animal belonging to class Nostochis, order Nostochis, family urechidaceae, genus urechis; is an important economic species distributed along the bank of the yellow Bohai sea in China, and has delicious meat quality and rich nutrition; it develops through embryo, larva (trochophore, larva of somite and worm-like larva) of each stage, and then is transformed into young urechis unicinctus.
(3) Trochophore: a free swimming planktonic larva is a larva specific to marine annelid and most mollusks, and has body shape similar to top with ciliated ring.
(4) And (3) larva of somite: the urechis animal develops into larva with segmented body through trochophore.
(5) RNA interference (RNA interference, RNAi): a phenomenon of gene silencing induced by Double-stranded RNA (dsRNA).
(6) Double-stranded RNA of Msx gene/Lox 5 gene/Antp gene: the artificially synthesized double-stranded RNA of the urechis unicinctus Msx gene/Lox 5 gene/Antp gene is used for RNA interference experiments.
The types and sources of the raw materials used in the invention are as follows:
(1) urechis unicinctus: the adult urechis unicinctus is purchased from Taidong aquatic product market in Qingdao, and the production place is Tai Lai Zhou.
(2) Urechis unicinctus larva: comprises trochophore and somite larvae which are artificially cultivated indoors.
(3) Indoor artificial breeding and conventional feeding of urechis unicinctus larvae: dissecting the male and female adult urechis unicinctus, obtaining mature sperms and eggs from the kidney tube of the adult urechis unicinctus, carrying out artificial insemination according to the ratio of the quantity of the sperms and the eggs to be 10:1, hatching the fertilized eggs in filtered seawater (the salinity is 30 per mill, the pH value is 8.0, and the water temperature is 18 +/-2 ℃), carrying out air-charging feeding on the hatched trochophores in the filtered seawater, feeding unicellular bait once in the morning and at the evening every day, and changing water 1/3 every day. The trochophore develops to the somite larva after about 30 days at the water temperature of 19 +/-2 ℃, the culture density of the trochophore is 10 to 5 per milliliter (decreasing along the development process), and the culture density of the somite larva is 5 to 3 per milliliter (decreasing along the development process).
(4) Double-stranded RNA synthesis: using Thermo Fisher Scientific
RNA kit synthesis.
(5) Detecting the gene expression level: the gene expression change is detected by a real-time fluorescence quantitative technology.
(6) Detecting the morphology of the nervous system: detecting the distribution of the nervous system marker molecule 5-hydroxytryptamine by using a fluorescence immunohistochemistry technology.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Sterilizing 500-mesh bolting silk, containers with the volumes of 20 ml and 250 ml and then drying; preparing double-stranded Msx gene RNA (shown as SEQ ID No.1, the sequence is 10 micromole per liter) and enough filtered seawater (the salinity is 30 per thousand, the pH value is 8.0, and the water temperature is 19 ℃); the ambient temperature is maintained at 20 ℃ and the relative humidity is greater than 50%.
Filtering out 67% seawater from normal bred urechis unicinctus trochophore (density: 5 per milliliter) by using 500-mesh bolting silk to ensure that the density of the larva reaches 15 per milliliter; then 10 ml of seawater (containing about 150 larvae) was taken in a container with a volume of 20 ml, 0.1 ml of double-stranded RNA was added thereto, mixed (final concentration of double-stranded RNA: 0.1. mu. mol/L) and allowed to stand for 12 hours; standing for 12 hours, transferring the larvae and seawater into a container with the volume of 250 ml, adding 20 ml of filtered seawater, and continuously breeding for 3 days according to conventional breeding conditions; after continuously raising for 3 days, 100 larvae are collected for detecting and analyzing gene expression quantity, and after continuously raising for 7 days, the rest larvae are collected for detecting the shape of the nervous system.
The test results are shown in FIGS. 1-2. Through detection, the expression of the Msx gene is reduced by 30% (figure 1), and the morphology of the urechis unicinctus larva nervous system is abnormal (figure 2, green (light color part) indicates the morphology of the larva nervous system, and red arrows (arrow part) indicate the abnormality of the nervous system).
Example 2
Sterilizing 500-mesh bolting silk, containers with the volumes of 20 ml and 250 ml and then drying; preparing double-stranded RNA (sequence shown in SEQ ID No.2, using concentration of 10 micromole per liter) of Lox5 gene and enough filtered seawater (salinity is 30 per thousand, pH is 8.0, water temperature is 20 ℃); the ambient temperature is maintained at 20 ℃ and the relative humidity is greater than 50%.
Filtering 92% seawater from normal bred urechis unicinctus larva (density: 4/ml) by using 500-mesh bolting silk to make the larva density reach 50/ml; then 10 ml of seawater (containing about 500 larvae) was taken in a container having a capacity of 20 ml, 0.05 ml of double-stranded RNA was added thereto, mixed (final concentration of double-stranded RNA: 0.05. mu. mol/l) and allowed to stand for 12 hours; standing for 12 hours, transferring the larvae and seawater into a container with the volume of 250 ml, adding 115 ml of filtered seawater, and continuously breeding for 3 days according to conventional breeding conditions; after continuously raising for 3 days, 100 larvae are collected and used for detecting and analyzing the gene expression quantity.
The test results are shown in FIG. 3. Through detection, Lox5 gene expression is reduced by 65% (FIG. 3).
Example 3
Sterilizing 500-mesh bolting silk, plastic containers with the volume of 20 ml and 100 ml and then drying; preparing Antp gene double-stranded RNA (the sequence is shown in SEQ ID No.3, the using concentration is 10 micromole per liter) and enough filtered seawater (the salinity is 30 per thousand, the pH value is 8.0, and the water temperature is 21 ℃); the ambient temperature is maintained at 20 ℃ and the relative humidity is greater than 50%.
Filtering 92% seawater from normally-raised urechis unicinctus larva (density: 4/mL) by using 500-mesh bolting silk to ensure that the larva density reaches 50/mL; then 10 ml of seawater (containing about 500 larvae) was taken in a container having a capacity of 20 ml, 0.05 ml of double-stranded RNA was added thereto, mixed (final concentration of double-stranded RNA: 0.05. mu. mol/l) and allowed to stand for 12 hours; standing for 12 hours, transferring the larvae and seawater into a container with the volume of 250 ml, adding 115 ml of filtered seawater, and continuously breeding for 3 days according to conventional breeding conditions; after continuously raising for 3 days, 100 larvae are collected and used for detecting and analyzing the gene expression quantity.
The test results are shown in FIG. 4. By detection, the Antp gene expression was reduced by 60% (FIG. 4).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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actacaccag tacctattta gctggtgaaa gcaataacaa cagtagtcca tatagcccta 240
tgcctgccca cacttccctg ggcagctcat atgaaagcta ccacgatcct gggtccattg 300
gaagtccgca tcatcacagt cgaggtggag cgtccgctgg ccacggactc ggaggaggct 360
atccaagctt cccgccttat gataggctcg aaagcatcca ccccatcgct tccgcaagcg 420
gatcttcctc atccggaagt agtcattcat attacaatag cattgggcat ggtcggta 478
Claims (2)
1. An RNA interference method of urechis unicinctus larvae is characterized by comprising the following steps:
(1) taking a culture water body in a normal breeding state of urechis unicinctus larvae, and filtering seawater in the culture water body by using bolting silk to obtain a concentrated water body with the density of the larvae of 25-60 per milliliter;
(2) mixing the concentrated water body with target gene double-stranded RNA to enable the final concentration of the target gene double-stranded RNA to be 0.05-0.1 micromole/liter, and standing for 10-15 hours to obtain an interference water body; the target gene is Lox5 and/or Antp;
(3) adding filtered seawater into the interference water body to the culture water body in the normal state in the step (1), and continuing to raise for more than or equal to 2 days;
the urechis unicinctus larvae are somniferous larvae.
2. Use of the RNA interference method of claim 1 for RNA interference in somites; the somnal larva is a somnal larva of an urechis animal; characterized in that when the period of continuing the breeding in the step (3) is more than 7 days, the operations of the step (1), the step (2) and the step (3) are repeated in sequence.
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