CN116769839A - RNA interference method for researching Abd-A gene regulation scylla paramamosain larva abdominal limb development - Google Patents
RNA interference method for researching Abd-A gene regulation scylla paramamosain larva abdominal limb development Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses an RNA interference method for researching Abd-A gene regulation scylla paramamosain larva abdominal limb development, which comprises the following steps: 1. specific primers for amplifying the Abd-A gene fragment and synthesizing dsRNA are designed according to the Abd-A gene sequence; 2. PCR amplification is carried out by taking plasmid with Abd-A gene fragment as a template, and dsRNA is synthesized by in vitro transcription; 3. placing the daphnia pustule in the scylla paramamosain IV on a special agarose gel plate, and introducing dsRNA from the gap between the head and the chest and the abdomen; 4. after the larvae grow to the V stage, the development change of the abdomen and limbs of the larvae is observed by adopting an electron microscope, and the invention relates to the technical field of aquatic biology. The RNA interference method for researching the Abd-A gene to regulate and control the development of the young abdominal limbs of scylla paramamosain is characterized in that after the expression of the Abd-A gene is interfered, the Abd-A gene is regulated and controlled to grow and develop the young abdominal limbs. The invention realizes the research of the functions of the Abd-A gene in the development of the scylla paramamosain larva abdominal limb, and is an effective means for exploring the functions of the genes related to the growth and development of the scylla paramamosain larva abdominal limb.
Description
Technical Field
The invention relates to the technical field of aquatic organisms, in particular to an RNA interference method for researching Abd-A gene regulation and control of scylla paramamosain larva abdominal limb development.
Background
Scylla paramamosain (scylaparamamosain) belongs to crustaceans (Crustaceae), decapoda, and Portulacaceae (Portulanidae), and is mainly distributed in the sea areas of the western Pacific ocean, southeast Asia, australia, indian ocean, and south Africa.
Early larval development of scylla paramamosain undergoes a growth phase from embryo hatching and rupture to daphnia larvae, to megalopum larvae, and to larval crabs, during which a series of limb development and specialization processes are accompanied. Among them, the growth of the abdominal limbs and the change of the morphology play an important role, and it is directly related to whether the larvae can smoothly complete the metamorphosis and enter the next growth stage. Therefore, the development stage of larvae becomes a critical part in the artificial cultivation process of scylla paramamosain. Analysis of fundamental theoretical problems of larval development and research on mechanisms of intrinsic driving factors are particularly necessary. The research on gene functions carried out in scylla paramamosain larvae at present is mainly limited to gene cloning and expression pattern analysis.
The daphnia scylla in the scylla paramamosain needs to undergo 4 molting times, the period is divided into 5 periods, the development time of each period of the daphnia scylla amosain is about 2-3 days, and the change of the belly development from the daphnia scylla amosain in the previous period to the next period can be smoothly observed in the period of the RNA interference, so that the function of the gene in the change of the form of the larva is revealed. But the scylla paramamosain larvae are small (2-3 mm), are not easy to observe by naked eyes, can not be subjected to RNA interference by adopting a conventional injection method, and can only be introduced into the larvae by adopting a microinjection mode. However, the daphnia larvae have cuticle shells, and capillary needles are not easy to penetrate, so that research on gene functions in scylla paramamosain larvae by using an RNA interference technology is hindered. Therefore, there is a need to develop a simple and efficient RNA interference technique for scylla paramamosain larvae.
The relevant literature reports: the Abd-A gene is an important member of the Hox gene family, is widely used in a plurality of species, is studied in insects at present, and is mainly involved in development processes such as generation of abdominal node of insects, differentiation of anterior and posterior axis of body node, development of gonad and fat body, differentiation of heart, formation of midgut and abdominal limb and the like. In crustaceans, a learner speculates that the Abd-A gene is responsible for establishing abdominal properties and controlling the morphological and functional differentiation of the appendages before and after the chest through the expression condition of Abd-A in the individual body segments of procambarus clarkia; in Litopenaeus vannamei, the Abd-A gene starts to be actively expressed in the daphnia larva stage, and by combining with the tissue and organ generation condition, the Abd-A is presumed to control the development and morphological characteristics of the thoracic and abdominal sections and the abdominal limbs of the Litopenaeus vannamei. What function the Abd-A gene plays in scylla paramamosain plays, whether it plays a key role in the development of the abdominal limb, is not reported at present.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides an RNA interference method for researching the Abd-A gene to regulate and control the development of the scylla paramamosain larvae limbs, and the function of the Abd-A gene in the development of the limbs is explored. The invention explores a method for injecting dsRNA into larvae from the back shell gap at the joint of the larva head and the chest and the abdomen of scylla paramamosain, and solves the technical problems that larvae are small in size and shells are not easy to puncture.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme: an RNA interference method for researching Abd-A gene regulation scylla paramamosain larva abdominal limb development takes scylla paramamosain larva as a research object and Abd-A as a target gene, which comprises the following steps:
s1, designing a specific primer for amplifying an Abd-A gene fragment and synthesizing dsRNA according to an Abd-A gene sequence, and adding a protective base and a T7 promoter sequence at the 5' end of a forward primer and a reverse primer for synthesizing the dsRNA;
s2, carrying out PCR amplification and product purification by taking a plasmid with an Abd-A gene sequence fragment as a template, and synthesizing dsRNA for gene interference by in vitro transcription by taking a PCR product (with a T7 promoter) as a template;
s3, placing the daphnia pustule in the scylla paramamosain IV stage on a special agarose gel plate, mixing dsRNA with an injection indicator, and injecting the mixture into the pustule by using a microinjection instrument, wherein the concentration of the injected dsRNA is 100 ng/. Mu.l, and the injection dosage is 1. Mu.g/g;
s4, observing the development change of the abdomen and limbs of the larvae by adopting an electron microscope after the larvae grow to the V-stage.
The plasmid with the Abd-A gene sequence fragment is used as a template for subsequent PCR reaction, the problem of mismatch possibly caused by high repetition rate of the scylla paramamosain genome sequence is solved, and the obtained PCR product has higher accuracy and meets the expected banding rate of 100 percent.
The total length of the Abd-A gene cDNA is 1,282bp, and the length for synthesizing dsRNA is 266bp.
Preferably, the construction of the plasmid with the target gene sequence fragment in the step S2 comprises the following steps:
t1, designing a primer clone Abd-A gene fragment by taking cDNA of a blue crab daphnia pseudomosla as a template, preparing a reaction system by adopting high-fidelity enzyme, designing an Open reading frame (Open ReadingFrame, ORF) of a target gene cloned by the primer, and adopting a high-fidelity enzyme premix, wherein the reaction system is as follows:
t2, adding the reaction system into a PCR tube, uniformly mixing and centrifuging, putting the mixture into a PCR instrument for amplification reaction, adding the system into a 200 mu L PCR tube, uniformly mixing and centrifuging, and putting the mixture into the PCR instrument, wherein the procedure is as follows:
after the reaction is finished, detecting a PCR product by using 1.5% agarose gel electrophoresis, performing gel cutting recovery on a strip meeting the expectations, purifying, and transferring into a pEASY-T1-simple carrier;
and T4, selecting a plasmid with a correct target fragment for extraction and purification to obtain the plasmid with a correct Abd-A gene sequence fragment.
Preferably, the PCR reaction system is 50. Mu.L, including dsAbd-A-F (10. Mu.M) 2.5. Mu.L, dsAbd-A-R (10. Mu.M) 2.5. Mu.L, 2 XHieffCanaceGoldPCRMasterMix 1.2. Mu.L, cDNA template (10 ng/. Mu.L) 2. Mu.L, and then ddH 2 O is added to 50 mu L;
the PCR reaction program used for PCR amplification is as follows: pre-denaturation at 98℃for 3min, denaturation at 98℃for 10s, extension at 68℃for 40s,35 cycles, extension at 72℃for 5min, and storage at 4 ℃.
Preferably, the dsRNA synthesis for gene interference comprises the steps of:
e1, adopting plasmid with Abd-A gene sequence as a template, and carrying out PCR reaction by using high-fidelity enzyme, wherein the system is as follows:
the reaction procedure was the same as described above.
After the reaction is finished, detecting the PCR product by using 1.5% agarose gel electrophoresis, and performing gel cutting recovery on the strip meeting the expectations and purifying the PCR product;
e3, in vitro transcription of dsRNA: the T7 in vitro transcription kit is adopted, and the system is as follows:
lightly mixing in a PCR tube, transferring to a PCR instrument, controlling the temperature at 42 ℃ for 5 hours, adding DNase, uniformly mixing, placing in the PCR instrument at 37 ℃ for 30 minutes, and removing unreacted DNA;
and E4, purifying dsRNA.
Preferably, the purification of the dsRNA comprises the steps of:
p1, adding 2.5 mu L of 4MLiCl and 75 mu L of absolute ethyl alcohol, uniformly mixing to obtain a mixed solution, and placing the mixed solution in an ultralow temperature refrigerator at-80 ℃ for 5 hours;
p2, pre-cooling to 4 ℃ by opening a refrigerated centrifuge in advance, putting the mixed solution into the centrifuge, centrifuging for 30min at 1,000rpm, and standing the dsRNA precipitation time for more than 5h or in an ultralow temperature refrigerator at-80 ℃ overnight;
p3, discarding supernatant, washing precipitate with 75% ethanol, centrifuging at 4deg.C, and repeating for several times;
p4, sucking out the ethanol by using a pipetting gun;
p5, placing the centrifuge tube on an ice box of an ultra-clean bench, and standing to volatilize ethanol;
p6, adding RNasefreewater into the centrifuge tube, and uniformly mixing;
p7, detecting the integrity of RNA through agarose gel, and simultaneously detecting the concentration and quality of the RNA by using a nucleic acid tester to obtain the dsRNA with qualified quality.
Preferably, the microinjection apparatus injection comprises the steps of:
w1, preparing an agarose gel table: preparing a 2% agarose gel plate by 1%DEPC water, and placing a groove plate on the gel;
w2, manufacturing a microinjection capillary needle: drawing a needle by using a microinjection capillary needle drawing instrument;
w3, fixing the blue crab daphnia pustule on an agarose gel table, and placing under an dissecting mirror;
w4, filling the mixed solution of the dsRNA for gene interference and the injection indicator into a capillary needle for injection;
and W5, transferring the daphnia scylla paramamosain larvae into the same cultivation barrel after the injection is finished, ensuring the consistency of the environment, and feeding at regular time.
Preferably, the agarose gel plate is prepared by using 1%DEPC water as sterilized seawater, the salinity is 30%, the salinity is consistent with the living salinity of larvae, and the diameter of a groove of the agarose gel plate is 2mm.
Preferably, the capillary needle diameter is 1mm, the needle opening for injection is about 0.2mm, and the microinjection dosage is 1 mug/g.
Preferably, the dsRNA is introduced into the daphnia pustule by microinjection at the interval between the daphnia pustule head pectoral girdle and the abdomen.
Preferably, it is used for exploring gene functions in scylla paramamosain larvae.
Preferably, the target gene is Abd-A, and the total sequence length is:
GGTCCACAGATGGCGATAGTGTCTTGAGTGTAGTCGCGCTCTCCCTCCCGGCCTTATCGCGCTGATAATGAAGGTCCGACCAAGGCGAGCGCTTATTGGTTCTCCGGGCCCCCTTACCTACTGATTGGGCAGACTTGTTTACCGCTCGGGGTTCTCGTGCCTGGTGTCTAATTCAGAAAAGGATTCATTTGCCATCACAGGTAGTCGAGGCTGGTGTGTACACAGTTGTTACTAAGTAGTTACCACTGGGAAGAATTATGTGAACAATGAGTTCAAATTATATTGATAGTATTCTGCCCAAGTACCAGGCGGAGTCGGCCGCGGCTAACCTGGTAAACTACAATACGCAAGCTAGAAGTATGTATCCGTACGTGAGTGTCACTTCACATCAGCTGTCCTCCACCCCCGCCACCAACATGTCGCCCTTCACTGCAATGACAGCCAACACAGACGGGGACAAGCAATGCCGCTACTCCCAGACTGGAGCCACGGATATGTCCCAATACGGCCTAAACCTGCAGAACTGTGCCACCACCAGCAACATGGCCCAGTACTTCCACCAGAACAACGTCACTAACCCACTGAACTCCTGTAGTCAGCCAACCCCGACGCCGCATATTCCTGACATCCCACGGTACCCATGGATGTCTATCACAGAAAACCAATGGCGGGGTCTGACAGCCAATTGGAACGGCCTGCCATGGAGTGAAAATCTGGTTCCAAAACCGGCGCATGAAGCTGAAGAAGGAGCTCCGAGCGGTGAAGGAAATCAGCGAACAGGTGCGACGGGAACGAGAAGAGCAGGATAAGCTCAAGCAGCAACAAGACGACAAAAAGGCAAACAAGGAACAACAGCCGTCAACAGCTAACGGCCAGCCTGCCTCAGCCACCAACGGCACCTCTTCATCGTCAGCGGGAGGTGGCACTGGGGACACCAAGGCTGCAACTTAACGCCAAGGTACTTGTTCCCCTCAAGGGCGTGCTGCTGCTGATACTGCTGCTGCATATGTAAGCAAATATCCTCACTTCTCGGCTACCAGAACCAATGATATGCACGCCCAACCAACCACTTTCACGTCCACACCTTCAGCAGAAGCTCCGCCCCCCGACCCCGACCCTCAGCCACGTCCTCACTCTGGAGGGCTCTCCGAGGAGACCTTGTTTACCCTACTTCAGCATCACTTCAATGCTTAAGACCAACCTACGAGTCTTACTGCTTTATGATTCTTAGTGATGGTGAAAATTTGATACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
preferably, the specific primer for dsRNA synthesis requires the addition of 6 protecting bases (GATCAC) and a T7 promoter sequence (TAATACGACTCACTTAGG) at the 5' end of the primer.
Preferably, the primer sequences for amplifying the Abd-A gene fragment are as follows:
dsAbd-A-F：5’CTGGTAAACTACAATACGCAAGCTA3’
dsAbd-A-R：5’CTACAGGAGTTCAGTGGGTTAGT3’。
the sequence of the synthesized gene fragment is as follows:
CTGGTAAACTACAATACGCAAGCTAGAAGTATGTATCCGTACGTGAGTGTCACTTCACATCAGCTGTCCTCCACCCCCGCCACCAACATGTCGCCCTTCACTGCAATGACAGCCAACACAGACGGGGACAAGCAATGCCGCTACTCCCAGACTGGAGCCACGGATATGTCCCAATACGGCCTAAACCTGCAGAACTGTGCCACCACCAGCAACATGGCCCAGTACTTCCACCAGAACAACGTCACTAACCCACTGAACTCCTGTAG。
preferably, the specific primer for synthesizing the dsRNA needs to add 6 protecting bases and a T7 promoter sequence at the 5' end of the primer, and the specific sequence is as follows:
dsAbd-A-T7F：
GATCACTAATACGACTCACTATAGGGCTGGTAAACTACAATACGCAAGCTA。
dsAbd-A-T7R：
GATCACTAATACGACTCACTATAGGGCTACAGGAGTTCAGTGGGTTAGT。
(III) beneficial effects
The invention provides an RNA interference method for researching Abd-A gene regulation and control of scylla paramamosain larva abdominal limb development. Compared with the prior art, the method has the following beneficial effects:
(1) The RNA interference method for researching Abd-A gene regulation scylla paramamosain larva abdominal limb development can successfully complete the expression interference of Abd-A gene and successfully observe the degeneration or the deletion of abdominal limb after the Abd-A gene interference by implementing RNA interference in the scylla paramamosain larva, thereby realizing the function of researching the morphological related gene and relieving the technical problem that the scylla paramamosain is difficult to implement gene editing.
(2) The RNA interference method for researching Abd-A gene regulation scylla paramamosain larva abdominal limb development is an effective means for exploring gene functions in the scylla paramamosain larva, because the development time of the daphnia larva in each period is about 2-3 days, the morphological change from the daphnia larva in the last period to the next period can be smoothly observed in the aging period of RNA interference by using the method of the invention.
Drawings
FIG. 1 is a schematic diagram of dsRNA introduction of scylla paramamosain larvae by microinjection technology;
FIG. 2 is a diagram showing the alignment of the sequences of the Abd-AdsRNA synthesized by the present invention with the gene sequences;
FIG. 3 is a dsRNA gel diagram of Abd-A and EGFP synthesized by the present invention;
FIG. 4 shows the detection result of the interference efficiency of the daphnia pseudovenosa stage IV Abd-ARNA;
FIG. 5 shows the morphological change result of the invention after the interference of the Abd-A gene in the daphnia pseudomosla larvae IV; wherein A-C: blank control group with normal abdominal limb; D-F: negative control group with normal abdominal limb; F-L: experimental groups, abdominal limb loss, circles are marked as abdominal limb loss positions of the experimental groups;
FIG. 6 shows the results of morphological changes after the interference of the Abd-A gene in the daphnia pseudomosla larvae V, specifically representing abdominal limb degeneration.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to fig. 1-5, embodiment 1 of the present invention provides a technical solution: an RNA interference method for researching Abd-A gene regulation scylla paramamosain larva abdominal limb development takes scylla paramamosain larva as a research object and Abd-A as a target gene, which comprises the following steps:
s1, designing a specific primer for amplifying an Abd-A gene fragment and synthesizing dsRNA according to an Abd-A gene sequence, and adding a protective base and a T7 promoter sequence at the 5' end of a forward primer and a reverse primer for synthesizing the dsRNA;
s2, carrying out PCR amplification and product purification by taking a plasmid with an Abd-A gene sequence fragment as a template, and synthesizing dsRNA for gene interference by in vitro transcription by taking a PCR product (with a T7 promoter) as a template;
s3, placing the daphnia pustule in the scylla paramamosain IV stage on a special agarose gel plate, mixing dsRNA with an injection indicator, and injecting the mixture into the pustule by using a microinjection instrument, wherein the concentration of the injected dsRNA is 100 ng/. Mu.l, and the injection dosage is 1. Mu.g/g;
s4, observing the development change of the abdomen and limbs of the larvae by adopting an electron microscope after the larvae grow to the V-stage.
In the embodiment of the invention, the construction of the plasmid with the target gene sequence fragment in the step S2 comprises the following steps:
t1, designing a primer clone Abd-A gene fragment by taking cDNA of the daphnia pseudocave larvae as a template, and preparing a reaction system by adopting high-fidelity enzyme;
t2, adding the reaction system into a PCR tube, uniformly mixing and centrifuging, and putting the mixture into a PCR instrument for amplification reaction;
after the reaction is finished, detecting a PCR product by using 1.5% agarose gel electrophoresis, performing gel cutting recovery on a strip meeting the expectations, purifying, and transferring into a pEASY-T1-simple carrier;
and T4, selecting a plasmid with a correct target fragment for extraction and purification to obtain the plasmid with a correct Abd-A gene sequence fragment.
In the present example, the PCR reaction system was 50. Mu.L, including dsAbd-A-F (10. Mu.M) 2.5. Mu.L, dsAbd-A-R (10. Mu.M) 2.5. Mu.L, 2 XHieffCanaceGoldPCRMasterMix 1.2. Mu.L, cDNA template (10 ng/. Mu.L) 2. Mu.L, and then ddH 2 O is added to 50 mu L;
the PCR reaction program used for PCR amplification is as follows: pre-denaturation at 98℃for 3min, denaturation at 98℃for 10s, extension at 68℃for 40s,35 cycles, extension at 72℃for 5min, and storage at 4 ℃.
In an embodiment of the invention, dsRNA synthesis for gene interference comprises the steps of:
e1, adopting plasmid with an Abd-A gene sequence as a template, and carrying out PCR reaction by using high-fidelity enzyme;
after the reaction is finished, detecting the PCR product by using 1.5% agarose gel electrophoresis, and performing gel cutting recovery on the strip meeting the expectations and purifying the PCR product;
e3, in vitro transcription of dsRNA: adopting a T7 in vitro transcription kit, lightly mixing in a PCR tube, transferring into a PCR instrument, controlling the temperature at 42 ℃ for 5 hours, adding DNase, uniformly mixing, placing in the PCR instrument at 37 ℃ for 30 minutes, and removing unreacted DNA;
and E4, purifying dsRNA.
In an embodiment of the invention, the purification of the dsRNA comprises the steps of:
p1, adding 2.5 mu L of 4MLiCl and 75 mu L of absolute ethyl alcohol, uniformly mixing to obtain a mixed solution, and placing the mixed solution in an ultralow temperature refrigerator at-80 ℃ for 5 hours;
p2, pre-cooling to 4 ℃ by opening a refrigerated centrifuge in advance, putting the mixed solution into the centrifuge, centrifuging for 30min at 1,000rpm, and standing the dsRNA precipitation time for more than 5h or in an ultralow temperature refrigerator at-80 ℃ overnight;
p3, discarding supernatant, washing precipitate with 75% ethanol, centrifuging at 4deg.C, and repeating for several times;
p4, sucking out the ethanol by using a pipetting gun;
p5, placing the centrifuge tube on an ice box of an ultra-clean bench, and standing to volatilize ethanol;
p6, adding RNasefreewater into the centrifuge tube, and uniformly mixing;
p7, detecting the integrity of RNA through agarose gel, and simultaneously detecting the concentration and quality of the RNA by using a nucleic acid tester to obtain the dsRNA with qualified quality.
In an embodiment of the present invention, microinjection apparatus injection comprises the steps of:
w1, preparing an agarose gel table: preparing a 2% agarose gel plate by 1%DEPC water, and placing a groove plate on the gel;
w2, manufacturing a microinjection capillary needle: drawing a needle by using a microinjection capillary needle drawing instrument;
w3, fixing the blue crab daphnia pustule on an agarose gel table, and placing under an dissecting mirror;
w4, filling the mixed solution of the dsRNA for gene interference and the injection indicator into a capillary needle for injection;
and W5, transferring the daphnia scylla paramamosain larvae into the same cultivation barrel after the injection is finished, ensuring the consistency of the environment, and feeding at regular time.
In the embodiment of the invention, 1 permillage DEPC water is used for preparing the agarose gel plate and is sterilized seawater, the salinity is 30 permillage, the salinity is consistent with the living salinity of larvae, and the diameter of a groove of the agarose gel plate is 2mm.
In the embodiment of the invention, the diameter of the capillary needle is 1mm, the needle opening for injection is about 0.2mm, and the microinjection dosage is 1 mug/g.
In the embodiment of the invention, the dsRNA is introduced into the daphnia larva body by microinjection at the interval between the daphnia larva head pectoral girdle and the abdomen.
The embodiment of the invention is used for exploring the gene function in scylla paramamosain larvae.
The specific implementation cases are as follows:
1. primer design
Primers were designed by PrimerPremier5.0 software, and synthesized by Shenzhen megagene Co., ltd. And PAGEplus mode was used. Primer sequence information is as follows:
dsAbd-A-T7F：
GATCACTAATACGACTCACTATAGGGCTGGTAAACTACAATACGCAAGCTA
dsAbd-A-T7R：
GATCACTAATACGACTCACTATAGGGCTACAGGAGTTCAGTGGGTTAGT
2. template preparation
1) The cDNA of the daphnia scylla in stage V is used as a template, an open reading frame of a primer clone Abd-A gene is designed, a high-fidelity enzyme premix of the next holothurian is adopted, and a reaction system is as follows:
the above system was added to a 200. Mu.L PCR tube, mixed well and centrifuged, and placed in a PCR instrument, the procedure was as follows:
after the reaction is finished, agarose gel (1.5%) electrophoresis is used for detecting the PCR product, the expected strip is cut and recovered, and the full-scale gold gel recovery kit is used, wherein the specific steps are as follows:
(1) the target strips in the agarose gel were cut, placed in a clean centrifuge tube, and weighed, 100mg was taken as 100 μl;
(2) adding gel with three times of volume of GSB solution, dissolving in water bath at 55deg.C for 10min, mixing every 3min to completely melt gel block (1 time of volume of isopropanol can be added into melted gel solution to increase DNA recovery rate);
(3) cooling the gel solution to room temperature, adding into a centrifugal column, standing at room temperature for 1min, centrifuging at 1,000Xg for 1min, and discarding effluent;
(4) 650 μLWB solution was added and centrifuged at 1,000Xg for 1min, and the effluent was discarded;
(5) centrifuging for 2min at 1,000Xg to remove the residual WB solution;
(6) placing the centrifugal column in a clean centrifuge tube, uncovering and standing for 1min, volatilizing residual ethanol, adding 40 μLEB (water bath at 70deg.C in advance) in the middle of the column, and standing for 1min at room temperature;
(7) 1,000Xg centrifugal 1min, eluting is purified DNA (repeated column loading can improve recovery rate), preserving at-20 ℃.
2) Tail adding: the DNA purified in 1) was subjected to tailing reaction using the reagent of the whole gold biology Co., ltd, the system being as follows:
the PCR tube was placed on a PCR instrument at 72℃for 10-15min.
3) Fragment of interest ligation
Ligating the above tailing product with pEASY-T1
Mixing the above materials in 200 μLPCR tube, and storing at 25deg.C for 7min and 4deg.C.
4) Transformation, screening and detection of fragments of interest
Transferring into Trans-T1 competent cells, lightly blowing and mixing, and incubating on ice for 25min; heat shock is carried out for 30s in a water bath kettle at the temperature of 42 ℃ and the incubation is carried out for 2min on ice; adding 300 mu L of LB culture medium at room temperature, and culturing at 200rpm and 37 ℃ for 2h; 200. Mu.L of the bacterial liquid was spread on a plate uniformly, and cultured overnight in an incubator at 37℃for 1min by centrifugation at 1,500 Xg to obtain more clones, and part of the supernatant was discarded to leave 100. Mu.L of the supernatant and the whole bacterial liquid was spread on the plate.
5) Positive clone detection
The PCR method identifies positive clones:
(1) white monoclonal was selected to 1mLLB/Amp + Liquid culture medium, 200rpm, 37 ℃ culture for 6 hours;
(2) 0.6 mu L of bacterial liquid is taken in a 12.5 mu LPCR system, and positive clones are identified by using M13Forward Primer and M13Reverse Primer;
(3) and (3) performing PCR amplification, wherein if the carrier is self-linked, the self-linked length is 199bp, and the bacterial liquid with single band and the same band size as the target band is reserved as the detection result. About 200 mu L of the strain is put into a centrifuge tube for sealing and is sent to Hua big gene biological company for sequencing, and the rest bacterial liquid is added with glycerol for uniform mixing and then frozen for storage.
6) Plasmid extraction
Performing blast comparison on the sequencing result and the original sequence, taking out bacterial liquid for comparing the correct sequence, performing expansion culture, and extracting plasmids by using a plasmid miniprep kit of the full gold biotechnology company, wherein the steps are as follows:
(1) taking bacterial liquid cultured for 14 hours, centrifuging for 1min at 10,000Xg, and discarding the supernatant;
(2) according to the above table, colorless solution RB (containing RNaseA) was added and the suspended bacteria were shaken to precipitate;
(3) according to the table, adding LB blue solution, gently turning up and down, mixing for 5 times, ensuring that the thalli are fully cracked, forming blue transparent solution, changing the color from semi-transparent blue to transparent, indicating complete cracking;
(4) according to the above table, adding NB yellow solution, gently turning up and down for 5-6 times (the color changes from blue to yellow, indicating uniform mixing, and complete neutralization) until compact yellow coagulated mass is formed, and standing at room temperature for 2min;
(5) centrifuging at 12,000Xg for 5min, carefully sucking the supernatant into a centrifuge column;
(6) centrifuging at 12,000Xg for 1min, discarding effluent, and adding the supernatant with volume greater than 800 μl into column for multiple times under the same conditions;
(7) adding 650 μl of solution WB, centrifuging at 12,000Xg for 1min, and discarding the effluent;
(8) centrifuging for 2min at 12,000Xg to remove the residual WB solution;
(9) placing the centrifugal column in a new centrifuge tube, adding 40 mu L of solution EB (heated in water bath at 65 ℃ in advance) in the center of the column, and standing at room temperature for 1min;
the DNA was eluted by centrifugation at 10,000Xg for 1min, and the eluted DNA was stored at-20 ℃.
3. In vitro transcription template preparation
1) Designing a dsRNA primer sequence with a T7 promoter sequence and a dsRNA primer sequence with a reverse T7 promoter sequence of the forward T7 promoter sequence of the Abd-A by using the full-length cDNA sequence of the Abd-A gene as a template, adopting the plasmid extracted in the step 2 as a PCR template, and also using the next holy high-fidelity enzyme, wherein the system is as follows:
the reaction procedure was as above.
2) After the reaction, the PCR products were detected by agarose gel (1.5%) electrophoresis, and the expected bands were cut and recovered, using the kit for the next holy gel recovery, on an ultra clean bench, the specific steps were as follows:
(1) weighing the gel;
(2) 150 mu L of sol BD is added to 100mg of agarose gel;
(3) the water bath of the water bath kettle is carried out for 10min at the temperature of 55 ℃, and the mixture is gently mixed every 3min until the glue block is completely melted, and 0.3 times of volume of isopropanol is added, so that the recovery rate can be improved;
(4) cooling the gel solution to room temperature, adding into DNA adsorption column, standing at room temperature for 1min, centrifuging at 12,000Xg for 1min, and discarding the effluent;
(5) 700. Mu.L of rinse solution (note the addition of alcohol) was added and centrifuged at 12,000Xg for 1min, and the effluent was discarded;
(6) 500. Mu.L of rinse solution (note the addition of alcohol) was added and centrifuged at 12,000Xg for 1min, and the effluent was discarded;
(7) placing the adsorption column back into a collecting pipe, centrifuging for 2min at 12,000Xg, air drying for 5min at room temperature, and removing residual alcohol;
(8) placing the adsorption column into a clean centrifuge tube, adding 40 mu LRNase-Freewater (water bath of 70 ℃ in advance) into the center of the column, standing for 2min at room temperature, centrifuging for 1min at 12,000Xg, and eluting DNA;
(9) the concentration was measured using a nucleic acid detector and stored at-20℃and the sequencing result of the obtained PCR product was shown in FIG. 2.
(3) In vitro transcription reaction
1) Here, T7 in vitro transcription kit from Takara was used, and the system was as follows:
2) Lightly mixing in 200 mu LPCR tube, transferring into PCR instrument, and controlling temperature at 42 deg.C for 5 hr;
3) Nuclease treatment: adding 2 mu LDNase, mixing well, and placing in a PCR instrument at 37 ℃ for 30min;
4) Adding 2.5 μl of 4MLiCl and 75 μl of absolute ethanol (pre-cooled at-20deg.C), mixing, and standing in a-80deg.C ultra-low temperature refrigerator for 5 hr;
5) The refrigerated centrifuge is started in advance, and the refrigerated centrifuge is centrifuged at 1,000rpm for 30min at 4 ℃;
6) Discarding supernatant, washing the precipitate with 75% ethanol (pre-cooling in advance), centrifuging at 4deg.C and 1,000rpm for 5min, and repeating for one time;
7) Centrifuging the sample at 1,000rpm and 4 ℃ for 1min, and sucking out ethanol by using a pipette;
8) Placing the centrifuge tube on an ice box of an ultra-clean bench, standing for 5min, and volatilizing ethanol;
9) Adding 50 mu LRNasefreesater into each centrifuge tube, and uniformly mixing;
10 RNA integrity was checked by 1.5% agarose gel, as shown in FIG. 3, lanes labeled 1 are Abd-AdsRNA, lanes labeled 2 are EGFPdsRNA, concentration and quality of RNA were checked by nucleic acid meter, and qualified dsRNA was selected for further experiments. OD260/280 is 1.8-2.0, and the qualification rate is more than 90%.
4. Microinjection of daphnia scylla stage IV individuals
(1) Agarose gel station was prepared: preparing 2% agarose gel plates by 1%DEPC water, placing groove plates on the gel, and conveniently fixing blue crab larvae;
(2) Manufacturing a microinjected capillary needle: drawing a needle by using a microinjection capillary needle drawing instrument;
(3) The experimental object is a daphnia scylla-like larva IV individual, the larva is fixed on an agarose gel table, the redundant water is wiped off, and the larva is placed under an anatomical lens;
(4) dsRNA is filled into a capillary needle for injection;
(5) The injection position is the gap between the back shells of the larvae, and 0.1% phenol red solution is used as an indicator;
(6) A blank group, a dsEGFP negative control group and a dsAbd-A experimental group are arranged, and each group comprises 400 groups;
(7) After injection is finished, transferring larvae into the same cultivation barrel, ensuring the consistency of the environment, and feeding at regular time;
(8) Samples were taken at 6h, 12h, 24h, 48h, 72h, and RNA extraction and electron microscopy were performed.
The interference efficiency detection result is shown in fig. 4, and the overall interference efficiency can reach 50%. The morphological observation results are shown in fig. 5, and after the expression of the Abd-A gene is interfered in the daphnia pseudocave larva stage IV, the change of the larva development to the abdomen morphology in stage V can be obtained, so that the Abd-A gene plays an important role in the abdomen development.
Example 2
By applying the technology of the invention, RNA interference of the Abd-A gene is also implemented on the daphnia pseudomosla larva V-stage individual, and the microinjection steps are as follows:
(1) Agarose gel station was prepared: preparing a 2% agarose gel plate by 1%DEPC water, and placing a groove plate on the gel;
(2) Manufacturing a microinjected capillary needle: drawing a needle by using a microinjection capillary needle drawing instrument;
(3) The experimental object is a young plant of the blue crab in the daphnia pseudocave stage V, the young plant is fixed on an agarose gel table, the redundant water is wiped off, and the young plant is placed under an dissecting mirror;
(4) dsRNA is filled into a capillary needle for injection;
(5) The injection position is the gap between the back shells of the larvae, and 0.1% phenol red solution is used as an indicator;
(6) Setting a dsEGFP negative control group and a dsAbd-A experimental group, wherein each group comprises 300 dsEGFP negative control groups and dsAbd-A experimental groups;
(7) After injection is finished, transferring larvae into the same cultivation barrel, ensuring the consistency of the environment, and feeding at regular time;
(8) Samples were taken at 6h, 12h, 24h, 48h, 72h, and RNA extraction and electron microscopy were performed.
The morphological observation results are shown in fig. 6, after the expression of the Abd-A gene is interfered in the daphnia pseudomosla stage V, the abdomens of the larvae develop for 72 hours, and then the abdomens of the larvae shrink and degenerate to a certain extent, so that the Abd-A gene can play an important role in the development of the abdomens of the abdomen.
And all that is not described in detail in this specification is well known to those skilled in the art.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. An RNA interference method for researching the development of the abdomen and limbs of scylla paramamosain larvae regulated by the Abd-A gene is characterized by comprising the following steps: the scylla paramamosain larva is taken as a study object, and the Abd-A is taken as a target gene, and the method specifically comprises the following steps:
s1, designing a specific primer for amplifying an Abd-A gene fragment and synthesizing dsRNA according to an Abd-A gene sequence, and adding a protective base and a T7 promoter sequence at the 5' end of a forward primer and a reverse primer for synthesizing the dsRNA;
s2, carrying out PCR amplification and product purification by taking a plasmid with an Abd-A gene sequence fragment as a template, and synthesizing dsRNA for gene interference by in vitro transcription by taking a PCR product (with a T7 promoter) as a template;
s3, placing the daphnia pustule in the scylla paramamosain IV stage on a special agarose gel plate, mixing dsRNA with an injection indicator, and injecting the mixture into the pustule by using a microinjection instrument, wherein the concentration of the injected dsRNA is 100 ng/. Mu.l, and the injection dosage is 1. Mu.g/g;
s4, observing the development change of the abdomen and limbs of the larvae by adopting an electron microscope after the larvae grow to the V-stage.
2. The method for researching the RNA interference of the Abd-A gene for regulating and controlling the development of the green scylla paramamosain larva abdominal limb according to claim 1, which is characterized in that: the construction of the plasmid with the target gene sequence fragment in the step S2 comprises the following steps:
t1, designing a primer clone Abd-A gene fragment by taking cDNA of the daphnia pseudocave larvae as a template, and preparing a reaction system by adopting high-fidelity enzyme;
t2, adding the reaction system into a PCR tube, uniformly mixing and centrifuging, and putting the mixture into a PCR instrument for amplification reaction;
after the reaction is finished, detecting a PCR product by using 1.5% agarose gel electrophoresis, performing gel cutting recovery on a strip meeting the expectations, purifying, and transferring into a pEASY-T1-simple carrier;
and T4, selecting a plasmid with a correct target fragment for extraction and purification to obtain the plasmid with a correct Abd-A gene sequence fragment.
3. The RNA interference method for studying Abd-a gene-regulated scylla paramamosain larva abdominal limb development according to claim 1 or 2, wherein: the PCR reaction system was 50. Mu.L, including dsAbd-A-F2.5. Mu.L, dsAbd-A-R2.5. Mu.L, 2 XHieffCanaceGoldPCRMaster Mix 1.2. Mu.L, cDNA template 2. Mu.L, and then ddH 2 O is added to 50 mu L;
the PCR reaction program used for PCR amplification is as follows: pre-denaturation at 98℃for 3min, denaturation at 98℃for 10s, extension at 68℃for 40s,35 cycles, extension at 72℃for 5min, and storage at 4 ℃.
4. The RNA interference method for studying Abd-a gene-regulated scylla paramamosain larva abdominal limb development according to claim 1 or 2, wherein: the synthesis of dsRNA for gene interference comprises the following steps:
e1, adopting plasmid with an Abd-A gene sequence as a template, and carrying out PCR reaction by using high-fidelity enzyme;
after the reaction is finished, detecting the PCR product by using 1.5% agarose gel electrophoresis, and performing gel cutting recovery on the strip meeting the expectations and purifying the PCR product;
e3, in vitro transcription of dsRNA: adopting a T7 in vitro transcription kit, lightly mixing in a PCR tube, transferring into a PCR instrument, controlling the temperature at 42 ℃ for 5 hours, adding DNase, uniformly mixing, placing in the PCR instrument at 37 ℃ for 30 minutes, and removing unreacted DNA;
and E4, purifying dsRNA.
5. The method for researching the RNA interference of the Abd-A gene for regulating and controlling the development of the green scylla paramamosain larva abdominal limb according to claim 1, which is characterized in that: the purification of the dsRNA comprises the following steps:
p1, adding 2.5 mu L of 4MLiCl and 75 mu L of absolute ethyl alcohol, uniformly mixing to obtain a mixed solution, and placing the mixed solution in an ultralow temperature refrigerator at-80 ℃ for 5 hours;
p2, pre-cooling to 4 ℃ by opening a refrigerated centrifuge in advance, putting the mixed solution into the centrifuge, centrifuging at 1,000rpm for 30min, and standing the dsRNA precipitation time for more than 5h or in an ultralow temperature refrigerator at-80 ℃ overnight;
p3, discarding supernatant, washing precipitate with 75% ethanol, centrifuging at 4deg.C, and repeating for several times;
p4, sucking out the ethanol by using a pipetting gun;
p5, placing the centrifuge tube on an ice box of an ultra-clean bench, and standing to volatilize ethanol;
p6, adding RNasefreewater into the centrifuge tube, and uniformly mixing;
p7, detecting the integrity of RNA through agarose gel, and simultaneously detecting the concentration and quality of the RNA by using a nucleic acid tester to obtain the dsRNA with qualified quality.
6. The method for researching the RNA interference of the Abd-A gene for regulating and controlling the development of the green scylla paramamosain larva abdominal limb according to claim 1, which is characterized in that: the microinjection instrument injection comprises the following steps:
w1, preparing an agarose gel table: preparing a 2% agarose gel plate by 1%DEPC water, and placing a groove plate on the gel;
w2, manufacturing a microinjection capillary needle: drawing a needle by using a microinjection capillary needle drawing instrument;
w3, fixing the blue crab daphnia pustule on an agarose gel table, and placing under an dissecting mirror;
w4, filling the mixed solution of the dsRNA for gene interference and the injection indicator into a capillary needle for injection;
and W5, transferring the daphnia scylla paramamosain larvae into the same cultivation barrel after the injection is finished, ensuring the consistency of the environment, and feeding at regular time.
7. The method for researching the RNA interference of the Abd-A gene for regulating and controlling the development of the green scylla paramamosain larva abdominal limb according to claim 1, which is characterized in that: the agarose gel plate is prepared by using 1%DEPC water as sterilized seawater, the salinity is 30%and is consistent with the living salinity of larvae, and the diameter of a groove of the agarose gel plate is 2mm.
8. The method for researching the RNA interference of the Abd-A gene for regulating and controlling the development of the green scylla paramamosain larva abdominal limb according to claim 6, which is characterized in that: the capillary needle diameter is 1mm, the needle opening for injection is about 0.2mm, and the microinjection dosage is 1 mug/g.
9. The method for researching the RNA interference of the Abd-A gene for regulating and controlling the development of the green scylla paramamosain larva abdominal limb according to claim 1 or 6, which is characterized in that: the dsRNA is introduced into the daphnia larva body by microinjection from the gap between the daphnia larva head and the chest and abdomen.
10. The method for researching the RNA interference of the Abd-A gene for regulating and controlling the development of the green scylla paramamosain larva abdominal limb according to claim 1, which is characterized in that: is used for exploring the gene function of scylla paramamosain larvae.
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