CN111387105B - Method for producing seeds of all-male macrobrachium rosenbergii - Google Patents
Method for producing seeds of all-male macrobrachium rosenbergii Download PDFInfo
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- CN111387105B CN111387105B CN202010179093.4A CN202010179093A CN111387105B CN 111387105 B CN111387105 B CN 111387105B CN 202010179093 A CN202010179093 A CN 202010179093A CN 111387105 B CN111387105 B CN 111387105B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/59—Culture of aquatic animals of shellfish of crustaceans, e.g. lobsters or shrimps
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C—CHEMISTRY; METALLURGY
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a method for producing seeds of full-male macrobrachium rosenbergii, which comprises the following steps: step one, designing and synthesizing siRNA targeting DMRT 1; transfecting the obtained siRNA prawn seedlings, extracting RNA of the interfered prawn seedlings, performing reverse transcription to prepare cDNA, and detecting the expression of DMRT1 and IAG genes after RNA interference by using real-time fluorescent quantitative PCR; step three, detecting and screening the false female parent; step four, cultivating and verifying all-male macrobrachium rosenbergii; step five, passage of the female parent 'false female' of the all-male macrobrachium rosenbergii; the method can solve the problems of low reversion rate of the all-male macrobrachium rosenbergii and high death rate in the interference process; the offspring of the shrimp has high male ratio and good culture effect.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for producing seeds of all-male macrobrachium rosenbergii.
Background
The macrobrachium rosenbergii has obvious male and female difference, the male shrimp grows fast, the individual is large, and the growth advantage is realized. In the market, prawns are popular in many places. Therefore, the all-male macrobrachium rosenbergii culture has wide market prospect. Although Israel has begun to use RNAi technology to interfere with IAG genes to produce "pseudofemale" Macrobrachium rosenbergii and the full male strains produced thereby. But the import cost is high, the intermediate process is long, the death rate is high, and the popularization of the Chinese market is limited; in the domestic existing gene seed production method, fertilized eggs are injected microscopically or the muscle of the young shrimps in the early stage is adopted, so that the death rate is high, the efficiency of preparing the false female shrimps is low, and the large-scale production is not facilitated; the method for removing the androgenic gland is complex to operate, low in reversing efficiency and high in mortality rate; the market needs a method for producing full male macrobrachium rosenbergii seeds which can meet the demand of full male macrobrachium rosenbergii seedlings in production.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a method for producing seeds of full-male macrobrachium rosenbergii, which can solve the problems of low reversion rate and high death rate in the interference process of the full-male macrobrachium rosenbergii; the offspring of the shrimp has high male ratio and good culture effect.
In order to achieve the above object, the present invention adopts the following technical solutions:
a method for producing seeds of full-male macrobrachium rosenbergii comprises the following steps:
step one, designing and synthesizing siRNA targeting DMRT 1;
cloning and verifying the sequence of Macrobrachium rosenbergii DMRT1 gene sequence disclosed by NCBI, and preparing siRNA and a negative control;
step two, RNA interference;
transfecting the obtained siRNA prawn fry, extracting RNA of the interfered prawn fry, performing reverse transcription to prepare cDNA, and detecting the expression of DMRT1 and IAG genes after RNA interference by utilizing real-time fluorescent quantitative PCR;
step three, detecting and screening the false female parent;
feeding the RNA-interfered macrobrachium rosenbergii in an indoor cement pond, capturing when the body length reaches 4 +/-0.5 cm, and identifying the sex conditions of male and female by using apparent characteristics and sex-specific molecular markers;
step four, cultivating and verifying all-male macrobrachium rosenbergii;
selecting the male and female ZZ parent shrimps which are verified by molecular markers to continue to be cultured, and mating and fertilizing the male and female shrimps after the ovaries are mature;
establishing families according to each female shrimp, and culturing the obtained juvenile shrimps of each family in separate ponds, and identifying the sex conditions of the female and male shrimps when the body length is 4 +/-0.5 cm; reserving all offspring as female parent of male shrimp for subsequent cultivation of all-male shrimp fries;
step five, passage of the female parent 'false female' of the all-male macrobrachium rosenbergii;
and (3) after the verified male and female parent shrimps are reserved, mating and fertilizing with male and female shrimps after the ovary is mature. After hatching, raising and desalting the fertilized eggs, carrying out RNA interference on the all-male shrimp fries; breeding the shrimp fries after the interference until the shrimp fries are sex differentiated, and screening the pseudo-female shrimps of which the phenotypes are female shrimps for subsequent continuous passage; and controlling the passage process.
The method for producing the seeds of the all-male macrobrachium rosenbergii,
step one, designing and synthesizing siRNA targeting DMRT1,
designing and synthesizing siRNA according to a Macrobrachium rosenbergii DMRT1 gene sequence published by NCBI; designing alternative siRNA sequences, screening the sequences from the candidate siRNAs, and synthesizing ten siRNAs and a negative control GFP in total; the ten sirnas included: siRNA-318, siRNA-489, siRNA-857, siRNA-914, siRNA-1013, siRNA-1179, siRNA-1245, siRNA-1489, siRNA-1510, siRNA-1535:
the negative control GFP sequence was GFP control.
In the method for producing the seeds of the all-male macrobrachium rosenbergii, dTdT is used for replacing two base overhangs at the 3' end of an siRNA sequence.
In the method for producing the seeds of the all-male macrobrachium rosenbergii, the sense strand or the antisense strand of the double-stranded siRNA is chemically modified by adopting the cholesterol locked nucleic acid dinitrophenol chemical group.
The method for producing the seeds of the all-male macrobrachium rosenbergii comprises the following specific steps of transfecting the shrimp fries with the obtained siRNA: the obtained siRNA nucleic acid and the in vivo transfection reagent are prepared into a transfection reagent compound, and the transfection reagent compound is injected into the shrimp larvae muscle with the body length of 2.0-2.5 cm.
In the method for producing the seeds of the all-male macrobrachium rosenbergii, the transfection reagent in vivo is EntransTM-in vivo; nucleic acid (. mu.g) of siRNA and EntransterTMIn vivo (μ l) configured as transfection reagent complexes according to 1: 2.
The method for producing the seeds of the all-male macrobrachium rosenbergii,
step three, detecting and screening the false female parent;
feeding the Macrobrachium rosenbergii subjected to RNA interference in an indoor cement pond, capturing when the body length reaches 4 +/-0.5 cm, and identifying the sex condition of male and female;
the method for identifying sex comprises the following steps:
male reproductive orifices of the macrobrachium rosenbergii are used as sex identification indexes of males and females, male shrimps are selected at the basal part of the fifth step with the reproductive orifices, and female shrimps are selected at the basal part of the fifth step without the reproductive orifices;
carrying out sex verification on a genome of female individuals with identified phenotypic sex in a population by using sex specific molecular markers of the macrobrachium rosenbergii; the method comprises the following steps: detecting a section of gene of male and female sex specific molecules in genome DNA of a muscle sample of the obtained macrobrachium rosenbergii; the presence of a female specific molecular fragment indicates that the individual is a female shrimp and the absence of a specific molecular fragment indicates that the individual is a genetically male shrimp.
The invention has the advantages that:
the invention designs and synthesizes siRNA according to the gene sequence of the Macrobrachium rosenbergii DMRT1, and the siRNA is introduced into the Macrobrachium rosenbergii body in the sex differentiation period to interfere the expression of the DMRT1 gene in the Macrobrachium rosenbergii body, reduce or silence the expression level of the DMRT1 protein in the Macrobrachium rosenbergii body, and obviously reduce the expression level of IAG gene; verifying through sex-specific molecular markers and family directional mating in the later period; the invention can realize the sex reversal of male shrimps to female shrimps;
compared with the method for removing the androgenic gland by an operation, the method effectively reduces the infection mortality rate and improves the success rate of sexual reversion; after breeding for 3-4 generations of passages, the reversion efficiency reaches about 30 percent;
the male proportion of the shrimps is high, and the male proportion of the offspring is 100%;
the full-male macrobrachium rosenbergii has fast growth speed, early marketing and high yield.
Drawings
FIG. 1 is a flow chart of the first generation of pseudo female shrimp production of the present invention;
FIG. 2 is a graph for detecting the interference efficiency of siRNA provided in the present invention; a graph of the expression levels of the DMRT1 gene (FIG. 2A) and IAG gene (FIG. 2B) in Macrobrachium rosenbergii relative to a control group;
fig. 3 is a molecular detection chart of sex of macrobrachium rosenbergii.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Macrobrachium rosenbergii sex control gene: macrobrachium rosenbergii DMRT1 mRNA
Amino acid sequence:
"MECGGYESEGKGKRKQHCTYCKNHGKKSRKTNHKCEHEECECLLCKLTRLSRLIMRHQQRLWRHLKDSKRRQNAAADAGGTAAGSGACEPSGEHTDSSGAAQGASSTKLQKCDMCRNHGEIMSKRAHKNACPYQDCPCELCSLTRKRRYIMXLQQRVRRSQVXSKQHNEVWEYVTQATAELEFLGLQDIHQTTEDHNASSSLSESSSSPSPDSSSTSGSLPHKPEVIXPNPVTPTXPAXTNRTRSKDTESSPVPMDMPPLEPLPECSPTRYSPRPPAPPFNVPSPAATSRIRTSYPSPPPPNPLPPNRGLTNVPPMEIEPAWMNLKREREEVLKMNPKVQTREENLFGHLDFMKDQLTMTGRLEPPVPPEGSRSFHNTAVAMNFGSSHVPPYKSLVPMIDTNHLPPPPLQKSRADCNLHFQYRSMVWGNVPTEPSVGFVPDDFQQQCYRSLLVNNADVRVQPPPGLIQIPRPQHPLRPRPLAPVRPHVLPVNVECDHVNLHYLAYFLQNQREHYRDTLSTLSTTRSFGLPREALFLHHPVP"
the nucleotide sequence is as follows:
cactgctacactgcgtctgcgcaaacgacctcgagatggagtgcgggggctacgagagcgagggcaagggcaagaggaagcagcactgcacctactgcaagaaccacgggaagaagagtcgcaagaccaaccacaagtgcgagcacgaggagtgcgagtgcctcctctgcaagctgaccagactctccaggctgatcatgaggcaccagcagaggctctggaggcaccttaaggactcgaagaggaggcagaacgccgctgcagacgcaggaggaacagcagcaggaagtggagcatgtgagccctcaggtgaacatactgactccagtggggctgcacaaggagccagtagcaccaaactgcagaaatgcgacatgtgccggaaccacggtgaaatcatgagcaagagagcgcacaagaacgcgtgcccttaccaagactgtccctgcgaactctgcagtctgacaagaaagcgccgctacatcatgrgactccagcagagagtcagaaggtctcaagtgrcgagcaaacagcacaacgaagtgtgggaatacgtgacccaagctacagctgagctggaattcctcgggcttcaggacatccaccagacgacagaggaccacaatgcatcttcctcattgtcagagtcgtcctcctcgccctctccagactcatcctccacctctgggtccctcccccacaaaccggaggtcatcycacccaatccagtcacycctacagyaccagccgakaccaatcgcacccgcagtaaggatacagaatcctctccagtccccatggatatgccccctctggaaccgcttccagaatgctctccaacaaggtacagtccaagaccgccagcacctccattcaatgtaccttcacctgctgcaacttctagaattagaacaagctacccttcaccccctcctccaaatccattgcctccaaataggggtttaacaaacgttccaccaatggaaatagaaccagcctggatgaacctcaaaagagaaagggaagaggttttgaaaatgaaccccaaagttcaaacaagagaagaaaatttattcggccacttggacttcatgaaagatcaactgaccatgacaggaaggttagagccaccagttccaccagagggcagcagaagttttcataacaccgccgttgcaatgaactttggtagctctcacgttccaccatacaagagtttagtgcccatgatagacacaaaccaccttccaccacctccactacagaaaagcagagcagattgcaatctacactttcagtacagatccatggtttggggtaacgtgccaactgagcccagtgtcggcttcgtcccagacgacttccagcaacagtgctatcgcagcttactcgtaaacaacgcagacgtaagggttcagcctccgccaggacttattcaaatcccacggcctcaacaccctctgcgcccacgtcctctcgcgcctgtcaggcctcacgtcctccctgtcaacgtggagtgtgatcacgttaacctgcattacctagcctatttcttacagaatcaaagagaacattaccgagataccttatctacgttgtccacaactaggtcctttggacttcccagagaggctctattccttcatcacccagtgccctaatctataatctataaacaaatcctactgagaacttcagtatcattattattatctttcaagtgatatcca；
a method for producing seeds of full-male macrobrachium rosenbergii,
establishing a pure-line macrobrachium rosenbergii pseudo female germplasm library, carrying out continuous five-generation population breeding on wild original macrobrachium rosenbergii, and establishing the pure-line macrobrachium rosenbergii pseudo female germplasm library on the basis of stable growth traits; the seed production flow chart of the 'false female' shrimp is shown in figure 1.
The method specifically comprises the following steps:
step one, siRNA design and synthesis targeting DMRT1
Designing and synthesizing siRNA according to a Macrobrachium rosenbergii DMRT1 gene sequence published by NCBI; on-line siRNA design software (http:// sidirect2.rnai. jp /) designs alternative siRNA sequences, selects sequences from the candidate siRNA, entrusts gene company to synthesize ten siRNA and one negative control GFP (see Table 1); in order to enhance the sequence stability, two base overhangs at the 3' end are replaced by dTdT; meanwhile, chemical groups such as cholesterol locked nucleic acid dinitrophenol and the like are adopted to chemically modify the sense or antisense strand of the double-stranded siRNA, so that the stability of the siRNA in the living body of a tested animal is enhanced. Powdery siDMRT1 and siGFP in centrifuge tubes were treated with RNase Free ddH according to the instructions2The stock solution with the dissolved O of 20 mu mol/L is stored in a refrigerator at the temperature of 20 ℃ below zero. The sequences are shown in Table 1;
TABLE 1
Step two, RNA interference;
transfecting the obtained siRNA prawn seedlings, extracting transfected RNA to detect the expression of DMRT1 after RNA interference, and continuously monitoring the sex state of the juvenile prawns;
nucleic acid (. mu.g) of the obtained siRNA and EntransterTMIn vivo (Engrenenbiosystem Co, Lid.) (μ l) was prepared as a transfection reagent complex at a ratio of 1:2, and siRNA and transfection reagent complex was injected into shrimp larvae muscle of 2.0-2.5cm (Mylabris chinensis, a kind of 5 generations of Mycoplasma onto which breeding was conducted) in accordance with a nucleic acid dosage of 0.8 μ g/g body weight, and a siGFP control group and a siDMRT1 interference group were set, respectively. It should be noted that the selection of transfection reagents is preferred, not exhaustive, and is suitable for use in the present invention as long as they can be used for siRNA transfection.
After 24h of injection, 6 of each group were randomly selected, the head and the chest of the young shrimp were dissected, immediately frozen in liquid nitrogen, and then transferred to-80 ℃ for total RNA extraction. After RNA extraction and reverse transcription, cDNA is obtained. The reaction conditions were set according to the standard instructions of the SYBR Green Realtime PCR Master Mix kit and the fluorescence signal was analyzed by Light Cycler (Roche). The PCR conditions were 95 ℃ for 5min, followed by 40 cycles of 95 ℃ for 15s, 0 ℃ for 20s and 72 ℃ for 20 s. All primers for qRT-PCR are shown in Table 2. By using 2－ΔΔCt(where Ct is the threshold cycle) the method compares them to the corresponding control to calculate the relative fold change. Three independent experiments were performed for statistical analysis.
TABLE 2 fluorescent quantitation primers
| Primer name | Upstream primer | Downstream primer |
| DMRT1 | AAGACTGTCCCTGCGAACTC | CGTATTCCCACACTTCGTTG |
| IAG | GCCTTGCAGTCATCCTTGA | AGGCCGGAGAGAAGAATGTT |
| β-actin | GAGACCTTCAACACCCCAGC | TAGGTGGTCTCGTGAATGCC |
We used intramuscular injection of siDMRT1 and siGFP, respectively, into macrobrachium rosenbergii in experimental groups, followed by detection of interference effects using real-time fluorescent quantitative PCR (figure 2). Experiments show that after 24 hours of interference, the expression levels of the Macrobrachium rosenbergii (siDMRT1) DMRT1 gene and IAG gene in the experimental group are respectively reduced by 90.8% (FIG. 2A) and 75.8% (FIG. 2B) (p is less than 0.05) compared with the control group (siGFP).
Step three, detecting and screening the false female parent;
feeding the RNA-interfered macrobrachium rosenbergii in an indoor cement pond, capturing when the body length reaches 4 +/-0.5 cm, and identifying the sex condition of male and female. The male reproductive orifices of the macrobrachium rosenbergii are used as sex identification indexes of males and females, male shrimps are used as the reproductive orifices at the base of the fifth step, and female shrimps are used in the opposite direction.
Experiment: the test is divided into a siDMRT1 experimental group and a siGFP control group, 200 shrimp larvae with the body length of 2.0-2.5cm are taken, the nucleic acid dosage is according to the standard of 0.8 mu g/g body weight, siRNA and a transfection reagent compound are injected from muscles, after 2 weeks, the siRNA and the transfection reagent compound are repeatedly introduced once by the existing transfection means, the Macrobrachium rosenbergii after RNA interference is raised in an indoor cement pond, and the apparent sex is identified when the shrimp bodies grow to 4 +/-0.5 cm. From the observation results, it can be seen that: the siDMRT trial group 124 survived, with 88 of these phenotypes being female shrimps, 36 being male phenotypes, with a proportion of female shrimps of 70.96%. In the siDMRT control group, 118 survived, 56 had a male shrimp phenotype, 62 had a female phenotype, and the hermaphroditism ratio was about 1: 1 (watch 3)
TABLE 3 proportion of interfering sex of juvenile Macrobrachium rosenbergii
| Interference with target gene | Number of disturbances | Number of survivors | Female shrimp | |
| siDMRT1 | ||||
| 200 | 124 | 88(70.96％) | 36(29.04％) | |
| |
200 | 118 | 62(52.54％) | 56(47.46％) |
And (3) carrying out sex verification on the genome of the female individual with the identified phenotype in the group by utilizing the sex specific molecular marker of the macrobrachium rosenbergii. The method comprises the following steps: extracting genome DNA from the muscle sample of the obtained macrobrachium rosenbergii. The female specific primer and beta-actin primer are used as internal reference for PCR amplification, the total volume of the amplification reaction is 25 mu L, and the amplification reaction contains about 200ng of template DNA, 0.2 mu L of primer, 0.5 unit of Taq enzyme (5U/. mu.L), 0.1 mu M dNTP and 1 xTaq reaction buffer solution. PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, and 35 cycles; extension at 72 ℃ for 10 min. And detecting by 1.0% agarose gel electrophoresis and photographing for analysis, and counting the female rate of the sample. The amplification product of the female specific primer is a strip of 220bp, and only the DNA of the shrimp with the genotype of ZW is amplified to form a strip; the beta-actin primer is an internal reference primer, and in order to eliminate experimental errors, all amplified macrobrachium rosenbergii samples have a 150bp band under the condition of experimental operation specifications (as shown in figure 3).
The experimental results show that: after the female shrimps are detected by the molecular marker, 18 female shrimps are detected, and 18 female shrimps are not provided with strips, which preliminarily shows that 18 male shrimps are subjected to sexual reversion and become female shrimps (refer to figure 3), and the success rate of the sexual reversion is 20.1%. In contrast, the control group showed no bands in all of the female shrimps, indicating that the control group did not show the gender-reversed female shrimps.
Step four, cultivating and verifying all-male macrobrachium rosenbergii;
and selecting the male and female parent shrimps subjected to molecular marker verification to continue to breed, and mating and fertilizing the male and female shrimps after the ovary is mature. Establishing a family according to each female shrimp, feeding bait every day after the giant freshwater prawn seedlings are hatched, regulating and controlling the water quality, and periodically detecting the chemical index and the biological index of the water quality in the water body of the seedlings and the development state of the larvae. Culturing the daphnia larvae of the macrobrachium rosenbergii for 20-22 days, and desalting the shrimp larvae when more than 90% of the larvae become juvenile shrimps. And breeding 300 shrimps of each family in each group in a pond. The sex of the male and female is determined by the body length of 4 plus or minus 0.5 cm. If the offspring is half of the male parent and half of the female parent, the maternal sex chromosome gene is female parent ZW, and the female parent is eliminated; if all the filial generations are male shrimps, the female parent gene is female ZZ. Then the male and female parent shrimps ZZ are reserved and are used for cultivating with the following full male shrimp seedlings.
Experiment: and selecting 18 'false female' parent shrimps after molecular marker verification to continue culturing until ovaries develop to maturity, and then mating and fertilizing with male shrimps. Among them, 11 eggs were carried and the seedlings were successfully raised. 300 shrimp fries were randomly extracted from each "false female" parent shrimp offspring and then independently cultured in 3 square meters of cement. Catching when the male and female bodies grow to 4 +/-0.5 cm, and identifying the sex condition of the male and female bodies.
From the observation results, it can be seen that: the male proportion of offspring of each family is 100 percent in 11 pools.
Step five, passage of all-male macrobrachium rosenbergii female parent 'false female' shrimp
Mating and fertilizing the verified male and female parent shrimps and male and ZZ parent shrimps, and then culturing the seedlings. And D, adopting the method of the step four to breed the full-male shrimp fries until the body length is 2.0-2.5cm, and adopting the method of the step 2 to carry out interference on the full-male shrimp fries injected with siDMRT 1. And (4) breeding the shrimp fries in a closed independent breeding pond after the interference, capturing when the shrimp fries are bred to the body length of 4 +/-0.5 cm, and identifying the sex conditions of the male and the female. Among them, pseudo female shrimps apparent as female shrimps were selected for the subsequent continuous passage. The operation flow is strictly controlled to prevent germplasm pollution.
The probability of the proportion of the male shrimps inverted into the female shrimps after the third generation of the macrobrachium rosenbergii in the experiment is as high as about 30 percent.
And (3) comprehensive analysis: according to the invention, siRNA is designed and synthesized according to the sequence of the gene segment DMRT1 of the macrobrachium rosenbergii, and is introduced into the macrobrachium rosenbergii body in the sex differentiation period to interfere the expression of the gene DMRT1 in the macrobrachium rosenbergii body, and after the interference of DMRT1 is found, the expression level of DMRT1 and IAG can be obviously reduced, and the sex specificity molecular marker and family directional mating verification are carried out; it was found that the present invention can achieve sex reversal of male shrimps to female shrimps. All of these "pseudofemale" shrimps were serially passaged. Compared with the method for removing the androgenetic gland by the operation, the method for producing the seeds of the all-male macrobrachium rosenbergii effectively reduces the infection mortality, and the reversion efficiency reaches about 30 percent after breeding for 3-4 generations.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Sequence listing
<110> research institute for fresh water aquatic products in Zhejiang province
<120> method for producing seeds of all-male macrobrachium rosenbergii
<141> 2020-03-12
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 541
<212> PRT
<213> Macrobrachium rosenbergii
<400> 1
Met Glu Cys Gly Gly Tyr Glu Ser Glu Gly Lys Gly Lys Arg Lys Gln
1 5 10 15
His Cys Thr Tyr Cys Lys Asn His Gly Lys Lys Ser Arg Lys Thr Asn
20 25 30
His Lys Cys Glu His Glu Glu Cys Glu Cys Leu Leu Cys Lys Leu Thr
35 40 45
Arg Leu Ser Arg Leu Ile Met Arg His Gln Gln Arg Leu Trp Arg His
50 55 60
Leu Lys Asp Ser Lys Arg Arg Gln Asn Ala Ala Ala Asp Ala Gly Gly
65 70 75 80
Thr Ala Ala Gly Ser Gly Ala Cys Glu Pro Ser Gly Glu His Thr Asp
85 90 95
Ser Ser Gly Ala Ala Gln Gly Ala Ser Ser Thr Lys Leu Gln Lys Cys
100 105 110
Asp Met Cys Arg Asn His Gly Glu Ile Met Ser Lys Arg Ala His Lys
115 120 125
Asn Ala Cys Pro Tyr Gln Asp Cys Pro Cys Glu Leu Cys Ser Leu Thr
130 135 140
Arg Lys Arg Arg Tyr Ile Met Xaa Leu Gln Gln Arg Val Arg Arg Ser
145 150 155 160
Gln Val Xaa Ser Lys Gln His Asn Glu Val Trp Glu Tyr Val Thr Gln
165 170 175
Ala Thr Ala Glu Leu Glu Phe Leu Gly Leu Gln Asp Ile His Gln Thr
180 185 190
Thr Glu Asp His Asn Ala Ser Ser Ser Leu Ser Glu Ser Ser Ser Ser
195 200 205
Pro Ser Pro Asp Ser Ser Ser Thr Ser Gly Ser Leu Pro His Lys Pro
210 215 220
Glu Val Ile Xaa Pro Asn Pro Val Thr Pro Thr Xaa Pro Ala Xaa Thr
225 230 235 240
Asn Arg Thr Arg Ser Lys Asp Thr Glu Ser Ser Pro Val Pro Met Asp
245 250 255
Met Pro Pro Leu Glu Pro Leu Pro Glu Cys Ser Pro Thr Arg Tyr Ser
260 265 270
Pro Arg Pro Pro Ala Pro Pro Phe Asn Val Pro Ser Pro Ala Ala Thr
275 280 285
Ser Arg Ile Arg Thr Ser Tyr Pro Ser Pro Pro Pro Pro Asn Pro Leu
290 295 300
Pro Pro Asn Arg Gly Leu Thr Asn Val Pro Pro Met Glu Ile Glu Pro
305 310 315 320
Ala Trp Met Asn Leu Lys Arg Glu Arg Glu Glu Val Leu Lys Met Asn
325 330 335
Pro Lys Val Gln Thr Arg Glu Glu Asn Leu Phe Gly His Leu Asp Phe
340 345 350
Met Lys Asp Gln Leu Thr Met Thr Gly Arg Leu Glu Pro Pro Val Pro
355 360 365
Pro Glu Gly Ser Arg Ser Phe His Asn Thr Ala Val Ala Met Asn Phe
370 375 380
Gly Ser Ser His Val Pro Pro Tyr Lys Ser Leu Val Pro Met Ile Asp
385 390 395 400
Thr Asn His Leu Pro Pro Pro Pro Leu Gln Lys Ser Arg Ala Asp Cys
405 410 415
Asn Leu His Phe Gln Tyr Arg Ser Met Val Trp Gly Asn Val Pro Thr
420 425 430
Glu Pro Ser Val Gly Phe Val Pro Asp Asp Phe Gln Gln Gln Cys Tyr
435 440 445
Arg Ser Leu Leu Val Asn Asn Ala Asp Val Arg Val Gln Pro Pro Pro
450 455 460
Gly Leu Ile Gln Ile Pro Arg Pro Gln His Pro Leu Arg Pro Arg Pro
465 470 475 480
Leu Ala Pro Val Arg Pro His Val Leu Pro Val Asn Val Glu Cys Asp
485 490 495
His Val Asn Leu His Tyr Leu Ala Tyr Phe Leu Gln Asn Gln Arg Glu
500 505 510
His Tyr Arg Asp Thr Leu Ser Thr Leu Ser Thr Thr Arg Ser Phe Gly
515 520 525
Leu Pro Arg Glu Ala Leu Phe Leu His His Pro Val Pro
530 535 540
<210> 2
<211> 1730
<212> DNA/RNA
<213> Macrobrachium rosenbergii
<400> 2
cactgctaca ctgcgtctgc gcaaacgacc tcgagatgga gtgcgggggc tacgagagcg 60
agggcaaggg caagaggaag cagcactgca cctactgcaa gaaccacggg aagaagagtc 120
gcaagaccaa ccacaagtgc gagcacgagg agtgcgagtg cctcctctgc aagctgacca 180
gactctccag gctgatcatg aggcaccagc agaggctctg gaggcacctt aaggactcga 240
agaggaggca gaacgccgct gcagacgcag gaggaacagc agcaggaagt ggagcatgtg 300
agccctcagg tgaacatact gactccagtg gggctgcaca aggagccagt agcaccaaac 360
tgcagaaatg cgacatgtgc cggaaccacg gtgaaatcat gagcaagaga gcgcacaaga 420
acgcgtgccc ttaccaagac tgtccctgcg aactctgcag tctgacaaga aagcgccgct 480
acatcatgrg actccagcag agagtcagaa ggtctcaagt grcgagcaaa cagcacaacg 540
aagtgtggga atacgtgacc caagctacag ctgagctgga attcctcggg cttcaggaca 600
tccaccagac gacagaggac cacaatgcat cttcctcatt gtcagagtcg tcctcctcgc 660
cctctccaga ctcatcctcc acctctgggt ccctccccca caaaccggag gtcatcycac 720
ccaatccagt cacycctaca gyaccagccg akaccaatcg cacccgcagt aaggatacag 780
aatcctctcc agtccccatg gatatgcccc ctctggaacc gcttccagaa tgctctccaa 840
caaggtacag tccaagaccg ccagcacctc cattcaatgt accttcacct gctgcaactt 900
ctagaattag aacaagctac ccttcacccc ctcctccaaa tccattgcct ccaaataggg 960
gtttaacaaa cgttccacca atggaaatag aaccagcctg gatgaacctc aaaagagaaa 1020
gggaagaggt tttgaaaatg aaccccaaag ttcaaacaag agaagaaaat ttattcggcc 1080
acttggactt catgaaagat caactgacca tgacaggaag gttagagcca ccagttccac 1140
cagagggcag cagaagtttt cataacaccg ccgttgcaat gaactttggt agctctcacg 1200
ttccaccata caagagttta gtgcccatga tagacacaaa ccaccttcca ccacctccac 1260
tacagaaaag cagagcagat tgcaatctac actttcagta cagatccatg gtttggggta 1320
acgtgccaac tgagcccagt gtcggcttcg tcccagacga cttccagcaa cagtgctatc 1380
gcagcttact cgtaaacaac gcagacgtaa gggttcagcc tccgccagga cttattcaaa 1440
tcccacggcc tcaacaccct ctgcgcccac gtcctctcgc gcctgtcagg cctcacgtcc 1500
tccctgtcaa cgtggagtgt gatcacgtta acctgcatta cctagcctat ttcttacaga 1560
atcaaagaga acattaccga gataccttat ctacgttgtc cacaactagg tcctttggac 1620
ttcccagaga ggctctattc cttcatcacc cagtgcccta atctataatc tataaacaaa 1680
tcctactgag aacttcagta tcattattat tatctttcaa gtgatatcca 1730
<210> 5
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 5
ucgcauuucu gcaguuuggt tccaaacugc agaaaugcga tt 42
<210> 4
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 4
acuucguugu gcuguuugct tgcaaacagc acaacgaagu tt 42
<210> 5
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 5
uucuaauucu agaaguugct tgcaacuucu agaauuagaa tt 42
<210> 6
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 6
uuguuaaacc ccuauuuggt tccaaauagg gguuuaacaa tt 42
<210> 7
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 7
uuucuucucu uguuugaact tguucaaaca agagaagaaa tt 42
<210> 8
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 8
ucuaucaugg gcacuaaact tguuuagugc ccaugauaga tt 42
<210> 9
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 9
uacugaaagu guagauugct tgcaaucuac acuuucagua tt 42
<210> 10
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 10
uagguaaugc agguuaacgt tcguuaaccu gcauuaccua tt 42
<210> 11
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 11
uugauucugu aagaaauagt tcuauuucuu acagaaucaa tt 42
<210> 12
<211> 42
<212> DNA/RNA
<213> Artificial Sequence
<400> 12
auaagguauc ucgguaaugt tcauuaccga gauaccuuau tt 42
<210> 13
<211> 46
<212> DNA/RNA
<213> Artificial Sequence
<400> 13
ucuugucugg guucuugggc uttcccaaga acccagacaa gaaatt 46
<210> 14
<211> 20
<212> DNA/RNA
<213> Artificial Sequence
<400> 14
aagactgtcc ctgcgaactc 20
<210> 15
<211> 20
<212> DNA/RNA
<213> Artificial Sequence
<400> 15
cgtattccca cacttcgttg 20
<210> 16
<211> 19
<212> DNA/RNA
<213> Artificial Sequence
<400> 16
gccttgcagt catccttga 19
<210> 17
<211> 20
<212> DNA/RNA
<213> Artificial Sequence
<400> 17
aggccggaga gaagaatgtt 20
<210> 18
<211> 20
<212> DNA/RNA
<213> Artificial Sequence
<400> 18
gagaccttca acaccccagc 20
<210> 19
<211> 20
<212> DNA/RNA
<213> Artificial Sequence
<400> 19
taggtggtct cgtgaatgcc 20
Claims (6)
1. A method for producing seeds of full-male macrobrachium rosenbergii is characterized by comprising the following steps:
step one, designing and synthesizing siRNA targeting DMRT1,
designing and synthesizing siRNA according to a Macrobrachium rosenbergii DMRT1 gene sequence published by NCBI; designing alternative siRNA sequences, screening the sequences from the candidate siRNAs, and synthesizing ten siRNAs and a negative control GFP in total; the ten sirnas included: siRNA-318, siRNA-489, siRNA-857, siRNA-914, siRNA-1013, siRNA-1179, siRNA-1245, siRNA-1489, siRNA-1510, siRNA-1535:
the negative control GFP sequence is GFP control;
step two, RNA interference;
transfecting the obtained siRNA prawn fry, extracting RNA of the interfered prawn fry, performing reverse transcription to prepare cDNA, and detecting the expression of DMRT1 and IAG genes after RNA interference by utilizing real-time fluorescent quantitative PCR;
step three, detecting and screening the false female parent;
feeding the RNA-interfered macrobrachium rosenbergii in an indoor cement pond, capturing when the body length reaches 4 +/-0.5 cm, and identifying the sex conditions of male and female by using apparent characteristics and sex-specific molecular markers;
step four, cultivating and verifying all-male macrobrachium rosenbergii;
selecting the male and female ZZ parent shrimps which are verified by molecular markers to continue to be cultured, and mating and fertilizing the male and female shrimps after the ovaries are mature;
establishing families according to each female shrimp, and culturing the obtained juvenile shrimps of each family in separate ponds, and identifying the sex conditions of the female and male shrimps when the body length is 4 +/-0.5 cm; reserving all offspring as female parent of male shrimp for subsequent cultivation of all-male shrimp fries;
step five, passage of the female parent 'false female' of the all-male macrobrachium rosenbergii;
after the verified male and female parent shrimps are reserved, mating and fertilization are carried out on the male and female shrimps after the ovary is mature;
after hatching, raising and desalting the fertilized eggs, carrying out RNA interference on the all-male shrimp fries; breeding the shrimp fries after the interference until the shrimp fries are sex differentiated, and screening the pseudo-female shrimps of which the phenotypes are female shrimps for subsequent continuous passage; and controlling the passage process.
2. The method for producing seeds of Macrobrachium andraeanum according to claim 1, wherein dTdT is substituted for the two base overhangs at the 3' end of the siRNA sequence.
3. The method for producing the seeds of the all-male macrobrachium rosenbergii as claimed in claim 1, wherein the sense strand or the antisense strand of the double-stranded siRNA is chemically modified by using a cholesterol locked nucleic acid dinitrophenol chemical group.
4. The method for producing the seeds of the all-male macrobrachium rosenbergii as claimed in claim 1, wherein the specific method for transfecting the shrimp larvae by the obtained siRNA comprises the following steps: the obtained siRNA nucleic acid and the in vivo transfection reagent are prepared into a transfection reagent compound, and the transfection reagent compound is injected into the shrimp larvae muscle with the body length of 2.0-2.5 cm.
5. The method for producing seeds of macrobrachium andraeanum according to claim 4, wherein the in vivo transfection reagent is Entranstm-in vivo; nucleic acid (. mu.g) of siRNA and EntransterTMIn vivo (μ l) configured as transfection reagent complexes according to 1: 2.
6. The method for producing seeds of the all-male macrobrachium rosenbergii according to claim 1,
the method for identifying the sex in the third step comprises the following steps:
male reproductive orifices of the macrobrachium rosenbergii are used as sex identification indexes of males and females, male shrimps are selected at the basal part of the fifth step with the reproductive orifices, and female shrimps are selected at the basal part of the fifth step without the reproductive orifices;
carrying out sex verification on a genome of female individuals with identified phenotypic sex in a population by using sex specific molecular markers of the macrobrachium rosenbergii; the method comprises the following steps: detecting a section of gene of male and female sex specific molecules in genome DNA of a muscle sample of the obtained macrobrachium rosenbergii; the presence of a female specific molecular fragment indicates that the individual is a female shrimp and the absence of a specific molecular fragment indicates that the individual is a genetically male shrimp.
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| CN112841096B (en) * | 2020-12-31 | 2022-08-05 | 浙江省淡水水产研究所 | Breeding method of all-male macrobrachium rosenbergii rapid-growth strain |
| CN113930429B (en) * | 2021-11-11 | 2023-10-27 | 上海海洋大学 | Short-chain ribonucleic acid for interfering expression of macrobrachium rosenbergii IAG gene and application thereof |
| CN114304012B (en) * | 2022-01-12 | 2023-04-21 | 湛江市渔正隆生物科技有限公司 | High-density aquaculture method |
| CN114375912B (en) * | 2022-01-28 | 2023-05-05 | 厦门后方水产科技有限公司 | Large-scale breeding method for all-male freshwater shrimps |
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