CN111387105A

CN111387105A - Method for producing seeds of all-male macrobrachium rosenbergii

Info

Publication number: CN111387105A
Application number: CN202010179093.4A
Authority: CN
Inventors: 徐宾朋; 陈雪峰; 高强; 彭菲; 程海华
Original assignee: Zhejiang Institute of Freshwater Fisheries
Current assignee: Zhejiang Institute of Freshwater Fisheries
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2020-07-10
Anticipated expiration: 2040-03-12
Also published as: CN111387105B

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

Method for producing seeds of all-male macrobrachium rosenbergii

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 Entranster^TMIn 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); for enhancement ofSequence 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 storage solution with the O dissolved at 20 mu mol/L is stored in a refrigerator at the temperature of-20 ℃, and the sequence is 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 Entranster^TMIn vivo (Engrenenbiosystem Co, L id.) (microliter) is prepared into a transfection reagent compound according to a ratio of 1:2, the nucleic acid dosage is 0.8 micrograms per gram of weight, siRNA and the transfection reagent compound are injected into shrimp larvae muscles with the body length of 2.0-2.5cm (Macrobrachium maindroni bred through 5 generations), and a siGFP control group and a siDMRT1 interference group are respectively arranged.

After 24h post-injection, 6 tails of each group were randomly selected, the head and the chest of the shrimp larvae were dissected, immediately frozen in liquid nitrogen, and then transferred to-80 ℃ for total RNA extraction, RNA extraction and reverse transcription were performed to obtain cDNA.the reaction conditions were set according to SYBR Green Realtime PCRMaster Mix kit Standard protocol, and the fluorescent signals were analyzed by L ig Cycler (Roche). the PCR conditions were 95 ℃ for 5min, then 40 cycles of 95 ℃ for 15s, 0 ℃ for 20s, and 72 ℃ for 20 s.qRT-PCR with all primers as shown in Table 2. by using 2, the PCR was performed by^－ΔΔCt(where Ct is the threshold cycle) the method willThey were compared to the corresponding controls to calculate relative fold changes. 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	Male shrimp
					siDMRT1
	200	124	88(70.96％)	36(29.04％)
					siGFP	200	118	62(52.54％)	56(47.46％)

A process for verifying the sex of female individual whose phenotype is identified in macrobrachium rosenbergii includes such steps as obtaining muscle sample of macrobrachium rosenbergii, extracting genome DNA, PCR amplification with female specific primer and β -actin primer as internal reference, the total volume of amplification reaction is 25 microns L and contains 200ng of template DNA, 0.2 microns L primer, 0.5 unit of Taq enzyme (5U/micron L), 0.1 microns of dNTP and 1 × Taq reaction buffer, PCR amplification procedure including pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30 sec, annealing at 55 deg.C for 30 sec, extension at 72 deg.C for 45 sec, 35 cycles, extension at 72 deg.C for 10 min, electrophoresis detection on 1.0% agarose gel and photographic analysis, and counting the female rate of sample, the amplification product of female specific primer is a 220bp band, only the DNA of shrimp whose genotype is ZW has band, and β -actin primer is a standard band for 150bp band of macrobrachium rosenbergii experimental operation.

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

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<170>SIPOSequenceListing 1.0

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Met Glu Cys Gly Gly Tyr Glu Ser Glu Gly Lys Gly Lys Arg Lys Gln

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His Cys Thr Tyr Cys Lys Asn His Gly Lys Lys Ser Arg Lys Thr Asn

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His Lys Cys Glu His Glu Glu Cys Glu Cys Leu Leu Cys Lys Leu Thr

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Arg Leu Ser Arg Leu Ile Met Arg His Gln Gln Arg Leu Trp Arg His

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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

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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

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Pro Lys Val Gln Thr Arg Glu Glu Asn Leu Phe Gly His Leu Asp Phe

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Met Lys Asp Gln Leu Thr Met Thr Gly Arg Leu Glu Pro Pro Val Pro

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Pro Glu Gly Ser Arg Ser Phe His Asn Thr Ala Val Ala Met Asn Phe

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Gly Ser Ser His Val Pro Pro Tyr Lys Ser Leu Val Pro Met Ile Asp

385390 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

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Gly Leu Ile Gln Ile Pro Arg Pro Gln His Pro Leu Arg Pro Arg Pro

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Leu Ala Pro Val Arg Pro His Val Leu Pro Val Asn Val Glu Cys Asp

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His Val Asn Leu His Tyr Leu Ala Tyr Phe Leu Gln Asn Gln Arg Glu

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<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

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 DMRT 1;

step two, RNA interference;

step three, detecting and screening the false female parent;

step four, cultivating and verifying 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 the all-male macrobrachium rosenbergii according to claim 1,

step one, designing and synthesizing siRNA targeting DMRT1,

the negative control GFP sequence was GFP control.

3. The method for producing seeds of Macrobrachium andraeanum according to claim 2, wherein dTdT is substituted for the two base overhangs at the 3' end of the siRNA sequence.

4. The method for producing the seeds of the all-male macrobrachium rosenbergii as claimed in claim 2, characterized in that the sense strand or the antisense strand of the double-stranded siRNA is chemically modified by a cholesterol locked nucleic acid dinitrophenol chemical group.

5. 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.

6. The method for producing seeds of macrobrachium andraeanum according to claim 5, wherein the in vivo transfection reagent is Entranstm-in vivo; nucleic acid (. mu.g) of siRNA and Entranster^TMIn vivo (μ l) configured as transfection reagent complexes according to 1: 2.

7. The method for producing seeds of the all-male macrobrachium rosenbergii according to claim 1,

step three, detecting and screening the false female parent;

the method for identifying sex comprises the following steps: