CN104193814A - Freshwater shrimp vitellogenin Vg gene, encoding protein and application of freshwater shrimp vitellogenin Vg gene - Google Patents

Abstract

The invention discloses a freshwater shrimp vitellogenin Vg gene, an encoding protein and an application of the freshwater shrimp vitellogenin Vg gene. The full-length cDNA sequence of the Vg gene is cloned from hepatopancreas of the freshwater shrimp by a primer group provided by the invention, and the expression of a vitellogenin gene in the ovary and the liver can be silenced after the dsRNA of the gene is injected to the pericardial cavity of a female freshwater shrimp, so that the ovary development of the freshwater shrimp is remarkably delayed. The invention provides a new thought and an effective means for solving the sexual precocity phenomenon of the freshwater shrimp and culturing good varieties.

Description

Freshwater shrimp vitellogenin Vg gene, proteins encoded and application

Technical field

The present invention discloses a kind of freshwater shrimp vitellogenin Vg gene, proteins encoded and application, relates to biotechnology and developmental regulation field, is specifically related to the clone of freshwater shrimp vitellogenin genes, also relates to the development rate of utilizing this gene dsRNA to regulate and control freshwater shrimp ovary.

Background technology

Freshwater shrimp, formal name used at school Macrobrachium nipponensis ( macrobrachium nipponense), be commonly called as river prawn, be subordinate to Decapoda (Decapoda), Palaemonidae (Palaemonidae), pond crayfish genus (Macrobrachium), be distributed widely in waters, China various places, growth is fast, strong adaptability, fanning economics are high, is the important freshwater aquiculture shrimps of China.Record according to 2012 " China Fisheries yearbook ", the annual production of whole nation cultivation freshwater shrimp exceedes 230,000 tons, and annual value of production exceedes 15,000,000,000 yuan, is one of kind the most rising in current freshwater aquaculture industry.In recent years, along with the falling sharply and cultivate the expansion of scale of its natural resources, there is the situation that production performance declines and germ plasm resource is degenerated in the freshwater shrimp of cultivation, and the phenomenon of the individual miniaturization of its Prawn and " sexual prematurity " is particularly outstanding." sexual prematurity " i.e. female, male sexual gland grows in advance, shrimp sexual gland is ripe too early aborning, lay eggs cause that individuality diminishes, pond culture density is excessive, the series of problems such as growth specification is uneven, inbreeding, the specification of final goods shrimp reduces, reproductive performance declines, have a strong impact on quality and the output value of freshwater shrimp, become a large bottleneck of shrimp culture industry development.For realizing the Sustainable development of freshwater shrimp industry, the solution of finding the female freshwater shrimp of solution " sexual prematurity " becomes very urgent.

Vitellogenin (Vitellogenin, Vg) is a kind of albumen being prevalent in oviparity nonmammalian blood, is the precursor of vitellin(Vt) (vitellin, Vn) in nearly all oviparous animal.Vitellin(Vt) is the vertebrates of all oviparity and the main component of the female yolk of invertebrates, in the run-up of yolk emergence period.It is considered to synthetic by the outer tissue of ovary, and secretion is discharged in blood, arrives ovocyte by blood transportation, and accumulate and store with the form of vitellin(Vt), be embryo and the main source of nutrition of young early development.Vitellin(Vt) and vitellogenin contain many fatty ingredients, can be the embryo who is growing nutrition and the functional substance such as amino acid, fat, carbohydrate, VITAMIN, p and s are provided.

RNAi(RNA interference, RNAi) be a kind of efficient sequence-specific gene knochout technique being extensively present in eukaryote.RNAi is brought out by the double-stranded RNA of different sources and length (double-stranded RNA, dsRNA) precursor, can effectively suppress the translation of target gene RNA.In recent years start to use in crustacean gene functional research.At present, crustaceans is expressed and be there is not yet report about the silence of Vg gene.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of freshwater shrimp vitellogenin Vg gene, proteins encoded and application, and this gene is used for regulating and controlling female freshwater shrimp development of ovary speed, provides fundamental basis for solving freshwater shrimp sexual prematurity.

Technical scheme:

A kind of freshwater shrimp vitellogenin, its aminoacid sequence is as shown in SEQ ID NO.2.

The freshwater shrimp vitellogenin Vg gene of the above-mentioned freshwater shrimp vitellogenin of encoding.

Described freshwater shrimp vitellogenin Vg gene, its nucleotide sequence is as shown in the 35th in SEQ ID NO.1～the 7645th.

The dsRNA of described freshwater shrimp vitellogenin Vg gene synthesized.

Described freshwater shrimp vitellogenin Vg gene is in the application delaying in the female freshwater shrimp development of ovary.

Described application, comprises the steps: the freshwater shrimp vitellogenin Vg gene in freshwater shrimp DNA sample to carry out pcr amplification, and amplified production is carried out to the synthetic dsRNA of in-vitro transcription, then by dsRNA to be expelled in the pericardial sac of freshwater shrimp.

Described application, the sequence of the upstream and downstream primer using in pcr amplification is as shown in SEQ ID NO.3～4.

Described application, the injected dose in injecting step is 4 μ g/g.

beneficial effect

Freshwater shrimp vitellogenin Vg gene provided by the invention can prepare dsRNA, mode by injection is injected in freshwater shrimp pericardial sac, can effectively delay the time of freshwater shrimp ovary maturity, prove that the expression of this gene has very important impact to the female development of ovary.

Brief description of the drawings

Fig. 1 is the schematic diagram of exogenous injection Vg-dsRNA on female freshwater shrimp ovary GSI gonad development index impact.

Note: 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d is respectively injection rear 1,3,5,7,9,13,15 and 17 days (sample size N >=3).

Fig. 2 is the schematic diagram of the expression amount impact of exogenous injection Vg-dsRNA on the endogenous Vg gene of female freshwater shrimp.

Note: 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d is respectively injection rear 1,3,5,7,9,13,15 and 17 days (sample size N >=3).The DEPC water of control group injection same dose.

Fig. 3 is the schematic diagram of the impact of the expression amount of exogenous injection Vg-dsRNA on the endogenous Vg gene of female freshwater shrimp in liver.

Note: 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d is respectively injection rear 1,3,5,7,9,13,15 and 17 days (sample size N >=3).The DEPC water of control group injection same dose.

Embodiment

Below by embodiment, the present invention is described in further detail.But it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (for example show with reference to J. Pehanorm Brooker etc. according to the described technology of the document in this area or condition, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

Approximate language used herein can be used for modifying any quantity statement in whole specification sheets and claims, and it can permit changing under the condition that does not cause its relevant basic function to change.The value of therefore, being modified by the term such as " approximately " is not limited to specified exact value.In at least some cases, approximate language can be corresponding with the precision of the instrument for measuring this value.Unless separately point out in context or statement, otherwise range limit can combine and/or exchange, and this scope is confirmed as and comprised included all subranges herein.Except in operation embodiment or indicating in elsewhere, the numeral of the amount of all expression compositions that use in specification sheets and claims, reaction conditions etc. or expression all should be understood to be subject to the modification of word " approximately " in all cases.

The object of this research is exactly by injecting in freshwater shrimp body with Vg-dsRNA, study expression variation and the impact on the development of ovary of this gene under dsRNA stimulates, the regulation mechanism of explaination dsRNA to the freshwater shrimp development of ovary, practically provides new approaches and reference frame for what utilize Vg-dsRNA perturbation technique resistance precocity.

embodiment 1: the acquisition of freshwater shrimp Vg full length gene cDNA

Total RNA extracts: choose the liver of 3～4 female shrimps of maturation, put into rapidly the precooling mortar that fills liquid nitrogen, grind rapidly.The extraction of total RNA is used the RNAiso Plus extraction reagent of Takara company to carry out in conjunction with the imitative extraction process of traditional phenol, and extracting method is consulted and used specification sheets.The quality that detects RNA through agarose gel electrophoresis, spectrophotometer is analyzed this sample OD260/280 ratio between 1.8～2.0, and measures the concentration of RNA.

CDNA the first chain is synthetic: carry out the synthetic of the first chain cDNA with reference to Takara M-MLV reverse transcription test kit.

The clone of freshwater shrimp Vg full length gene cDNA sequence:

According to acquired Vg full length gene cDNA sequence (Macrobrachium rosenbergii macrobrachium rosenbergii, BAB69831; The imitative long volume shrimp of Japan pandalopsis japonica, ACU51164; The long volume shrimp of the high back of the body pandalus hypsinotusbAD11098; ) coding region compare, according to comparison result, at conservative region design primer, utilizing primer sets one (SEQ ID NO:5～SEQ ID NO:14) and the cDNA of above-mentioned reverse transcription is the clone that template is carried out intermediate segment.

More specifically, above-mentioned primer sets is composed as follows:

The nucleotide sequence of forward primer VGAF1 is as shown in SEQ ID NO.5;

The nucleotide sequence of forward primer VGBF2 is as shown in SEQ ID NO.6;

The nucleotide sequence of forward primer VGCF3 is as shown in SEQ ID NO.7;

The nucleotide sequence of forward primer VGDF4 is as shown in SEQ ID NO.8;

The nucleotide sequence of forward primer VGEF5 is as shown in SEQ ID NO.9;

The nucleotide sequence of reverse primer VGAR1 is as shown in SEQ ID NO.10;

The nucleotide sequence of reverse primer VGBR2 is as shown in SEQ ID NO.11;

The nucleotide sequence of reverse primer VGCR3 is as shown in SEQ ID NO.12;

The nucleotide sequence of reverse primer VGDR4 is as shown in SEQ ID NO.13;

The nucleotide sequence of reverse primer VGER5 is as shown in SEQ ID NO.14.

Carry out pcr amplification reaction system as follows:

PCR response procedures is: 94 DEG C of denaturation 3min, then enter following circulation: 94 DEG C of 30s, and 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations, last 72 DEG C are extended 10min, 4 DEG C of preservations.1.5% agarose gel electrophoresis detects.

Utilize the pillar DNA glue recovery test kit of Shanghai Sheng Gong biotechnology company limited (Sangon) to carry out the recovery of object fragment, product is connected to pMD18-T carrier (Takara), and be transformed into bacillus coli DH 5 alpha, carry out blue hickie screening, after picking mono-clonal hickie amplification cultivation, the positive colony that inserts object fragment is delivered to Shanghai Bo Shang biotech firm and carry out sequencing analysis.Several sections of products that obtain are spliced, obtain the intermediate segment of freshwater shrimp Vg gene.

According to Vg gene cDNA intermediate segment sequence, design again specificity forward primer 3VGF1(SEQ ID NO.15), 3VGF2(SEQ ID NO.16); With reverse primer 5VGR1(SEQ ID NO.17), 5VGR2(SEQ ID NO.18) carry out respectively cDNA 3 ' and 5 ' rapid amplifying, operation steps is undertaken by TaKaRa 3 '-RACE and TaKaRa5 '-RACE test kit specification sheets.By above-mentioned clone's step carry out subsequently cut glue, recovery, conversion, Clone and sequence finally obtain Vg gene 3 ' and 5 ' end sequence.The sequencing result of intermediate segment and 3 ' and 5 ' end is compared, and splicing obtains freshwater shrimp Vg full length gene cDNA sequence.Show through sequence alignment, freshwater shrimp Vg gene and other crustacean homology are more than 70%.Its nucleotide sequence is (1st～34 and 7646th～7800 is the non-coding region of 3 ' and 5 ' end) as shown in the 35th in SEQ ID NO.1～the 7645th, and the aminoacid sequence of its proteins encoded is as shown in SEQ ID NO.2.

embodiment 2: freshwater shrimp Vg gene dsRNA's is synthetic

Classify foundation as with SEQ ID NO.1 nucleotides sequence, in open reading frame, adopt the online dsRNA primer-design software of NCBI (http://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl) design to prepare the primer (SEQ ID NO.3-SEQ ID NO.4) of double-stranded RNA, according to Transcript AidTM T7 High Yield Transcription kit (Fermentas, Inc., USA) test kit explanation carries out the synthetic dsRNA of in-vitro transcription.The dsRNA preparing is dissolved in DEPC water, measures its concentration and on 1.5% sepharose, detect its purity.Above-mentioned dsRNA solution is expelled in the pericardial sac of freshwater shrimp with the dosage of 4 μ g/g.The DEPC water of control group injection same volume, tests female shrimp used all in development of ovary initial stage (firm drained ovum).The 1st, 3,5,7,9,11,13,15 and 17 days collection samples (N >=3) after injection, and tissue is kept in RNA preservation liquid (Takara).Finally extract total RNA of each sample and be inverted to cDNA, adopting Real Time PCR to detect Vg gene and change with respect to the expression amount of control group, calculating with this efficiency that Vg gene disturbs.Sexual gland index is the important indicator of evaluating animal sexual gland development condition.According to formula GSI=sexual gland weight in wet base/body weight weight in wet base × 100%, calculate the GSI gonad development index (Gonad Somatic Index, GSI) of two groups of female freshwater shrimps.

experimental result

Shown in Fig. 1, for the schematic diagram of the gonad development index contrast of the GSI gonad development exponential sum injection group of control group, can find out growth that Vg-dsRNA injection significantly delayed freshwater shrimp ovary the control group development of ovary in the 11st day in ripening stage experimental group still in yolk accumulation period.

As shown in Figure 2, in ovary, with respect to control group, latter the 1st day of injection, the expression amount of freshwater shrimp Vg gene has significantly declined 76%, has declined 92% at the 5th day, along with the growth of control group ovary, Vg gene has presented the expression rule that first raises and reduce afterwards in ovary.But obviously delayed in this rule of control group, the peak period that treatment group occurs in yolk in the time of the 15th day, and control group ovary has completed one and takes turns growth cycle, emptying.

As shown in Figure 3, in liver, with respect to control group, latter the 1st day of injection, the expression amount of freshwater shrimp Vg gene has significantly declined 74%, has declined 97% at the 5th day, along with the growth of control group ovary, Vg gene has presented equally the expression rule that first raises and reduce afterwards in liver.Obviously be delayed according to the growth of expression rule control group.Result proves external source injection Vg-dsRNA, can effectively delay gonad development, and this result of study provides theoretical basis for solving this difficult problem of freshwater shrimp sexual prematurity, also the method is applied to production practice directive significance is provided for follow-up.

SEQUENCE LISTING

<110> China Aquatic Science Research Academy Fresh Water Fishery Research Center

<120> freshwater shrimp vitellogenin Vg gene, proteins encoded and application

<130> without

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 7800

<212> DNA

<213> vg

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aactgtgtat gaagtgcaac atgagggaga aaaggttgtt gtgaccaagt tgaaaaacca 600

tcgcttctgc caagatcatt acgctaaccg agctgagaca ccaaaagctt ggatgaaggc 660

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caccagcatt acttgtaagg acaagaatgt cataaaacca gcctatggtt cttacaagta 780

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tggatcatat cttgcagcaa ttaaatgtgc taaacatgaa gaactgcaca agattactga 1740

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gctgaaagca cagagtgaaa cttaccttat gaagagagta aagggtcaac aggagaccaa 2820

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gatgaccaaa gcagatcttg tttggccaga aggtcgacaa tctatagtaa acttagaagc 4740

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gctcctgaag gcgatagatc acctcatatc ttctagctct ttgtccatgt ctgatagccc 5040

tcggcgtctg ctctttatcg tgctcactac aaacatcatg accaccattc ttattccctg 5100

ttgcctgatg tcaccctctc gtacaatgga aggaggatgt ggatgcaata cttgcacagg 5160

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atatgagatt gccttccgcc gatctcattc cgattggagt ggagaatcca gatatgaagg 5280

tcgtgtcagc catccaggac tagagaggga ccgtcgaatt gatattcagc ttagaagaga 5340

ggaacagaag gtcacaggaa gtgttgagct cgatattttc ctcagaccag aagataagat 5400

cactggaaag ttagaatcac tcatttatgc tcgaaacact gtcatcgttg aagcaaaact 5460

tgaaggaaag gtactgaagg atcaccccaa ggtcattgta cgtggagcct atggtccaag 5520

caatgttgga tttgatatta aattgcacaa gactcactct tctcctacat ccttcaggat 5580

gtcgggaaga tatgagaaga ggactgccag aaatgccgct gcttccttca tcatagagaa 5640

tgaacatcaa aaaaccattg acatttctat agcacttcat cctcatcaga gtccttggtg 5700

ttatggagta cgagctgaag ctaaggcaaa gtcgtccatc attggaactt acgacatcta 5760

ttctgagctt tgcaagccag gatttgtatc attgaccaca aagaaacatg gaagtgacaa 5820

gatgtacata actaaactcg gtatagaagg aatgaagaac atcgaagcca gtattttgga 5880

agctaaccca caaaccatgg agaaaagacc acttggcatg gctcgtatgg tgctaacttc 5940

tcccacaatg atgaagctgg aaactaagta tgaaggtgat aagctacatg aaatcaagag 6000

tactgtacgc gatatgtggt ggaggcttgt ttcatctgca ggtaacggga tggattcttt 6060

gtgcagtgaa gttttacaag agggctctgg atcaccttct ggccacatgt cttcggtatg 6120

gcgggaaatt aagaatgaag cctcaagaat ctattctgat ttggaaaacg atttagtcat 6180

tcctaaatat gagaagctca gggaatggct tcatggtggc attctctcta acattgccga 6240

gggatgttca aaggtttggt ctcattatgc tcacttccaa agcagtttat catcatctgt 6300

atccaacatg ataagaacta ttcgagagga attccctgga ttaactagag tagtcactga 6360

agttgtaatg ggaactgctc gtggcctgca aactggtgaa atgccggagg tgttccgtcg 6420

atggtggaac gatttccttg agagttcttt ctgcagggct atagcatctg actttgaatc 6480

actctggagt gaacaccaag aggagtacca gggattgcag cagatctgga ggaaaatcaa 6540

gaacactctt gcaaaggacg ttaacagaca gagacgtaat cttatgcact acaagaaacc 6600

caggcatctt gttaactgga tcgtccacga catgaacttt gaacgaatgg ttttcaaggg 6660

tgtagataag gttatcaaga atgttgtgca gaaagctctc ttcgtacctt ttcagatcga 6720

tggtaaccac ttccagcttc agttacctat tcgtcgacca gtgcactcac ttcctcaagc 6780

tttgtcttat gtatcaatga atccaactcc agttgttgac cgtgcattat ggtggtttga 6840

agctcttatg cccactccag tcgataacat tgtatgggct tattataaat tccttcctcg 6900

ccatgcacgt tatcttctgc caccctacaa tcacacagcc atggttgttg atggatcaga 6960

gatcctcacc tttgatggag tagttctgcg agtacctcat tctccctgca aagtcttgct 7020

tgcacagtac aagactcact ccttgattat gcaaaatcag cagcctggcc atttgccaca 7080

cttcattttg aaggctgctg gagctacagt ggaggtcaag cctgacttcg ttgttaccgt 7140

caatggaaat ccagtaagtg ggcccaggga agtgcaaggt gaagtcgtaa tcatgaaaga 7200

aaatgagaag atcaaggtca ggactccatt cataacactc cgtgtctaca aagacagccg 7260

caccgcttca gtcgaggtgt ctggctggac atttggtaga atagctggtc ttttgggcat 7320

ctatgatggg gaaaaggcaa ctgataggtt gacacctcag ggtacaaggg catctaatct 7380

tcaagagctg gtcaagtcct ggcaagagaa tcctcagtgc gagactcctc acgtagcccc 7440

catgagtcct atgcaggttc cagtggtcca tatgctccac tgccaaaccc tcttcggagt 7500

tcggtctcgc tgcaacccag tcgtccatcc agggccattc aggaagatgt gcttcgccag 7560

caagaacgcc tgtcacgttg ctaaggccta tcgggccatg tgcgagacga agggcattaa 7620

ggagaccttc cctctgggat gctaatgacc aaacctatcc atggaagtca ttgtgtaaat 7680

gagtgactcc aagcctagtg acctcattgt tctagttttt aagaaatgca acgaaaatgt 7740

acaaaaaata ataaatttct agatcgataa taaatatgag atgcataaca agaaaaaaaa 7800

<210> 2

<211> 2536

<212> PRT

<213> vg-prt

<400> 2

Met Thr Ser Leu Ala Leu Leu Leu Leu Ala Ile Ala Ser Phe Ser Pro

1 5 10 15

Ala Pro Trp Pro Ser Gly Thr Asn Leu Cys Ser Lys Glu Cys Pro Val

20 25 30

Ala Gly Ser Pro Lys Leu Phe Tyr Ala Pro Glu Lys Thr Tyr Val Tyr

35 40 45

Ser Tyr Thr Gly Lys Ser Arg Ile His Leu Lys Asp Val Glu Gly Ala

50 55 60

Asn Val Asp Met Glu Trp Asn Ser Gln Val Glu Leu Ser Trp Leu Ser

65 70 75 80

Pro Cys Asp Met Ala Ile Thr Met Lys Asn Pro Ser Ile Gly Gly Gly

85 90 95

Ser Gly Ser Ala Glu Ala Arg Phe Leu Glu Arg Tyr Pro Leu Val Val

100 105 110

Ser Ile Ile Asp Gly Arg Val Lys Ala Ala Cys Gly His Pro Asp Asp

115 120 125

Asp Thr Trp Ser Ile Asn Leu Lys Lys Gly Ile Ala Ser Ala Phe Gln

130 135 140

Asn Thr Leu Pro Ser Asn Ser Thr Ile Asn Ser Gly Leu Asn Phe Thr

145 150 155 160

Glu Thr Asp Ile Ile Gly Asn Cys Ser Thr Val Tyr Glu Val Gln His

165 170 175

Glu Gly Glu Lys Val Val Val Thr Lys Leu Lys Asn His Arg Phe Cys

180 185 190

Gln Asp His Tyr Ala Asn Arg Ala Glu Thr Pro Lys Ala Trp Met Lys

195 200 205

Ala Pro Leu Pro Met Glu Glu Ser Tyr Ser Glu Cys Lys Gln Glu Ile

210 215 220

Thr Asn Gly Ile Tyr Thr Ser Ile Thr Cys Lys Asp Lys Asn Val Ile

225 230 235 240

Lys Pro Ala Tyr Gly Ser Tyr Lys Tyr Val Glu Ala His Gln Glu Ser

245 250 255

Val Leu Arg Phe Gln Ser Glu Thr Asp Gln Ile Pro Pro Ser Val Ser

260 265 270

Gln Leu Pro Ser Arg Phe Ile Arg Lys Thr Leu Arg Tyr Asp Gln His

275 280 285

Thr Leu Lys Lys Asp Pro Ser Met Ala Ala Lys Leu Asp Glu Met Leu

290 295 300

Lys Gln Val Cys Glu Lys Val Lys His Gly Val His Glu His Ala Ala

305 310 315 320

Ser Gln Phe Ala Gln Ala Leu His Phe Leu Arg Arg Val Pro Glu Glu

325 330 335

Ala Ile Pro Gln Thr Leu Glu Lys Ile Arg Gly Gly Gln Ile Cys Glu

340 345 350

Gln Arg Gln Lys Met Glu Ser Met Phe Leu Asp Gly Leu Ala Phe Val

355 360 365

Tyr Glu Ser Gly Ala Val Lys Val Met Val Gln Glu Leu Ile Ser Gly

370 375 380

Lys Ala Thr Gly Gly Arg Ala Ala Leu Tyr Ala Ala Ser Met Tyr Phe

385 390 395 400

Met Pro Arg Pro Cys Ile His Ser Ile Glu Ala Leu Lys Pro Leu Phe

405 410 415

Glu Asn Phe Gln Arg Phe Pro Arg Thr Thr Leu Ala Gly Ala Thr Met

420 425 430

Val His Thr Tyr Cys Arg Gln Asn Pro Lys Cys Gln Glu Lys Ala Pro

435 440 445

Val Arg Gln Leu Ala Glu Thr Leu Ser Ser Lys Val Gly Gln Met Cys

450 455 460

Thr Ala Ser Pro Asn Glu Glu Thr Arg Glu His Ala Leu Ala Leu Leu

465 470 475 480

Lys Ser Leu Gly Asn Met Gly Val Met Asn Ser Glu Ile Ala Arg Pro

485 490 495

Ile Leu Gln Cys Ile Glu Asn Pro Glu Ala Asp Lys Gly Ile Arg Arg

500 505 510

Ala Ala Thr Gln Thr Phe Arg Asn Val His Cys His Gln Glu Ile His

515 520 525

Pro Ile Met Lys Gln Leu Ile Asn Val Ala Leu Asp His Arg Lys Arg

530 535 540

Thr Glu Val Arg Ile Gly Ser Tyr Leu Ala Ala Ile Lys Cys Ala Lys

545 550 555 560

His Glu Glu Leu His Lys Ile Thr Glu Lys Ile Ala Lys Glu Glu Asn

565 570 575

Thr Gln Val Arg Ser Phe Ile Leu Ser His Leu His Asn Ile Lys Glu

580 585 590

Ser Thr Val Pro Tyr Lys Ala Asn Leu Lys Ser Arg Leu Glu Ser Ile

595 600 605

Val Leu Pro Ser Asn Phe Thr Lys Asp Trp Arg Lys His Ser Arg Asn

610 615 620

Ile Asp Leu Ser Tyr Tyr Ala Pro Thr Phe Gly Val Gly Ala Gly Val

625 630 635 640

Glu Ser Asn Val Ile Tyr Ala Pro Gly Ser Tyr Val Pro Arg Ser Val

645 650 655

Asn Leu Asn Leu Thr Ala Ala Leu Gly Ala Thr Pro Phe Asn Ile Gly

660 665 670

Glu Ile Gly Ala Arg Phe Glu Gly Leu Glu Thr Ile Ile Glu Glu Leu

675 680 685

Phe Gly Pro His Ser Tyr Phe Lys Arg Thr Pro Ala Arg Gln Ile Trp

690 695 700

Glu Asp Leu Lys Glu Thr Leu Ser Lys Ile Ser Glu Arg Leu Glu Gly

705 710 715 720

Ser Phe Arg Gln Arg Arg Ser Ile Asp Leu Ser Gln Ile Ser His Leu

725 730 735

Phe Asp Lys Leu Tyr Gly Asn Arg His Met Gln Lys Ala Asp Ile Tyr

740 745 750

Ala Arg Ile Asn Asn Gln Glu Met Ala Phe Gly Ser Trp Glu Gly Asn

755 760 765

Met Lys Asn Ile Lys Ile Asp Glu Leu Ile Asn Asn Leu Phe Asp Lys

770 775 780

Phe Asp Asn Val Ile Asn Arg Ala Ala Thr Thr Asn Ile Asp Thr Val

785 790 795 800

Arg Thr Ala Gln Phe Tyr Met Asn Asn His Leu Pro Thr Ile Gln Gly

805 810 815

Leu Pro Ile Lys Ile Lys Leu Glu Gly Thr Ala Val Ala Gly Ile Lys

820 825 830

Met Glu Thr His Val Arg Gly Leu Thr Ser Gly Thr Pro Gly Val Val

835 840 845

Lys Phe Leu Pro Ser Leu Ser Thr Gln Ile Asp Ala Phe Ile Gly Tyr

850 855 860

Asp Cys His Ile Val Arg Ala Gly Ile Lys Met Lys Asn His Ile Ala

865 870 875 880

Thr Asn Tyr Gly Ala Ser Ile Asn Ala Lys Tyr Thr Ser Gly Gln Gly

885 890 895

Phe Glu Val Glu Leu Glu Met Pro Glu Lys Met Glu Leu Leu Lys Ala

900 905 910

Gln Ser Glu Thr Tyr Leu Met Lys Arg Val Lys Gly Gln Gln Glu Thr

915 920 925

Lys Val His Pro Ser Ser Val Gln Ser Thr Arg Phe Gln Thr Gln Ser

930 935 940

Cys Leu Thr Lys Met Glu Pro Val Leu Gly Leu Lys Leu Cys Tyr Asp

945 950 955 960

Val Asn Met Pro Asn Ile Phe Arg Ser Gln Gly Leu Pro Leu Gly Pro

965 970 975

Pro Ala Ile Phe Lys Val Val Leu Glu Lys Ala Glu Ser Gly Met Arg

980 985 990

Gly Phe Leu Val Lys Gly Arg Glu Glu His Val Gly Gly Lys Lys Val

995 1000 1005

Ile Lys Val Glu Leu Glu Thr Pro Gly Thr Ser Ser Pro Lys Lys

1010 1015 1020

Ala Thr Ala Ile Ile Thr Ser Ala Asn Glu Gly Glu Glu Lys Lys

1025 1030 1035

Ile Ser Ala Thr Phe Glu Cys Glu Arg Leu Gly Gly Ile Ser Ile

1040 1045 1050

His Met Met Arg Lys Trp Thr Gln Ser Glu Lys Gln Val Glu Met

1055 1060 1065

Ile Ala Tyr Phe Ser Glu Ser Arg Gln Tyr Asn Pro Ser Thr Lys

1070 1075 1080

Gly Met Glu Ala Lys Phe Leu Trp Asn Gly Glu Gly Glu Glu Val

1085 1090 1095

Lys Leu Asp Met Ala Leu Asn Thr Leu Ala Ala Ile Arg Glu Trp

1100 1105 1110

Val Gln Leu Asn Phe Glu Val Ser Gly Asp Leu Arg Tyr His Glu

1115 1120 1125

Trp Cys Arg Ile Pro Leu Pro Gln Arg Leu Arg Lys Phe Glu Thr

1130 1135 1140

Lys Val Ala Leu Arg Asn Trp His Ile Ile Ser Phe Met Arg Lys

1145 1150 1155

Glu Gly Glu Ser Gln Tyr Lys Ser Thr Leu Lys Ile Gly Gln Arg

1160 1165 1170

Gly Asn Glu Lys Ile Asp Ile Ile Gly Asn His Val Met Glu Gly

1175 1180 1185

Ser Ser Tyr Arg Asp Met Thr Phe Lys Ser Asn Ile Glu Ala Lys

1190 1195 1200

Ile Gly Asn Ala Arg Tyr Lys Lys Asn Phe Val Leu Tyr Asn Gln

1205 1210 1215

Glu Ser Lys Met Gly Val Glu Trp Gln Val Ala Ser Gln Glu Gly

1220 1225 1230

Gly Lys Val Ile Glu Leu Glu Met Met Leu Ile Arg Ser Gly Glu

1235 1240 1245

Thr Ser Gln Val Lys Phe Leu Leu Asp Ile Pro Glu Arg Met Lys

1250 1255 1260

Lys Val Leu Leu Glu Ala Ser Ala Ala Gly Gln Gly Ser Ser Gln

1265 1270 1275

Tyr Gln Val Arg Ala Met Ala Lys Tyr Gly Glu Ser Pro Ile Phe

1280 1285 1290

His Val Glu Gly Pro Val Thr Ala Met Leu Ser Ser Lys Asn Thr

1295 1300 1305

His Leu Lys Thr Glu Leu Arg Thr Val Leu Leu Arg Ser Gln Pro

1310 1315 1320

Tyr Thr Val Thr Thr Ser Leu Val Met Met His Gly Lys Gln Ala

1325 1330 1335

Val Val Phe Glu Leu Lys Asp Arg Glu Glu His Leu Ile Thr Val

1340 1345 1350

Gln Trp Asn Met Ala Thr Arg Asp Gly Gln Glu Thr His Leu Asp

1355 1360 1365

Phe Lys Met Leu Ile Pro Ser Met Ile Glu Lys Thr Met Asn Ala

1370 1375 1380

Ile Val Ser Glu Lys Val Phe His Leu Ser Val Asn Gln Leu Ile

1385 1390 1395

Gly Pro Lys Ser Ser Ser Pro Leu Arg Arg Lys Gly Phe Leu Asp

1400 1405 1410

Val Asp Phe Glu Gly Lys Arg Ala Asn Val Glu Phe Ala Trp Asp

1415 1420 1425

Ala Asp Arg Asn Pro Asn Lys Lys Ile Lys Ala Glu Val Lys Met

1430 1435 1440

Val Asn Pro Leu Ser Thr Leu Arg Asp Cys Val Val Gln Gly Asn

1445 1450 1455

Trp Ala Tyr Leu Glu Arg Gln His Gln Phe Lys Ala Glu Leu Lys

1460 1465 1470

Leu Ser Asp Ile Arg Ser Trp Phe Val Gly Arg Asn Ser Leu Met

1475 1480 1485

Leu Glu Val Thr Asn Pro Asn Gln Gln Met Tyr Lys Met Asn Ser

1490 1495 1500

Ile Leu Met Val Glu Lys Lys Gly Ser Gly Pro Lys Val Gly Ala

1505 1510 1515

Asp Ile Thr Phe Arg Thr Pro Glu Asn Arg Glu Tyr Lys Trp Asn

1520 1525 1530

Ser Glu Ser Ser Leu Glu Cys Arg Glu Gly Leu Gln Asn Val Arg

1535 1540 1545

Leu Met Thr Lys Ala Asp Leu Val Trp Pro Glu Gly Arg Gln Ser

1550 1555 1560

Ile Val Asn Leu Glu Ala Met His Arg Trp Thr Pro Ser Gln Arg

1565 1570 1575

Glu Ala Glu Leu Lys Ile Glu Val Thr Cys Pro Ser Leu Pro Gln

1580 1585 1590

Pro Ile Lys Thr Lys Leu Gln Leu Asn Asn Gln Gln Gly Glu Tyr

1595 1600 1605

Ser Thr Lys Trp Val Ile Glu Met Gly Phe Pro Val Asn Gly Ala

1610 1615 1620

Met Tyr Lys Leu Lys Leu Ser Pro Glu His Gly Val Gln Gly Phe

1625 1630 1635

Glu Val Glu Leu Asn Leu Glu Glu Val Leu Lys Leu Leu Lys Ala

1640 1645 1650

Ile Asp His Leu Ile Ser Ser Ser Ser Leu Ser Met Ser Asp Ser

1655 1660 1665

Pro Arg Arg Leu Leu Phe Ile Val Leu Thr Thr Asn Ile Met Thr

1670 1675 1680

Thr Ile Leu Ile Pro Cys Cys Leu Met Ser Pro Ser Arg Thr Met

1685 1690 1695

Glu Gly Gly Cys Gly Cys Asn Thr Cys Thr Gly Pro Ser Cys Ala

1700 1705 1710

Asp Cys His Cys Ser Phe Val Pro His Lys Gly Leu Ser Asp Ser

1715 1720 1725

Lys Tyr Glu Ile Ala Phe Arg Arg Ser His Ser Asp Trp Ser Gly

1730 1735 1740

Glu Ser Arg Tyr Glu Gly Arg Val Ser His Pro Gly Leu Glu Arg

1745 1750 1755

Asp Arg Arg Ile Asp Ile Gln Leu Arg Arg Glu Glu Gln Lys Val

1760 1765 1770

Thr Gly Ser Val Glu Leu Asp Ile Phe Leu Arg Pro Glu Asp Lys

1775 1780 1785

Ile Thr Gly Lys Leu Glu Ser Leu Ile Tyr Ala Arg Asn Thr Val

1790 1795 1800

Ile Val Glu Ala Lys Leu Glu Gly Lys Val Leu Lys Asp His Pro

1805 1810 1815

Lys Val Ile Val Arg Gly Ala Tyr Gly Pro Ser Asn Val Gly Phe

1820 1825 1830

Asp Ile Lys Leu His Lys Thr His Ser Ser Pro Thr Ser Phe Arg

1835 1840 1845

Met Ser Gly Arg Tyr Glu Lys Arg Thr Ala Arg Asn Ala Ala Ala

1850 1855 1860

Ser Phe Ile Ile Glu Asn Glu His Gln Lys Thr Ile Asp Ile Ser

1865 1870 1875

Ile Ala Leu His Pro His Gln Ser Pro Trp Cys Tyr Gly Val Arg

1880 1885 1890

Ala Glu Ala Lys Ala Lys Ser Ser Ile Ile Gly Thr Tyr Asp Ile

1895 1900 1905

Tyr Ser Glu Leu Cys Lys Pro Gly Phe Val Ser Leu Thr Thr Lys

1910 1915 1920

Lys His Gly Ser Asp Lys Met Tyr Ile Thr Lys Leu Gly Ile Glu

1925 1930 1935

Gly Met Lys Asn Ile Glu Ala Ser Ile Leu Glu Ala Asn Pro Gln

1940 1945 1950

Thr Met Glu Lys Arg Pro Leu Gly Met Ala Arg Met Val Leu Thr

1955 1960 1965

Ser Pro Thr Met Met Lys Leu Glu Thr Lys Tyr Glu Gly Asp Lys

1970 1975 1980

Leu His Glu Ile Lys Ser Thr Val Arg Asp Met Trp Trp Arg Leu

1985 1990 1995

Val Ser Ser Ala Gly Asn Gly Met Asp Ser Leu Cys Ser Glu Val

2000 2005 2010

Leu Gln Glu Gly Ser Gly Ser Pro Ser Gly His Met Ser Ser Val

2015 2020 2025

Trp Arg Glu Ile Lys Asn Glu Ala Ser Arg Ile Tyr Ser Asp Leu

2030 2035 2040

Glu Asn Asp Leu Val Ile Pro Lys Tyr Glu Lys Leu Arg Glu Trp

2045 2050 2055

Leu His Gly Gly Ile Leu Ser Asn Ile Ala Glu Gly Cys Ser Lys

2060 2065 2070

Val Trp Ser His Tyr Ala His Phe Gln Ser Ser Leu Ser Ser Ser

2075 2080 2085

Val Ser Asn Met Ile Arg Thr Ile Arg Glu Glu Phe Pro Gly Leu

2090 2095 2100

Thr Arg Val Val Thr Glu Val Val Met Gly Thr Ala Arg Gly Leu

2105 2110 2115

Gln Thr Gly Glu Met Pro Glu Val Phe Arg Arg Trp Trp Asn Asp

2120 2125 2130

Phe Leu Glu Ser Ser Phe Cys Arg Ala Ile Ala Ser Asp Phe Glu

2135 2140 2145

Ser Leu Trp Ser Glu His Gln Glu Glu Tyr Gln Gly Leu Gln Gln

2150 2155 2160

Ile Trp Arg Lys Ile Lys Asn Thr Leu Ala Lys Asp Val Asn Arg

2165 2170 2175

Gln Arg Arg Asn Leu Met His Tyr Lys Lys Pro Arg His Leu Val

2180 2185 2190

Asn Trp Ile Val His Asp Met Asn Phe Glu Arg Met Val Phe Lys

2195 2200 2205

Gly Val Asp Lys Val Ile Lys Asn Val Val Gln Lys Ala Leu Phe

2210 2215 2220

Val Pro Phe Gln Ile Asp Gly Asn His Phe Gln Leu Gln Leu Pro

2225 2230 2235

Ile Arg Arg Pro Val His Ser Leu Pro Gln Ala Leu Ser Tyr Val

2240 2245 2250

Ser Met Asn Pro Thr Pro Val Val Asp Arg Ala Leu Trp Trp Phe

2255 2260 2265

Glu Ala Leu Met Pro Thr Pro Val Asp Asn Ile Val Trp Ala Tyr

2270 2275 2280

Tyr Lys Phe Leu Pro Arg His Ala Arg Tyr Leu Leu Pro Pro Tyr

2285 2290 2295

Asn His Thr Ala Met Val Val Asp Gly Ser Glu Ile Leu Thr Phe

2300 2305 2310

Asp Gly Val Val Leu Arg Val Pro His Ser Pro Cys Lys Val Leu

2315 2320 2325

Leu Ala Gln Tyr Lys Thr His Ser Leu Ile Met Gln Asn Gln Gln

2330 2335 2340

Pro Gly His Leu Pro His Phe Ile Leu Lys Ala Ala Gly Ala Thr

2345 2350 2355

Val Glu Val Lys Pro Asp Phe Val Val Thr Val Asn Gly Asn Pro

2360 2365 2370

Val Ser Gly Pro Arg Glu Val Gln Gly Glu Val Val Ile Met Lys

2375 2380 2385

Glu Asn Glu Lys Ile Lys Val Arg Thr Pro Phe Ile Thr Leu Arg

2390 2395 2400

Val Tyr Lys Asp Ser Arg Thr Ala Ser Val Glu Val Ser Gly Trp

2405 2410 2415

Thr Phe Gly Arg Ile Ala Gly Leu Leu Gly Ile Tyr Asp Gly Glu

2420 2425 2430

Lys Ala Thr Asp Arg Leu Thr Pro Gln Gly Thr Arg Ala Ser Asn

2435 2440 2445

Leu Gln Glu Leu Val Lys Ser Trp Gln Glu Asn Pro Gln Cys Glu

2450 2455 2460

Thr Pro His Val Ala Pro Met Ser Pro Met Gln Val Pro Val Val

2465 2470 2475

His Met Leu His Cys Gln Thr Leu Phe Gly Val Arg Ser Arg Cys

2480 2485 2490

Asn Pro Val Val His Pro Gly Pro Phe Arg Lys Met Cys Phe Ala

2495 2500 2505

Ser Lys Asn Ala Cys His Val Ala Lys Ala Tyr Arg Ala Met Cys

2510 2515 2520

Glu Thr Lys Gly Ile Lys Glu Thr Phe Pro Leu Gly Cys

2525 2530 2535

<210> 3

<211> 39

<212> DNA

<213> artificial sequence

<400> 3

taatacgact cactataggt gccaagaaaa agctcctgt 39

<210> 4

<211> 39

<212> DNA

<213> artificial sequence

<400> 4

taatacgact cactataggg ccaaaggttg gtgcatagt 39

<210> 5

<211> 22

<212> DNA

<213> artificial sequence

<400> 5

tctggcgaca gcctcagctg gt 22

<210> 6

<211> 22

<212> DNA

<213> artificial sequence

<400> 6

ctggagcagt caaggttatg gt 22

<210> 7

<211> 22

<212> DNA

<213> artificial sequence

<400> 7

gccagagaaa atggagttgg tg 22

<210> 8

<211> 22

<212> DNA

<213> artificial sequence

<400> 8

gtcaggcgaa acatcacaag tc 22

<210> 9

<211> 23

<212> DNA

<213> artificial sequence

<400> 9

ctgttgcttg atgtcaccct ctc 23

<210> 10

<211> 23

<212> DNA

<213> artificial sequence

<400> 10

ttgatgacag tgaacgttcc tga 23

<210> 11

<211> 22

<212> DNA

<213> artificial sequence

<400> 11

tacagagcac acgattccag ac 22

<210> 12

<211> 22

<212> DNA

<213> artificial sequence

<400> 12

ttggtactga gagcttcctt gg 22

<210> 13

<211> 22

<212> DNA

<213> artificial sequence

<400> 13

cctgtgacct tctgttcctc tc 22

<210> 14

<211> 22

<212> DNA

<213> artificial sequence

<400> 14

accgtgcatt atggtggctt ga 22

<210> 15

<211> 23

<212> DNA

<213> artificial sequence

<400> 15

tcttgttaac tggatcgtcc acg 23

<210> 16

<211> 23

<212> DNA

<213> artificial sequence

<400> 16

aagctctctt cgtacctgtt cag 23

<210> 17

<211> 24

<212> DNA

<213> artificial sequence

<400> 17

attacaggtg tgcagagttc cctc 24

<210> 18

<211> 22

<212> DNA

<213> artificial sequence

<400> 18

aagtacccta cctgaaccac ct 22

Claims (8)

1. a freshwater shrimp vitellogenin, its aminoacid sequence is as shown in SEQ ID NO.2.

2. the freshwater shrimp vitellogenin Vg gene of coding freshwater shrimp vitellogenin claimed in claim 1.

3. freshwater shrimp vitellogenin Vg gene according to claim 2, is characterized in that: its nucleotide sequence is as shown in the 35th in SEQ ID NO.1～the 7645th.

4. by the dsRNA of the freshwater shrimp vitellogenin Vg gene synthesized described in claim 2 or 3.

5. the freshwater shrimp vitellogenin Vg gene described in claim 2 or 3 is in the application delaying in the female freshwater shrimp development of ovary.

6. application according to claim 5, it is characterized in that, comprise the steps: the freshwater shrimp vitellogenin Vg gene in freshwater shrimp DNA sample to carry out pcr amplification, amplified production carried out to the synthetic dsRNA of in-vitro transcription, then by dsRNA to be expelled in the pericardial sac of freshwater shrimp.

7. application according to claim 6, is characterized in that: the sequence of the upstream and downstream primer using in pcr amplification is as shown in SEQ ID NO.3～4.

8. application according to claim 6, is characterized in that: the injected dose in described injecting step is 4 μ g/g.