CN104313031A - Freshwater shrimp molt-inhibiting hormone gene and application thereof in accelerating molting and growing of freshwater shrimps - Google Patents

Abstract

The invention discloses a freshwater shrimp molt-inhibiting hormone gene and application thereof in accelerating molting and growing of freshwater shrimps. The nucleic acid sequence of the freshwater shrimp molt-inhibiting hormone gene is shown by SEQ ID NO:1. A RNA interfering primer is designed by using an RNA interference technology according to an open reading frame of the sequence of the freshwater shrimp molt-inhibiting hormone gene, then PCR amplification is performed by taking freshwater shrimp total cDNA as a template to obtain a sequence shown by SEQ ID NO:4, in vitro transcription is performed according to SEQ ID NO:4 to synthesize dsRNA, and the dsRNA is injected into the hemocelom of a freshwater shrimp to accelerate the molting and growing of the freshwater shrimp. A RNAi method is used, causes small damages to the freshwater shrimps, cannot influence normal ingesting, inhabiting and mating of the freshwater shrimps, is convenient to perform long-term researches and has outstanding effects. The invention provides a new train of thought and an effective means for adjusting and controlling growth and development of the freshwater shrimps and breeding an excellent variety of the freshwater shrimps.

Description

Freshwater shrimp molt-inhibiting hormone gene and the application in acceleration freshwater shrimp casts off a skin and grows thereof

Technical field

The present invention relates to biotechnology and developmental regulation field, be specifically related to a kind of freshwater shrimp molt-inhibiting hormone gene and the application in acceleration freshwater shrimp casts off a skin and grows thereof.

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 is more than 23.7 ten thousand tons, and annual value of production is more than 15,000,000,000 yuan, and shrimp culture has played very important effect in China's aquaculture.In recent years, along with the continuous expansion of large-scale cultivation and improving constantly of consumers demand, cultivate the key that the more excellent new variety of proterties are current freshwater shrimp genetic breedings.

MIH (molt-inhibting hormone, MIH), crustacean hyperglycemic hormone (the crustacean hyperglycemic hormone of synthesis secretion in crustacean optic stalk, CHH) a kind of important neuropeptide in family, has that regulation and control crustacean casts off a skin, the effect of growth activity.Multinomial research in crustacean finds, MIH can suppress the generation of casting off a skin of crustacean, is a kind of important hormone in the regulation and control of crustacean reproduction and development.

RNAi(RNA interference, RNAi) technology is a kind of gene silent technology brought out by double-stranded RNA.The degraded of the mRNA of homologous complementary with it can be induced specifically in cell, the expression of corresponding gene is closed.RNAi to be considered to be in gene functional research one of most effective means, has become the important tool in a lot of fields such as the research of the function of crustacean important gene.But at present, the application in the research of freshwater shrimp important gene not yet has report.

Summary of the invention

The object of the present invention is to provide a kind of freshwater shrimp molt-inhibiting hormone gene and the application in acceleration freshwater shrimp casts off a skin and grows thereof.Utilize the dsRNA of freshwater shrimp molt-inhibiting hormone gene design and synthesis provided by the invention, effectively can accelerate generation that freshwater shrimp casts off a skin thus accelerate the g and D of freshwater shrimp, growing freshwater shrimp improved seeds that are fast, that grown for cultivating new thinking and effective means are provided.

Freshwater shrimp molt-inhibiting hormone gene, its nucleotide sequence is as shown in SEQ ID NO:1.

The application of described freshwater shrimp molt-inhibiting hormone gene in acceleration freshwater shrimp casts off a skin and grows.

Described application, utilize RNA perturbation technique, according to the open reading frame designated rna interference primer of freshwater shrimp molt-inhibiting hormone gene sequence, then with the total cDNA of freshwater shrimp for template carries out pcr amplification, obtain sequence as shown in SEQ ID NO:4, carry out in-vitro transcription synthesis dsRNA with SEQ ID NO:4, after dsRNA being expelled to the pericardial sac of freshwater shrimp, accelerating freshwater shrimp and cast off a skin and grow.

Described RNA disturbs the upstream primer nucleotide sequence of primer as shown in SEQ ID NO:2, and downstream primer nucleotide sequence is as shown in SEQ ID NO:3.

DsRNA be the PCR primer SEQ ID NO:4 that obtained by pcr amplification with upstream primer SEQ ID NO:2 and downstream primer SEQ ID NO:3 purified after, carry out that in-vitro transcription synthesis obtains.

Auricular injected dose dsRNA being expelled to freshwater shrimp is every gram of body weight injection 2 ~ 6 μ g dsRNA, is preferably every gram of body weight and injects 4 μ g dsRNA.

The concrete steps of application are as follows:

First according to freshwater shrimp molt-inhibiting hormone gene (MIH gene) sequence SEQ ID NO:1, in its open reading frame designated rna interference primer, and add before above-mentioned primer T7 promoter sequence ( tAATACGACTCACTATAGGG), determine upstream primer SEQ ID NO:2 and downstream primer SEQ ID NO:3, sequence is respectively: tAATACGACTCACTATAGGGgTAAACCTGACAGCGCAAGG

, tAATACGACTCACTATAGGGcCATCTGTTGAGCTGTTCCA(underscore is T7 promoter sequence).

PCR primer (sequence is as shown in SEQ ID NO:4) is obtained by pcr amplification with above-mentioned upstream primer SEQ ID NO:2 containing T7 promotor and downstream primer SEQ ID NO:3, according to Transcript AidTM T7 High Yield Transcription kit (Fermentas after purified, Inc., USA) test kit illustrates and carries out in-vitro transcription synthesis dsRNA, and its nucleotide sequence is as shown in SEQ ID NO:5.

DsRNA to be casted off a skin developmental application freshwater shrimp: inject after above-mentioned dsRNA divides the pericardial sac being clipped to the female and male shrimp freshwater shrimp that the same period grows and carried out Continuous Observation and data statistics in six weeks.Result shows, and: dsRNA can specific reticent MIH gene mRNA expression, and can accelerate the increase of generation that male and female freshwater shrimp casts off a skin and male shrimp body weight significantly.

Beneficial effect: shedding determines the growth of freshwater shrimp and the key factor of reproduction, to cast off a skin and the main method of growing is realized by eyestalk ablation for accelerating freshwater shrimp at present.But the physical abuse that eyestalk ablation causes freshwater shrimp is very large, and especially juvenile prawn, usually causes high mortality.Utilize the damage of the method for RNAi to freshwater shrimp little, do not affect it and search for food normally, perch and mating, be convenient to carry out long-term research, and action effect is remarkable.

Accompanying drawing explanation

Fig. 1. after adult freshwater shrimp injection dsRNA, in optic stalk, MIH gene is in the mrna expression amount of different sampling point time, and freshwater shrimp β-Actin gene is as reference gene.2,4,6,8,10,12 are respectively latter 2 days of injection, 4 days, 6 days, 8 days, 10 days and 12 days (N=3).**P < 0.01。The DEPC water of control group injection same dose.

Fig. 2. during continuity RNAi, freshwater shrimp casts off a skin the audio-visual picture of change of the frequency (a) and body weight (b).Wherein: group 1, group 2, group 3, group 4 is respectively that group disturbed by male dried shrimps, group disturbed by male shrimp control group, female dried shrimps, the DEPC water of female shrimp control group control group injection same dose.

embodiment

Embodiment 1: the acquisition of freshwater shrimp MIH full length gene cDNA

Total RNAs extraction: choose ripe freshwater shrimp two, aseptic Dissecting scissors Eyestalk tissue, in RNA conserving liquid (TaKaRa Bio Inc. Japan), carry out optic stalk pigment part to clip broken, carry out freshwater shrimp optic stalk Total RNAs extraction after rinsing pre-treatment, concrete operation step is with reference to Takara Trizol test kit.

First chain cDNA synthesizes: with above-mentioned total serum IgE, obtains the first chain cDNA with reference to after the reverse transcription of Takara M-MLV Reverse Transcription box.

The acquisition of freshwater shrimp MIH full length gene cDNA:

Foundation Macrobrachium rosenbergii ( macrobrachium rosenbergii, KC990939.1) and the conservative region of MIH cDNA, design two, to middle primer, is shown in SEQ ID NO:6 ~ 9,

（ middle F1：5′-CCGGCATTCCTTTGTTTACCTG-3, middle R1：5′-ATATTCTGGCGTGTGGTCCTG-3′;

MiddleF2:5 '-CCTGAGTGTCTGTCCAGTTTGA-3 ', middle R2:5 '-TGGTCCTGGAGCTTATTTTAGAGG-3 '), carry out the clone of intermediate segment with above-mentioned first chain cDNA for template, it is as follows to carry out pcr amplification reaction system:

RT liquid	2 μL
		10×PCR Buffer	2.5 μL
MgCl 2(25mM)	2 μL
		dNTP Mixture(25mM each)	0.2 μL
Forward Primer(10μM)	1 μL
		Reverse Primer(10μM)	1 μL
Taq DNA Polymerse(5U/μL)	0.2 μL
		dH 2O	up to 25 μL

PCR response procedures is: 94 DEG C of denaturation 3min, then enter following circulation: 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations, and last 72 DEG C extend 10min, 4 DEG C of preservations.1.2% agarose gel electrophoresis detects.

The pillar DNA glue of Shanghai Sheng Gong biotechnology company limited (Sangon) recovery test kit is utilized 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 inserting object fragment is delivered to Shanghai Bo Shang biotech firm and carries out sequencing analysis, obtain the intermediate segment of freshwater shrimp MIH gene cDNA.

According to the intermediate segment sequence of the freshwater shrimp MIH gene cDNA obtained, utilize RACE technology amplification 3 ' end, 5 ' end, operation steps is undertaken by TaKaRa 3 '-RACE and TaKaRa5 '-RACE test kit specification sheets.PCR primer, through cutting glue recovery, purifying, clone, order-checking, then carries out with the intermediate segment of acquired freshwater shrimp MIH gene cDNA splicing rear acquisition freshwater shrimp MIH full length gene cDNA sequence, as shown in SEQ ID NO:1.

Embodiment 2: the acquisition of the dsRNA of freshwater shrimp MIH gene

According to the freshwater shrimp MIH gene (Accession number:KF878973) of cloning acquisition, the online sequence analysis software of employing NCBI ( http:// www.ncbi.nlm.nih.gov/gorf/gorf.html) determine that the open reading frame position of MIH gene is 436-795bp.According to the above results, adopt online dsRNA primer-design software ( http:// www.flyrnai.org/cgi-bin/RNAi_find_primers.pl), dsRNA primer is designed in freshwater shrimp MIH open reading frame, and T7 promoter sequence 5 '-TAATACGACTCACTATAGGG-3 ' is added before every bar primer, composition dsRNA synthetic primer, its primer sequence is respectively upstream primer SEQ ID NO:2 and downstream primer SEQ ID NO:3.All primers all synthesize in Bo Shang biotechnology (Shanghai) Co., Ltd..

Adopt above-mentioned freshwater shrimp dsRNA primer enterprising performing PCR amplification (condition is with embodiment 1) of the total cDNA of freshwater shrimp, obtain SEQ ID NO:4, after the SanPrep pillar PCR primer Purification Kit of Sangon Biotech (Shanghai) Co., Ltd., according to TranscriptAid ^tMt7 High Yield Transcription kit (Fermentas, Inc., USA) test kit illustrates and carries out in-vitro transcription synthesis dsRNA, and sequence is as shown in SEQ ID NO:5.DsRNA concentration adopts BioPhotometer (Eppendorf, Hamburg, Germany) to measure at 260 nm, and regulates final concentration to be 4 μ g/ μ l.

Embodiment 3: the dsRNA of injection freshwater shrimp MIH gene to be casted off a skin the impact of growing on freshwater shrimp

1. the test selection of shrimp

For the silencing efficiency of research dsMIH gene, first this research carried out disposable RNAi experiment and selected vigor comparatively by force, evenly individual, body weight is the female shrimp of the adult of about 1.50 ± 0.35g 50, be divided into two groups, one group is injection dsRNA group, and another group is injection DEPC water group (control group).Before experiment, in the vinyon white bucket of 500L, inflation supports 72 h temporarily, makes it adapt to Laboratory culture environment, 1 spiral shell of sooner or later respectively throwing something and feeding every day.

According to the result design continuity RNAi experiment that disposable RNAi tests, continuity RNAi tests the juvenile prawn that shrimp used is the same batch of membrane of being hatched by this laboratory, male and female are distinguished under Stereo microscope, select female, the male shrimp each 30 of body weight at about 0.60 ± 0.25g, be divided into four groups (n=15): group disturbed by male dried shrimps and group injection dsRNA disturbed by female dried shrimps; The DEPC water of two control group injection equivalent.Experiment is carried out in the winter time 11 ~ December, and water temperature is progressively risen to 25 DEG C by experiment first two weeks, makes it adapt to this temperature, 1 spiral shell of sooner or later respectively throwing something and feeding every day.

2. the injection of the dsRNA of freshwater shrimp MIH gene and detection

Inject the dosage of 4 μ g dsRNA according to every gram of body weight, dsRNA solution, as entry needle, injects in pericardial sac from freshwater shrimp carapace base portion by the capillary tip drawing-down taking internal diameter as 1.2mm, control group injection DEPC water.The sampling of disposable RNAi experiment was respectively at after injection the 2nd day, the 4th day, the 6th day, the 8th day, the 10th day and the 12nd day, and each sampling point designs 3 and biologically to repeat, and gathered optic stalk and was organized in RNA conserving liquid the extraction carried out for RNA after pre-treatment.Continuity RNAi carried out same substance, same dose after-teeming every 7 days to each group of shrimp, whole process lasts 6 time-of-week.In experimentation, every day is added up the shrimp that casts off a skin in each group, measures and record respectively, the results are shown in Figure 1 and table 1 before experiment and at the end of experiment to the body weight of each group of shrimp.

The freshwater shrimp that table 1. is added up after being in continuous six weeks of female, the male freshwater shrimp injection dsRNA of same developmental stage casts off a skin the change of the frequency and body weight.

Group variable	Group disturbed by male dried shrimps	Male shrimp control group	Group disturbed by female dried shrimps	Female shrimp control group
					The frequency of casting off a skin (number of times)	17	2	12	5
Body weight evolution (g)	0.26	0.06	0.08	0.04

3. the detection of freshwater shrimp MIH gene silencing efficiency

Extract the total serum IgE of each sample and be inverted to cDNA, adopting Real Time PCR to detect the expression amount of MIH gene relative to reference gene (freshwater shrimp β-Actin), to calculate the efficiency of MIH gene silencing.

4. freshwater shrimp MIH gene dsRNA is casted off a skin on freshwater shrimp and the impact of body weight

In experimentation, every day is added up the shrimp that casts off a skin in each group, measures respectively, record and carry out statistical study before experiment and at the end of experiment to the body weight of each group of shrimp.

5. interpretation of result

As shown in Figure 1, relative to control group, latter 2nd day of injection, the expression amount of freshwater shrimp MIH gene significantly have dropped 61%, have dropped 93% at the 6th day, have dropped 91% at the 8th day.67% and 62% is have dropped respectively the 10th day and the 12nd day.

As shown in table 1 and Fig. 2, during carrying out continuity RNAi, there is significant difference in the frequency of casting off a skin for the treatment of group and control group, and the frequency of casting off a skin for the treatment of group is significantly higher than control group.In six weeks, the male shrimp of experimental group and female shrimp have 17 times, 12 times records of casting off a skin respectively, and corresponding control group is only 2 times, 5 times.The male shrimp weight average of experimental group increases 0.26g, increase compared with before experiment significantly ( p<0.05) body weight increases are not remarkable, and before and after other three groups experiments.

From above result, compared with control group, the dosage of the dsRNA of 4 μ g is injected with every gram of body weight, after the dsRNA of shot freshwater shrimp MIH gene, the expression amount of freshwater shrimp MIH gene significantly declines, the 6th day time, interference effect reaches maximum, and this interference effect time length is long, and when the 12nd day, difference is still remarkable.This experimental studies results shows, the dsRNA of freshwater shrimp molt-inhibiting hormone gene provided by the invention effectively can accelerate the frequency of casting off a skin of female, male freshwater shrimp, and the increase Be very effective to male shrimp body weight.New thinking and effective means is provided for cultivating freshwater shrimp proterties improved seeds.

Sequence table

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Claims (6)

1. freshwater shrimp molt-inhibiting hormone gene, is characterized in that: its nucleotide sequence is as shown in SEQ ID NO:1.

2. the application of freshwater shrimp molt-inhibiting hormone gene described in claim 1 in acceleration freshwater shrimp casts off a skin and grows.

3. application according to claim 2, it is characterized in that: utilize RNA perturbation technique, according to the open reading frame designated rna interference primer of freshwater shrimp molt-inhibiting hormone gene sequence, then with the total cDNA of freshwater shrimp for template carries out pcr amplification, obtain sequence as shown in SEQ ID NO:4, carry out in-vitro transcription synthesis dsRNA with SEQ ID NO:4, after dsRNA being expelled to the pericardial sac of freshwater shrimp, accelerating freshwater shrimp and cast off a skin and grow.

4. application according to claim 3, is characterized in that: described RNA disturbs the upstream primer nucleotide sequence of primer as shown in SEQ ID NO:2, and downstream primer nucleotide sequence is as shown in SEQ ID NO:3.

5. application according to claim 3, is characterized in that: described dsRNA nucleotide sequence is as shown in SEQ ID NO:5.

6. application according to claim 3, is characterized in that: auricular injected dose dsRNA being expelled to freshwater shrimp is every gram of body weight injection 2 ~ 6 μ g dsRNA.