CN108728445B - CARP-1 gene and application thereof - Google Patents

CARP-1 gene and application thereof Download PDF

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CN108728445B
CN108728445B CN201810575733.6A CN201810575733A CN108728445B CN 108728445 B CN108728445 B CN 108728445B CN 201810575733 A CN201810575733 A CN 201810575733A CN 108728445 B CN108728445 B CN 108728445B
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李法君
付春鹏
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Weifang University of Science and Technology
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Abstract

The invention relates to the field of crustacean growth and development, in particular to a CARP-1 gene and application thereof in promoting the growth of macrobrachium nipponense. The Macrobrachium nipponensis CARP-1 gene has a base sequence shown in SEQ ID NO. 1. The primer provided by the invention clones a CARP-1 gene from the muscle of macrobrachium nipponensis; an RNA interference primer is designed according to an open reading frame of the CARP-1 gene, and double-stranded RNA is injected into the macrobrachium nipponense body to silence the CARP-1 gene, so that the molting cycle of the macrobrachium nipponense is shortened, the growth speed of the macrobrachium nipponense is accelerated, and theoretical reference is provided for improving the production performance of the macrobrachium nipponense. The invention firstly clarifies that the CARP-1 gene has the function of regulating and controlling the growth and development of crustaceans.

Description

CARP-1 gene and application thereof
Technical Field
The invention relates to the field of crustacean growth and development, in particular to a CARP-1 gene and application thereof in promoting the growth of macrobrachium nipponense.
Background
Growth and development are the most basic biological processes of animals, and crustaceans are no exception. Due to the existence of chitin exoskeletons, researches on the growth and development of crustaceans mostly focus on the regulation and control of molting mechanisms, and a lot of research results are obtained. It is undeniable that the molting mechanism does play an important role in the growth and development of crustaceans. It is also to be clearly understood that the growth of crustacean carcasses is responsible for molting. From this perspective, molting is merely an outward manifestation of crustacean growth. Therefore, the identification of important genes of crustacean body growth not only helps to understand the basic biological process of crustacean growth and development, but also has important production significance for improving the yield and quality of crustaceans by molecular means.
Macrobrachium nipponensis (Macrobrachium nipponense) commonly called freshwater shrimp and river shrimp belongs to Macrobrachium, brachialidae and Macrobrachium. Has the characteristics of strong adaptability, high growth speed, strong fecundity, large individual, rich nutrition and the like, and is an important freshwater economic shrimp in China. According to the 'annual book of Chinese fishery statistics in 2017', the annual output of freshwater shrimps cultured in China is 272,592 tons. The cultivation of the macrobrachium nipponensis becomes one of the important means for increasing the production and creating income for fishermen, and plays an important role in the aquiculture in China. With the recent continuous expansion of the culture scale, macrobrachium nipponensis has the phenomena of production performance reduction and germplasm resource degradation, wherein the individual miniaturization is particularly prominent, and finally, the specification of commercial macrobrachium nipponensis is reduced, the quality of the macrobrachium nipponensis is seriously influenced, and the macrobrachium nipponensis becomes one of the bottlenecks of the development of the macrobrachium nipponensis culture industry. In order to breed good varieties of large-size macrobrachium nipponensis, the identification of important genes for regulating the growth and development of macrobrachium nipponensis becomes a problem to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a CARP-1 gene and application thereof in promoting growth of macrobrachium nipponense.
In order to achieve the purpose, the invention adopts the technical scheme that:
a CARP-1 gene, Macrobrachium nipponensis CARP-1 gene has a base sequence shown in SEQ ID NO. 1.
The Macrobrachium nipponensis CARP-1 gene is a sequence which has 90 percent of homology with a base sequence shown by SEQ ID NO. 1.
The double-stranded RNA of the Macrobrachium nipponensis CARP-1 gene is a base sequence shown in SEQ I NO: 5.
An application of a CARP-1 gene, namely an application of double-stranded RNA of the CARP-1 gene of macrobrachium nipponensis in promoting the growth of the macrobrachium nipponensis.
A preparation for promoting growth of Macrobrachium nipponensis comprises solution of double-stranded RNA (dsRNA) synthesized in vitro based on the CARP-1 gene of Macrobrachium nipponensis; solution refers to DEPC water or sterile water.
By using RNA interference (RNAi) technology, an interference primer is designed according to an open reading frame of a base sequence CARP-1 gene shown in SEQ ID NO. 1, PCR reaction is carried out by taking total muscle RNA of the macrobrachium nipponensis as a template, then double-stranded RNA (dsRNA) shown in SEQ I NO. 5 is synthesized in vitro by taking PCR reaction liquid as the template, and the obtained double-stranded RNA (dsRNA) is dissolved in water to obtain the preparation (dsRNA-CARP-1) for promoting the growth of the macrobrachium nipponensis. The water is DEPC water or sterile water.
Further, the following steps are carried out:
designing an interference primer according to an open reading frame of the CARP-1 gene, carrying out PCR reaction by taking total muscle RNA of the macrobrachium nipponensis as a template (the obtained sequence is shown as SEQ ID NO: 4), carrying out in vitro synthesis of dsRNA-CARP-1 by taking PCR reaction liquid as the template, dissolving the dsRNA-CARP-1 in water, injecting the dissolved dsRNA-CARP-1 into the pericardial cavity of the macrobrachium nipponensis with the dosage of 5 mu g/g, and further promoting the growth of the macrobrachium nipponensis.
The primer is a forward upstream primer sequence shown as SEQ ID NO. 2, and a reverse downstream primer sequence shown as SEQ ID NO. 3.
The application of a preparation in interfering the CARP-1 gene to promote the growth of the macrobrachium nipponense; wherein the dosage used is 5. mu.g/g.
The invention has the advantages that:
the invention firstly identifies the CARP-1 gene related to the growth and development of the macrobrachium nipponense, the growth of the macrobrachium nipponense can be accelerated by the CARP-1 gene under the condition of reduced expression level, and the CARP-1 gene is proved to be a negative regulation factor for the growth and development of the macrobrachium nipponense. The invention provides dsRNA-CARP-1 to the bodies of macrobrachium nipponensis, the expression change of the dsRNA-CARP-1 under the stimulation of the dsRNA and the change of the molting cycle and the body weight of the macrobrachium nipponensis on the same day clarify the function of the CARP-1 gene and promote the research of the CARP-1 in other crustaceans.
Drawings
FIG. 1 is a graph showing the interference efficiency of male Macrobrachium nipponensis after injection of dsRNA-CARP-1 according to an embodiment of the present invention, in which: 7d, 14d, 21d and 28d refer to day 7, 14, 21 and 28, respectively, post-injection. P <0.01, the interfering group was injected with dsRNA-CARP-1, and the control group was injected with the same dose of DEPC water.
FIG. 2 is a graph showing the interference efficiency of female Macrobrachium nipponensis after injection of dsRNA-CARP-1 according to an embodiment of the present invention, in which: 7d, 14d, 21d and 28d refer to day 7, 14, 21 and 28, respectively, post-injection. P <0.01, the interfering group was injected with dsRNA-CARP-1, and the control group was injected with the same dose of DEPC water.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention.
The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as "about," is not to be limited to the precise value specified. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Unless context or language indicates otherwise, range limitations may be combined and/or interchanged, and such ranges are identified and include all the sub-ranges included herein. Other than in the operating examples, or where otherwise indicated, all numbers or expressions referring to quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as modified in all instances by the word "about".
The primer provided by the invention clones a CARP-1 gene from the muscle of macrobrachium nipponensis; an RNA interference primer is designed according to an open reading frame of the CARP-1 gene, and double-stranded RNA is injected into the macrobrachium nipponense body to silence the CARP-1 gene, so that the molting cycle of the macrobrachium nipponense is shortened, the growth speed of the macrobrachium nipponense is accelerated, and theoretical reference is provided for improving the production performance of the macrobrachium nipponense. The invention firstly clarifies that the CARP-1 gene has the function of regulating and controlling the growth and development of crustaceans.
Example 1: obtaining of Macrobrachium nipponense CARP-1 gene full-length cDNA
Total RNA extraction: selecting about 3g of macrobrachium nipponensis, taking the muscle of the macrobrachium nipponensis, putting the macrobrachium nipponensis into a precooled mortar containing liquid nitrogen, and quickly grinding. Muscle total RNA was extracted using the Takara RNAiso Plus extraction reagent in combination with a traditional phenol-mimetic extraction method. Detecting the quality of RNA through 1.2% agarose gel electrophoresis, analyzing the OD260/280 ratio of the sample to be 1.8-2.0 by a spectrophotometer, and determining the concentration of the RNA.
First strand cDNA Synthesis: first strand cDNA was synthesized using the above RNA as a template according to the Takara M-MLV reverse transcription kit instructions.
Cloning of the full-length cDNA sequence of Macrobrachium nipponensis CARP-1: the verification of the intermediate fragments was carried out by designing forward primers MF1(SEQ ID NO:6), MR1(SEQ ID NO:7) and MF2(SEQ ID NO:8) and MR2(SEQ ID NO:9) based on the intermediate fragments of the muscle transcriptome of Macrobrachium nipponensis. The PCR amplification reaction system is as follows:
Figure BDA0001686839820000031
the PCR reaction program is: pre-denaturation at 94 ℃ for 3min, then entering the following cycle: 30s at 94 ℃, 30s at 55 ℃, 90s at 72 ℃, 30 cycles, final extension for 5min at 72 ℃ and storage at 4 ℃. Detection by 1.5% agarose gel electrophoresis.
The method comprises the steps of utilizing a column type DNA gel recovery kit of Shanghai biological engineering Co., Ltd (Sangon) to recover target fragments, connecting products to a pMD18-T vector (Takara), transforming the vectors into escherichia coli DH5 α, carrying out blue-white spot screening, picking out single-clone white spot amplification culture, and sending positive clones inserted with the target fragments to Shanghai platinum-Shang biological company for sequencing analysis.
According to the cDNA intermediate fragment sequence of the CARP-1 gene, specific forward primers 3F (SEQ ID NO:11) and 5R (SEQ ID NO:12) are designed to carry out 3 'and 5' rapid amplification respectively, and the operation steps are as follows
Figure BDA0001686839820000041
RACE 5 '/3' Kit instructions. The subsequent steps of cutting gel, recovering, transforming, cloning and sequencing are carried out according to the cloning steps to finally obtain the 3 'end sequence and the 5' end sequence of the CARP-1 gene. The sequencing results of the intermediate fragment and the 3 'and 5' ends are compared and spliced to obtain the full-length cDNA sequence (SEQ ID NO:1) of the Macrobrachium nipponensis CARP-1 gene.
Example 2: synthesis of Macrobrachium nipponensis CARP-1 gene dsRNA
Based on the nucleotide sequence of SEQ ID NO:1, a primer (SEQ ID NO: 2; SEQ ID NO:3) for preparing double-stranded RNA is designed in the open reading frame of the CARP-1 gene, and then the following PCR reaction is carried out by taking the total muscle RNA of the macrobrachium nipponensis as a template,
Figure BDA0001686839820000042
the obtained sequence is shown in SEQ ID NO. 4.
Then, in vitro Transcription was performed according to the instruction of the Transcript AidTM T7High Yield Transcription kit (Fermentas, Inc., USA) kit to synthesize dsRNA, and in vitro synthesis of double-stranded RNA (dsRNA) using the PCR reaction solution as a template was performed as shown in SEQ ID NO: 5.
The in vitro synthesized double-stranded RNA (dsRNA) obtained above was identified by 1.5% agarose gel, and then the dsRNA sequence (dsRNA-CARP-1) was dissolved in DEPC water for use.
Example 3: effect of dsRNA-CARP-1 injection on growth and development of Macrobrachium nipponense
1. Selection of experimental shrimps
Selecting 60 adult macrobrachium nipponensis with strong activity, uniform individuals and weight of about 2g respectively, evenly dividing the adult macrobrachium nipponensis into two groups (30 per group), wherein one group is an experimental group (RNAi group), namely the obtained dsRNA-CARP-1 solution, and the other group is a DEPC water group control group. The injection dose of the experimental group and the control group is 5 mug/g; air is filled in the glass jar before the experiment for temporary culture, the water temperature is 25 ℃, so that the culture environment of the laboratory is adapted, and the snail and artificial bait are fed in the morning and at night every day.
dsRNA injection and growth parameter detection
At an injection dose of 5. mu.g/g, dsRNA-CARP-1 solution was injected into the pericardial cavity from the basal part of the cephalic turbinate of Macrobrachium nipponensis, and DEPC water was injected into the control group. The intervention time was 4 weeks, once weekly. Muscle tissues were taken at 7d, 14d, 21d and 28d, respectively, and stored in RNA storage solutions. Finally, the total RNA of each sample is extracted and inverted into cDNA. The interference efficiency is calculated by detecting the change of the expression level of CARP-1 relative to a control group by adopting a fluorescent quantitative PCR technology. The body weights of the control group and the experimental group were measured at the end of the experiment, and the data were analyzed (see fig. 1, fig. 2, and table 1).
Interference efficiency is expressed amount after interference/expression amount before interference
As shown in FIG. 1, the expression level of CARP-1 in male Macrobrachium nipponensis in the experimental group was decreased by 88.81%, 86.72%, 85.55% and 91.80% at 7d, 14d, 21d and 28d after the interference, respectively, as compared with the expression level of muscle in the control group.
As shown in FIG. 2, the expression level of female Macrobrachium nipponensis CARP-1 in the experimental group was decreased by 87.52%, 88.82%, 89.522% and 86.95% at 7d, 14d, 21d and 28d after the interference, respectively, as compared with the expression level of muscle of Macrobrachium nipponensis in the control group.
The results show that the dsRNA-CARP-1 dissolving solution effectively reduces the expression level of CARP-1.
TABLE 1 changes in molting individuals and mean body weight in Macrobrachium nipponensis RNAi experiments
Figure BDA0001686839820000051
As shown in table 1, the numbers of molts of male and female shrimps in the RNAi-interfered group were 25 and 22, respectively, while the numbers of molts of male and female shrimps in the control group were 12 and 10, respectively. Compared with a control group, the average body weight of male and female shrimps in the RNAi interference group is increased by 0.31g and 0.29g respectively.
The results show that the interference of the CARP-1 gene can shorten the molting cycle and accelerate the growth of the macrobrachium nipponense, so the CARP-1 plays an important regulation and control function in the growth and development process of the macrobrachium nipponense.
SEQ ID NO:1
Figure BDA0001686839820000052
Figure BDA0001686839820000061
Figure BDA0001686839820000071
SEQ ID NO:2
TAATACGACT CACTATAGGG AGCGTGTATT CACTGGGACC
SEQ ID NO:3
TAATACGACT CACTATAGGG TCCCGTCTAG GTGATCTTGG
SEQ ID NO:4
Figure BDA0001686839820000072
SEQ ID NO:5
Figure BDA0001686839820000073
SEQ ID NO:6
GAAGTCTAAT TGGACAGCCT CC
SEQ ID NO:7
AGACCTTTGC TGTTAAGCAT CC
SEQ ID NO:8
GGATGTCCGC AGCAGCTCGA GG
SEQ ID NO:9
CGTGAGATTT CCGAGCTTCG TC
SEQ ID NO:10
GATGGTGAAG TGACTGTCAA TACAGGAAG
SEQ ID NO:11
GAGGCTGGAT TTGTGCACTA GTCATCTGG
SEQ ID NO:11。
Sequence listing
<110> Weifang science and technology college
<120> CARP-1 gene and application thereof
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<213> Macrobrachium nipponense CARP-1 gene (Macrobrachium nipponense)
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cagccatcat caatactaca gcggccatat tgaagtaaac aagcgacccg tattcgtgcg 60
atctggagga tatattcgct cggggtctta ggtgagggag gatggcacaa tttggtggtg 120
ggaaagcacc accaccatgg cagcagaggc cgcagacaac tcccacacct caactgcagc 180
catcgctcct tggccagcat ccatctgcac ctactcagca aattgtgata ggaggagcac 240
cacagcaacc tcagtatgct cctgtacagc agagtattat ccagcctcca ggcttgggtg 300
gactacaaac tttgggccaa cccactataa cacagcaaac cataggtcaa ccaactacag 360
gtggaagtct aattggacag cctcctatca catgtcaggc tctacaacct cctagtctta 420
ttaatccagc ccagatgact agtgcacaaa tccagcctca gattcagcct cagatacaac 480
ctcaggtctc tgctgtaaca tacccaacac caagagctct tgctcctcca ggtgctttta 540
atccccctgc tgctccaaac aatttaggaa atagtattgc aaataatata caaacaaatt 600
tacaaactaa tctcactacc aacaatgcag cccaacaacc tcagaaacag cgtgtattca 660
ctgggaccat aacaaaactt tgtgctgact ttggatttgt tgatgaggat gtgttcttcc 720
agaccagttg tgtcaaaggt caggcaccga aagtttgtga tagggtattg gtggaagcta 780
cctataatcc taatatgcca tttaaatgga atgccaaccg cgttcaagtg attccagggc 840
ccactgtggc ccctgcccag accccaagta gaagtggtcc tcctgctaga gaaataaaca 900
gtcgtggggg atcttacaat gctgtacctc caccaaattt cacatctgaa tcacaagttg 960
gaaattttag tggtcgagga ggtataggcc cacggccaag atcacctaga cgggaacgac 1020
gggaagaacg cagccgtaca gcgagggaca gtgaacgtga ccgtagtgaa aaagagaaag 1080
aggaaaagag agaaagtcgg aagagaagta gaagtagagg gcggtcgcgt tccccacctc 1140
gtcgaagaat gagagtagtt cctagatata atgttcaagt tcctaagatt ttattacata 1200
tgaaagaggc aaatgtttta gaattgaaaa aacgatataa caacctttat gttccatcgg 1260
atgtattctt ggcccgaccg ttatggtcag atacctttcc tgcacacagg tcctttcagt 1320
ttccccgtcc tgtctctttt catataatga ataaggaagt ggaacctgtt ctagaaaatg 1380
atgccatata tgatccccct gatgtcaacc attgctatgc tgccaaggta atgttgatgg 1440
cagtaccagc tgtggaagac attttcagaa gatcgtgtgc attgagtgaa gaccatgcgg 1500
atgttcgcga ctgctttgtt catccaacac gtcttataaa ctttttggtc ggtttgaaag 1560
gcaagaatga aactatggct attggtggtt actggagccc atcgcttgat ggtcccaacc 1620
ctgagaagga tccgaatgtc ctcatacaca cggccattag gacctgcaaa gcactgaccg 1680
gcattgactt agcagggtgc actacctggt accggtttgc ggagatttac tatcgacgag 1740
gagaatccac acacaagggc cggagctcgc ctgcccgggt tgaaaccact gtactatttc 1800
tcccagatgt gtggaacgtt tgcccaacta aactcgagtg ggacggcctc cacctcaact 1860
ataagcggca gttggacaag aagctctcct cctcggcagg atgtccgcag cagctcgagg 1920
atctagaggg ggaggaggaa gacaaggaag aagctgttag gaagaatccc agccattatg 1980
ccagcttaga cccaaagacc atgaaggtgg ttgaactgcg atctgaatta gatgcacgga 2040
tgcttaacag caaaggtctc aaatcacagc tgatcgctcg tttaacaaag attttgaaaa 2100
atgagcagga aaaggaggaa atggagaaag ctgcagcagc agaaagtgca gaagtagatg 2160
aaaccaaaaa gaaggaagag gaggaaagga aaagagaaga agagaagaaa agaaaagaag 2220
aagaagagag aaagaaagaa gaggaggaga aaaagaaagc tgaagaacgg gagagaaacc 2280
tgctagaaaa acgttacacc cttcctgatc aacccatgat tgttgttcat ccatcacgaa 2340
ctgcaaagtc tggaaaattt gactgtacaa ccatgtcctt gtcggtgttg ttagattaca 2400
gacaggagga taataaagag cactcgtttg aagtaagcct ttttgctgag ctcttcaatg 2460
aaatgctgat gagggacttt gcttttcgca tttacagagc actgtatgaa tctccagaaa 2520
agccaaataa agaagagaag aaagacaagg aggacaaaaa agaaaaagac aaagacaaaa 2580
gagagaaaga caagaaagat gaggaaaagg acaaaaagga ggataaggat aaaaaggaag 2640
aatcagatga gaagatggaa gaggaaaaat cagctgacaa gaaggaaaag aaaacagctg 2700
aaaaggatga ggaaaagaag gaagaggagg aagaagatga agaaatggag gaagacgaag 2760
aggaggagat ggatgatgaa gattctaagg atgaaatgcc aagtacaaag cgaggggaca 2820
gcaaagaaag gaaagaaagc aaagaaggaa ggagaaaaga tgataaaaaa gacagggaaa 2880
gaagagaaaa gaaagagaag aagaaaatgt ttacaattga tccctatctc ctcctttctt 2940
ttgtatactt tgaccaaacg catacgggtt atattattga cagggacttg gaagacataa 3000
ttaacatgct gggactaaat ctctcgagag cccagcaaaa gaagttgatg tgtaaagtta 3060
tgtcgcgtga tgtcctccac tatcgcaaac tgaccgatgt tccactggag gataaggaca 3120
agcctaggga ccctgcacca actattgaca tggagagctt ggcaaaagga aataacatgg 3180
tgatgcctgt gttcttggat gctacaaatg ctcccagtgc agtagatata aaatcactct 3240
ccaagaaaaa cataagaatc aaagaggaac cactatcatc agttaatgat ggctcccaga 3300
acaaagagca gaatgatggt gaagtgactg tcaatacagg aagtcaaggt gttgtgaagt 3360
atggaggatc tcttgtcgac attggaaaat taatacagca attagaaaaa tctgagaatg 3420
ctagaaacag cacagaggag aagctgagag agttgcagaa agaactaatg gccagtaaag 3480
acgaagctcg gaaatctcac gaaaagtgtc tgaaattaag taaagaagtc aaagcatcca 3540
actctagtct caagacaatg gaagaggagc ttcagaaagc cattgaggac aataatgtgt 3600
atcaaaaagc cctgaatcaa atcaagactg tgataaaacc tctggtagcc ggagttgatc 3660
tggacgatga ggacatccca ataaaagtga aagtggagcc caaggaaaat gggactgttg 3720
agtgatatgc aaacagcata gctgtaacac ggtgcaaaga tagcatggac aattttttat 3780
ttaaatagtt caaaatgaat attttagtga gtagagattt attgaaacct gttgcaaggt 3840
gtgtgactct tatccagtgc aaaagagtaa tatttctata tttatttcgg tataaataga 3900
gaaacaggga atattgagtg gtggaatgct gtgtaacaaa ctgaagttgt aataccaagt 3960
tgcaccaaac cataatgcag gggaatttca ttacagtact tatgattttc aaatgcagta 4020
gcacttagaa gctcagggta ttgttagtgt ttgttgttgt agattttgtg taccattgtt 4080
tcatttgata ctgtctcttg atatcttctc tcaaaaggaa ttacattggt atacgtaatt 4140
aagtacaatg tgattatatt cagattgttt ttgtttattc atgtatttgt gacacaatat 4200
ttcagtaaca tttatcctgt gtataagtga ttgaaaagtg aattaattta atgttatttc 4260
actcaacagt gtacagtaat acgtgacaat tttgtaacat atatcctcaa attatatttt 4320
catttggcta tgcaataaat catgacaaaa aattattgaa ttttagttgt ttgtaggttt 4380
gtccactgct atatgtgtgt gttccgtatg ttattgttct gaggtgatgt cttgattgcc 4440
tctttatttt ttatggaatg ctaatgattg tatttattag agagtatgag tagtgcttga 4500
tattcagttt ctaaattgag catctaagtg ctaatatgat gcccatccct tccactcaaa 4560
agttgtgttg aacacgagaa tgggtgcagt tgctccaggg aaatgtagaa agatgattga 4620
tatgtagcat 4630
<210>2
<211>1240
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
cagccatcat caatactaca gcggccatat tgaagtaaac aagcgacccg tattcgtgcg 60
atctggagga tatattcgct cggggtctta ggtgagggag gatggcacaa tttggtggtg 120
ggaaagcacc accaccatgg cagcagaggc cgcagacaac tcccacacct caactgcagc 180
catcgctcct tggccagcat ccatctgcac ctactcagca aattgtgata ggaggagcac 240
cacagcaacc tcagtatgct cctgtacagc agagtattat ccagcctcca ggcttgggtg 300
gactacaaac tttgggccaa cccactataa cacagcaaac cataggtcaa ccaactacag 360
gtggaagtct aattggacag cctcctatca catgtcaggc tctacaacct cctagtctta 420
ttaatccagc ccagatgact agtgcacaaa tccagcctca gattcagcct cagatacaac 480
ctcaggtctc tgctgtaaca tacccaacac caagagctct tgctcctcca ggtgctttta 540
atccccctgc tgctccaaac aatttaggaa atagtattgc aaataatata caaacaaatt 600
tacaaactaa tctcactacc aacaatgcag cccaacaacc tcagaaacag cgtgtattca 660
ctgggaccat aacaaaactt tgtgctgact ttggatttgt tgatgaggat gtgttcttcc 720
agaccagttg tgtcaaaggt caggcaccga aagtttgtga tagggtattg gtggaagcta 780
cctataatcc taatatgcca tttaaatgga atgccaaccg cgttcaagtg attccagggc840
ccactgtggc ccctgcccag accccaagta gaagtggtcc tcctgctaga gaaataaaca 900
gtcgtggggg atcttacaat gctgtacctc caccaaattt cacatctgaa tcacaagttg 960
gaaattttag tggtcgagga ggtataggcc cacggccaag atcacctaga cgggaacgac 1020
gggaagaacg cagccgtaca gcgagggaca gtgaacgtga ccgtagtgaa aaagagaaag 1080
aggaaaagag agaaagtcgg aagagaagta gaagtagagg gcggtcgcgt tccccacctc 1140
gtcgaagaat gagagtagtt cctagatata atgttcaagt tcctaagatt ttattacata 1200
taatacgact cactataggg agcgtgtatt cactgggacc 1240
<210>3
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
taatacgact cactataggg tcccgtctag gtgatcttgg 40
<210>4
<211>367
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
agcgtgtatt cactgggacc ataacaaaac tttgtgctga ctttggattt gttgatgagg 60
atgtgttctt ccagaccagt tgtgtcaaag gtcaggcacc gaaagtttgt gatagggtat 120
tggtggaagc tacctataat cctaatatgc catttaaatg gaatgccaac cgcgttcaag 180
tgattccagg gcccactgtg gcccctgccc agaccccaag tagaagtggt cctcctgcta 240
gagaaataaa cagtcgtggg ggatcttaca atgctgtacc tccaccaaat ttcacatctg 300
aatcacaagt tggaaatttt agtggtcgag gaggtatagg cccacggcca agatcaccta 360
gacggga 367

Claims (5)

1. An application of double-stranded RNA of the CARP-1 gene of macrobrachium nipponensis in promoting the growth of the macrobrachium nipponensis;
the base sequence of the double-stranded RNA of the Macrobrachium nipponensis CARP-1 gene is shown as SEQ ID NO. 5;
the base sequence of the Macrobrachium nipponensis CARP-1 gene is shown as SEQ ID NO: 1.
2. A preparation for promoting the growth of macrobrachium nipponensis is characterized in that: a solution of double-stranded RNA (dsRNA) containing the CARP-1 gene of Macrobrachium nipponensis according to claim 1.
3. The agent for promoting the growth of macrobrachium nipponensis according to claim 2, wherein: by utilizing an RNA interference (RNAi) technology, an interference primer is designed according to an open reading frame of a base sequence CARP-1 gene shown in SEQ ID NO. 1, the total muscle RNA of the macrobrachium nipponensis is taken as a template to carry out PCR reaction, then the PCR reaction solution is taken as the template to carry out in vitro synthesis of double-stranded RNA (dsRNA) shown in SEQ ID NO. 5, and the obtained double-stranded RNA (dsRNA) is dissolved in DEPC water to obtain the preparation for promoting the growth of the macrobrachium nipponensis.
4. The agent for promoting the growth of macrobrachium nipponensis according to claim 3, wherein: the primer is a forward upstream primer sequence shown as SEQ ID NO. 2, and a reverse downstream primer sequence shown as SEQ ID NO. 3.
5. Use of a formulation according to claim 4, wherein: the preparation is applied to the promotion of the growth of the macrobrachium nipponense by interfering the CARP-1 gene.
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CN109536506B (en) * 2018-12-10 2022-03-22 潍坊科技学院 Insulin-like receptor gene for regulating growth and development of macrobrachium nipponense and application thereof

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WO2007101222A3 (en) * 2006-02-28 2007-11-01 Univ Wayne State Cancer treatment
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WO2007101222A3 (en) * 2006-02-28 2007-11-01 Univ Wayne State Cancer treatment
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