CN114395572A - Freshwater shrimp legumain-like gene and application of dsRNA thereof - Google Patents

Freshwater shrimp legumain-like gene and application of dsRNA thereof Download PDF

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CN114395572A
CN114395572A CN202210031747.8A CN202210031747A CN114395572A CN 114395572 A CN114395572 A CN 114395572A CN 202210031747 A CN202210031747 A CN 202210031747A CN 114395572 A CN114395572 A CN 114395572A
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legumain
gene
dsrna
freshwater
freshwater shrimp
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CN114395572B (en
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乔慧
傅洪拓
程丹
张文宜
蒋速飞
熊贻伟
金舒博
龚永生
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Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences
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Abstract

The invention discloses a freshwater shrimp Legumain-like gene and application of dsRNA thereof, belonging to the technical field of biology. The invention provides an application of freshwater shrimp Legumain-like gene and dsRNA thereof, which comprises the freshwater shrimp Legumain-like gene, a gene segment, the dsRNA thereof and the application of the dsRNA in inhibiting the development of freshwater shrimp ovary. Firstly, a complete sequence of Legumain-like is obtained through gene cloning, wherein the nucleotide sequence is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO. 2. An RNA interference primer is designed by taking the full sequence as a template to obtain an interference gene segment SEQ ID NO. 3. Then, dsRNA is synthesized by the primer. The dsRNA and the dsGFP are injected into the pericardial cavity of female freshwater shrimps for interference, and the detection finds that the development of ovaries of the female freshwater shrimps is effectively inhibited.

Description

Freshwater shrimp legumain-like gene and application of dsRNA thereof
Technical Field
The invention relates to a freshwater shrimp legumain-like gene and application of dsRNA thereof, belonging to the technical field of biology.
Background
Freshwater shrimps, the school name Macrobrachium nipponense (Macrobrachium nipponense), belong to the brachiaridae (Palaemonidae) and Macrobrachium genus (Macrobrachium), are named because their body color is grayish and has brown patches, and are also commonly called river shrimps. Naturally distributed in china, japan, korea, laos, malaysia, mainma (mainma mainland), vietnam and russian far east, gradually spread to singapore, philippines, uzbekistan, irakan, iran, etc. Freshwater shrimps are important economic freshwater aquaculture species in China, but are always puzzled by the problem of 'sexual maturity rapidly'. "sexual fast maturity" refers to the breeding of female shrimp for many generations during the breeding season. The phenomenon can cause the problems of multi-generation paradise, overhigh culture density, increased feed consumption, serious oxygen deficiency and the like. Therefore, the method for solving the problem has important significance for improving the yield of the freshwater shrimps and promoting the healthy and rapid development of the freshwater shrimp industry.
Asparagine endopeptidases (LGMN) are novel members of the cysteine protease family. Originally found in the plants canavalia and cowpea, researchers subsequently found legumain expression in both vertebrates and mammals. legumain, like other families of cathepsins, is primarily localized to the lysosomes of cells and is involved in a variety of physiological processes such as degradation of matrix proteins. However, relatively few studies have been made on the role of legumain in normal development in the body and in normal tissues. The effect of legumain-like in the ovarian maturation development process is researched by taking freshwater shrimps as a research object.
RNAi (RNA interference) technology, which refers to the phenomenon in which small double-stranded RNA molecules can specifically degrade or inhibit the expression of homologous mRNA, thereby inhibiting or turning off the expression of a particular gene. In published researches, the inhibition of the development of ovaries by interfering with genes such as vitellin and the like in the bodies of freshwater shrimps is mentioned, so that the technology has important significance for solving the problem of 'fast maturity' of the freshwater shrimps.
Disclosure of Invention
The invention aims to solve the problem of 'fast maturity' of female freshwater shrimps, slow down the mature speed of ovary development by a biotechnology means, and provide a target gene related to the ovary development of the female freshwater shrimps and application of dsRNA thereof in inhibiting the ovary development of the freshwater shrimps.
The invention provides a Legumain-like gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The invention also provides cysteine protease coded by the Legumain-like gene, and the amino acid sequence of the cysteine protease is shown as SEQ ID NO. 2.
The invention also provides application of the Legumain-like gene of the freshwater shrimps or cysteine protease of the freshwater shrimps as a target in inhibiting the development of the ovary of the freshwater shrimps or preparing an inhibitor for the development of the ovary of the freshwater shrimps.
The invention provides a high-efficiency freshwater shrimp Legumain-like interference gene fragment, and the nucleotide sequence of the fragment is SEQ ID NO. 3.
The invention provides an application of a freshwater shrimp Legumain-like interference gene fragment in preparation of an inhibitor for the development of freshwater shrimp ovary.
The invention provides a method for preparing a freshwater shrimp Legumain-like interference gene fragment, which comprises the following steps: the total cDNA of freshwater shrimp is used as a template, and an interference gene segment with a nucleotide sequence shown as SEQ ID NO.3 is obtained by amplifying an upstream primer SEQ ID NO.4 and a downstream primer SEQ ID NO. 5.
The invention also provides dsRNA for inhibiting the development of the ovary of the freshwater shrimp, and the dsRNA is obtained by in vitro transcription by using the freshwater shrimp Legumain-like interfering gene fragment as a template.
The invention also provides application of the dsRNA in inhibiting the mRNA expression of the Legumain-like gene of the freshwater shrimp.
The invention also provides application of the dsRNA in inhibiting the development of the ovary of the freshwater shrimp or preparing a product for inhibiting the development of the ovary of the freshwater shrimp.
The invention also provides a method for preparing dsRNA for inhibiting the development of the ovary of the freshwater shrimp, which is to obtain the dsRNA by taking the gene segment with the nucleotide sequence shown as SEQ ID NO.3 as a template for transcription.
The invention also provides a biological inhibitor containing the dsRNA for inhibiting the development of the ovary of the freshwater shrimp.
The invention also provides a method for inhibiting the development of the ovary of the freshwater shrimp, which is to inject the dsRNA or the biological inhibitor into the pericardial cavity of the freshwater shrimp, wherein the dsRNA or the biological inhibitor can silence/inhibit the expression of the Legumain-like gene.
In one embodiment of the invention, the injection dosage of the dsRNA or the biological inhibitor is 3.5-4.5 mu g/g.
The invention also provides a kit for inhibiting the development of the ovary of the freshwater shrimp, which contains the dsRNA or the biological inhibitor.
The freshwater shrimp Legumain-like gene, cysteine protease encoded by the freshwater shrimp Legumain-like gene, and the freshwater shrimp Legumain-like gene fragment are applied to the breeding of freshwater shrimps.
Has the advantages that:
1. the silencing efficiency of the dsRNA1 in the hepatopancreas and the ovary reaches 88.51-99.41%, and the expression of the freshwater shrimp Legumain-like gene is effectively inhibited.
2. The dsRNA1 can effectively reduce the mRNA expression of the Legumain-like gene of the freshwater shrimps, the gonadal index of the freshwater shrimps in 17 days is reduced by 50 percent compared with that of a control group, and the development speed of the ovary of the freshwater shrimps is inhibited.
Drawings
FIG. 1 is a graph showing the relative expression level changes of Legumain-like gene in ovary and hepatopancreas after dsRNA injection. Panel A shows mRNA expression of Legumain-like gene at different sampling time points in ovary. Panel B shows mRNA expression of Legumain-like gene at different sampling time points in the hepatopancreata. The freshwater shrimp EIF gene is used as an internal reference gene. 1. 9, 17 are day 1, day 9, day 17 after injection, respectively (n-9). Different letters represent significant p <0.05 difference.
FIG. 2 is the graph of the change of the ovarian gonad development index of freshwater shrimps. 1. 9, 17 are day 1, day 9, day 17 after injection, respectively (n-9). Different letters represent significant p <0.05 difference.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Unless otherwise indicated, the reagents and materials used in the following examples are all commercially available or may be prepared by known methods.
Example 1: obtaining the full-length nucleotide sequence of the freshwater shrimp Legumain-like gene.
The full-length sequence of the gene Legumain-like is obtained by performing transcriptome comparative analysis on each development period of female freshwater shrimp nests.
Example 2: freshwater shrimp Legumain-like gene fragment and acquisition of dsRNA thereof
Based on the freshwater shrimp Legumain-like gene with the nucleotide sequence shown as SEQ ID NO.1, specific primers (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7) for RNA interference are designed in an open reading frame by adopting NCBI online dsRNA primer design software (https:// www.flyrnai.org/cgi-bin/RNAi _ find _ primers. pl), and a T7 promoter sequence is added in front of the primers: TAATACGACTCACTATAGG.
PCR products (sequences shown as SEQ ID NO.3 and SEQ ID NO. 8) were obtained by PCR amplification using the upstream primer SEQ ID NO.4 and the downstream primer SEQ ID NO.5 containing the T7 promoter and the upstream primer SEQ ID NO.6 and the downstream primer SEQ ID NO.7, purified and recovered by a PCR product purification kit, the recovered PCR products were purified as templates for in vitro Transcription of dsRNA1 and dsRNA2, in vitro Transcription was performed using TranscriptaID TM T7 High Yield Transcription kit (Fermentas, Inc., USA) to synthesize dsRNA1 and dsRNA2 according to the manufacturer's instructions, and then the purity and integrity of dsRNA1 and dsRNA2 were checked by 1.2% agarose gel electrophoresis, and the concentrations of dsRNA1 and dsRNA2 were measured at 260nm using an ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany) and then kept at-80 ℃ until use.
PCR amplification System: 10 XEX Taq Buffer 5. mu.L, TaKaRa EX Taq 0.25. mu.L, dNTP mix 4. mu.L, upstream primer (10. mu. moL/L) 1. mu.L, downstream primer (10. mu. moL/L) 1. mu. L, cDNA/GFP plasmid 1. mu.L, ddH2The content of O is filled to 50 mu L.
PCR amplification procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; extension at 72 ℃ for 5 min. The amplification product was stored at 4 ℃.
The dsGFP of the green fluorescent protein GFP gene was prepared in the same manner as described above (the nucleotide sequence of the GFP gene is referred to as NCBI accession No. MN 114103.1).
Example 3: experiment for inhibiting development of female freshwater shrimp ovary by injecting dsRNA synthesized by Legumain-like gene fragment
(1) Injection of dsRNA
According to the appearance characteristics of the freshwater shrimp ovary development, 0.60 +/-0.12 g of 180 healthy freshwater shrimps with the ovary development at the IV stage (the proliferation stage of the egg protocyte and white and transparent) is selected to ensure the consistency of the initial ovary development period, and a dsGFP control group, a blank control group, an experiment group of dsRNA1 and an experiment group of dsRNA2 are arranged, wherein each group comprises 60 freshwater shrimps and 3 parallel repeats (n is 20) are arranged. Temporarily culturing in a glass jar for three days before injection to adapt to the culture environment of a laboratory (water temperature is 25 +/-1 ℃), and feeding fresh spiral shells once every morning and evening.
Before injection, the freshwater shrimps are placed in a glass jar for temporary culture for three days to adapt to the culture environment of a laboratory (the water temperature is 25 +/-1 ℃), and fresh snails are fed in the morning and at the evening every day. According to different experimental groups, the dsRNA obtained in example 2 was injected into the pericardial cavity of freshwater shrimp by using a microinjector at a dose of 4. mu.g/g.
(2) Detection of Legumain-like gene silencing efficiency
At day 1, 9, and 17 after injection, 3 random samples were collected from each jar, and the ovary and the hepatopancreas of the freshwater shrimps were dissected out. Total RNA was extracted using RNAioso Plus (TaKaRa, Japan) reagent, and template cDNA was obtained by reverse transcription using Primer script II1st Strand cDNA Synthesis reverse transcription kit (Bio-Rad) and M-MLVkit (TaKaRa, Japan), and then Real Time PCR was performed using this as a template to detect the relative expression level of Legumain-like, and internal reference gene (EIF) was used to calculate the silencing efficiency of the target gene. The results show that compared with the dsGFP control group, the dsRNA1 experimental group has 92.66% and 96.18% of hepatopancreas silencing efficiency and 88.51% and 99.41% of ovary silencing efficiency respectively at 9 days and 17 days after injection. (FIG. 1).
(3) Changes in the gonadal development Index (GSI) of freshwater shrimps
According to the formula of gonad development index (GSI), namely the wet weight of gonad/wet weight of body weight multiplied by 100%, the gonad development indexes of the freshwater shrimps of three groups of dsGFP, dsRNA1 and dsRNA2 are calculated.
Through comparison of the GSI gonad development index of the control group with the gonad development indexes of the experiment group injected with dsRNA1 and the experiment group injected with dsRNA2, the GSI of the control group is 3.91% after being emptied from 12.68% of the GSI of the ovary development stage IV after being cultured for 1 to 9 days and then is developed to the II stage (secondary vitellogenesis stage, light green); the dsRNA1 experimental group develops to the II stage after the emptying of 12.00 percent of GSI in the IV stage of ovarian development, and the GSI is 4.01 percent; the dsRNA2 group developed to stage II after emptying from 11.68% of GSI in stage IV of ovarian development, and the GSI was 3.96%. Culturing for 9-17 days, and the GSI of the control group is 10.45% when the control group develops to the IV stage; after 17 days of culture, the gonad development index of the dsRNA1 experimental group is 6.94%, and the gonad development reaches the stage III; the development conditions of the dsRNA2 group are similar to the trend of the control group, and are not obviously different, and the gonad development index is 11.45 percent after 17 days of culture (figure 2). Therefore, the dsRNA1 has a certain inhibiting effect on gonad development. The gonad development index result shows that the dsRNA1 exogenously injected with Legumain-like can effectively inhibit the development speed of the ovary of the freshwater shrimp, and the dsRNA2 does not inhibit the development speed of the ovary.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> research center of freshwater fishery of Chinese aquatic science research institute
<120> freshwater shrimp legumain-like gene and application of dsRNA thereof
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Asp Asn Ile Gly Phe Tyr Val Thr Thr Ser Ala Asp Pro Ser Glu Ser
195 200 205
Ser Tyr Ala Cys Tyr Phe Asp Glu Leu Arg Asp Thr Tyr Ile Gly Asp
210 215 220
Gln Phe Gly Cys Cys Trp Met Glu Asn Ser Asp Glu Ser Asp Leu Thr
225 230 235 240
Thr Leu Thr Leu Gly Asp Gln Tyr Gln Ile Ile Tyr Glu Cys Asn Tyr
245 250 255
Gln Ser His Ser Pro Trp Tyr Gly Gln Pro Ser Met Arg Asp Glu Val
260 265 270
Val Gly Ala Phe Leu Gly Met Gly Lys Thr Gln Gln Arg Lys Ser Ser
275 280 285
Gly Gly Ser His Ser Lys Asp Asp Ala Val Pro Ser Val Asp Val Pro
290 295 300
Leu Phe Ile Leu Lys Asn Lys Met Lys Lys Ala Asn Gly Asp Glu Lys
305 310 315 320
Ile Ala Leu Gln Ala Gln Ile Asn Gly Leu Phe Lys Lys Arg Glu Phe
325 330 335
Val Gln Asp Ile Thr Lys Asn Ile Ala Thr Leu Val Thr Gly Asp Glu
340 345 350
Glu Leu Ala Glu Tyr Tyr Ile Glu Asn Pro Met Arg Lys Val Asn Asp
355 360 365
Trp Ala Cys Tyr Lys Arg Ala Val Asp Ala Phe Asn Asp Lys Cys Phe
370 375 380
Arg Phe Gly Glu Asn Pro Phe Ala Met Thr Met Thr Pro Thr Leu Leu
385 390 395 400
Asn Leu Cys Glu Asn Asn Phe Gly Ala Asp Asn Ile Val Arg Arg Ile
405 410 415
Glu Gln Val Cys Asn Phe Gly Phe Val Ser Gly Ile Glu
420 425
<210> 3
<211> 368
<212> DNA
<213> Artificial sequence
<400> 3
tggaggatct cattccaagg acgatgctgt gccaagcgtg gatgtacctc tcttcatctt 60
gaaaaataag atgaaaaaag ccaatggtga cgagaagatc gcgcttcagg ctcagattaa 120
cggcttgttt aagaaacgag aattcgttca agacataacc aagaacatcg caacactggt 180
gactggcgat gaagagttgg cggaatatta cattgaaaat ccgatgagaa aagtcaacga 240
ctgggcctgt tacaaaagag ccgtcgatgc cttcaacgat aagtgcttcc ggtttggaga 300
gaatcctttc gccatgacaa tgaccccgac gctgttgaac ctctgcgaga acaacttcgg 360
agccgata 368
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
tggaggatct cattccaagg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
tatcggctcc gaagttgttc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
atcaaggaca tgcacgacaa 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
tcgtccttgg aatgagatcc 20
<210> 8
<211> 405
<212> DNA
<213> Artificial sequence
<400> 8
atcaaggaca tgcacgacaa caaccagtac aaagagatgg tgatgtacat ggaatcatgt 60
gaatcagggt ccctgtacga tggcatctat ccagataata ttgggtttta tgtaacgacc 120
tcggctgacc cctccgagtc ttcctacgct tgctatttcg acgaactcag agacacgtac 180
attggcgacc agttcggctg ttgctggatg gagaattctg acgagagtga cctcactaca 240
ctgaccctag gggaccagta tcaaatcata tacgagtgca attaccaatc acactctcca 300
tggtatggac agccatctat gcgggacgag gtcgtcggtg ctttcctggg aatgggaaag 360
acccagcaga ggaagtcttc tggaggatct cattccaagg acgat 405

Claims (10)

1. The application of the Legumain-like gene of freshwater shrimps or cysteine protease of the freshwater shrimps as a target in inhibiting the development of the ovary of the freshwater shrimps or preparing an inhibitor for the development of the ovary of the freshwater shrimps;
the nucleotide sequence of the Legumain-like gene is shown in SEQ ID NO. 1;
the amino acid sequence of the cysteine protease is shown in SEQ ID NO. 2.
2. The application of the freshwater shrimp Legumain-like interference gene fragment in preparing the inhibitor for the development of the ovary of the freshwater shrimp;
the nucleotide sequence of the Legumain-like interference gene fragment is SEQ ID NO. 3.
3. The dsRNA is obtained by in vitro transcription by using a freshwater shrimp Legumain-like interfering gene fragment as a template;
the nucleotide sequence of the Legumain-like interference gene fragment is SEQ ID NO. 3.
4. The use of the dsRNA of claim 3 to inhibit the mRNA expression of the Legumain-like gene of freshwater shrimp.
5. The dsRNA of claim 3, which inhibits the development of the ovary of freshwater shrimp or is used for preparing a product for inhibiting the development of the ovary of freshwater shrimp.
6. A biostatic agent, comprising the dsRNA of claim 3.
7. A method of inhibiting the development of the ovary of a freshwater shrimp by injecting the dsRNA of claim 3 or the biological inhibitor of claim 6 into the pericardial cavity of the freshwater shrimp.
8. The method of claim 7, wherein the injected dose of dsRNA or biostatic agent is 3.5-4.5 μ g/g.
9. A kit for inhibiting the development of the ovary of freshwater shrimp, comprising the dsRNA of claim 3 or the biological inhibitor of claim 6.
10. The application of the freshwater shrimp Legumain-like gene, cysteine protease coded by the freshwater shrimp Legumain-like gene and freshwater shrimp Legumain-like interference gene fragment in the breeding of freshwater shrimps;
the nucleotide sequence of the Legumain-like gene is shown in SEQ ID NO. 1;
the amino acid sequence of the cysteine protease is shown as SEQ ID NO. 2;
the nucleotide sequence of the Legumain-like interference gene fragment is SEQ ID NO. 3.
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Citations (3)

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