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CN109536506B - An insulin-like receptor gene regulating the growth and development of Macrobrachium japonicus and its application - Google Patents

An insulin-like receptor gene regulating the growth and development of Macrobrachium japonicus and its application Download PDF

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CN109536506B
CN109536506B CN201811502966.XA CN201811502966A CN109536506B CN 109536506 B CN109536506 B CN 109536506B CN 201811502966 A CN201811502966 A CN 201811502966A CN 109536506 B CN109536506 B CN 109536506B
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李法君
李婷婷
付春鹏
张玉兰
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Abstract

本发明涉及生物技术和甲壳动物生长发育调控领域，具体涉及一种调控日本沼虾生长发育的类胰岛素受体基因及其应用。类胰岛素受体基因(ILPR基因)如SEQ ID NO:1中核酸所示。根据ILPR基因的开放阅读框设计RNA干扰引物，将双链RNA(dsRNA)注射到日本沼虾体内，可有效降低ILPR基因的表达水平；同时使日本沼虾生长显著变缓。本发明首次在甲壳动物中克隆ILPR基因的mRNA序列，首次明确了ILPR基因具有调控日本沼虾生长发育的功能，为日本沼虾的良种选育和生产性能的开发利用提供了重要的经济性状候选基因。The invention relates to the fields of biotechnology and crustacean growth and development regulation, in particular to an insulin-like receptor gene for regulating the growth and development of Macrobrachium japonicus and its application. The insulin-like receptor gene (ILPR gene) is shown in the nucleic acid in SEQ ID NO:1. RNA interference primers were designed according to the open reading frame of the ILPR gene, and the double-stranded RNA (dsRNA) was injected into the body of Macrobrachium japonicus, which could effectively reduce the expression level of the ILPR gene; meanwhile, the growth of Macrobrachium japonicus was significantly slowed down. The present invention clones the mRNA sequence of the ILPR gene in crustaceans for the first time, clarifies for the first time that the ILPR gene has the function of regulating the growth and development of Macrobrachium japonicus, and provides an important candidate for economic traits for the selection of superior species and the development and utilization of the production performance of Macrobrachium japonicus Gene.

Description

Insulin-like receptor gene for regulating growth and development of macrobrachium nipponense and application thereof
Technical Field
The invention relates to the field of biotechnology and crustacean growth and development regulation, in particular to an insulin-like receptor gene for regulating and controlling the growth and development of macrobrachium nipponense and application thereof.
Background
Macrobrachium nipponensis (Macrobrachium nipponense), commonly called freshwater shrimp and river shrimp, belongs to Crustacea, Tenpoda, brachycarpidae and Macrobrachium. Has the characteristics of strong adaptability, high growth speed, strong fecundity, large individual, rich nutrition and the like, and is the only native variety in the freshwater economic shrimps cultured in large scale in China. According to the statistics of 'annual book for fishery statistics in China in 2018', the annual output of macrobrachium nipponensis in China in 2017 is 240,739 tons. The cultivation of the macrobrachium nipponensis becomes one of the important means for increasing the production and creating income for the majority of fishermen, and plays an important role in the aquiculture in China. In recent years, with the continuous expansion of the culture scale and the culture area, the phenomena of the reduction of the production performance of the macrobrachium nipponensis and the degradation of germplasm resources are increasingly prominent, wherein the miniaturization of individuals is particularly obvious, and finally, the specification of commercial macrobrachium nipponensis is reduced, and the quality and the economic benefit of the macrobrachium nipponensis are seriously influenced. Therefore, the development of important genes for regulating the growth and development of macrobrachium nipponense and further elucidation of the growth and development mechanism of the macrobrachium nipponense become an urgent problem to be solved in the current macrobrachium nipponense fine breed breeding research.
In the case of crustacean growth, due to the presence of chitin exoskeletons, research on crustacean growth has focused on the regulation of the molting mechanism. Admittedly, the molting mechanism plays an important role in the growth and development of crustaceans. However, it is clearly recognized that growth genes and signal pathways are the leading factors in the growth and development of crustaceans, and few growth-related genes have been reported. Therefore, the exploration of the macrobrachium nipponensis growth related gene not only can provide an important target gene for fine breed breeding of macrobrachium nipponensis, but also is helpful for enriching the growth and development theory of crustacean.
Research shows that the biological processes regulated by the protein hormones are firstly combined with receptors thereof, so that a series of related signal paths are opened, and the due biological functions are finally exerted. For example, in vertebrates, regulation of blood glucose levels is primarily dependent on Insulin signaling pathways mediated by Insulin receptors (inrs). In invertebrates, such as nematodes, molluscs and insects, proteins similar to vertebrate Insulin sequences are also present, often termed Insulin-like peptides (ILP). An Insulin-like receptor (ILPR) is used as a receptor thereof, mediates ILP to open an Insulin Signaling Pathway (ISP), a Mitogen Activated Protein Kinase (MAPK) pathway signal, a rapamycin Target protein (TOR) signaling pathway, and regulates various life processes such as metabolism, reproduction, immunity and the like. It can be seen that ILPR is an effector of ILP biological function. Thus, elucidating the function of the ILPR gene will undoubtedly help to fully elucidate the molecular mechanism behind the "ILP/ILPR" regulatory pathway ". However, no report has been made on the mRNA sequence of crustacean ILPR gene and the biological function "assumed" thereof.
Disclosure of Invention
The invention aims to solve the technical problem of providing an insulin-like receptor gene for regulating and controlling the growth and development of macrobrachium nipponense and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
an insulin-like receptor gene for regulating the growth of Macrobrachium nipponense, wherein the insulin-like receptor gene (ILPR gene) is shown by the nucleic acid in SEQ ID NO. 1.
The application of the insulin-like receptor gene in regulating the growth and development of macrobrachium nipponense.
Furthermore, the ILPR gene shown by the SEQ ID NO:1 sequence utilizes RNA interference (RNAi) technology to synthesize double-stranded RNA (dsRNA) shown by the SEQ ID NO:5 in vitro, and the obtained double-stranded RNA (dsRNA) is applied to the regulation of the growth and development of the Macrobrachium nipponense.
Still further, the double-stranded RNA (dsRNA) is prepared by designing an interference primer according to an open reading frame of an ILPR gene shown in a sequence of SEQ ID NO. 1 by using an RNA interference technology, and performing PCR reaction by using total muscle RNA of macrobrachium nipponense as a template to obtain a sequence shown in SEQ ID NO. 4; then, the obtained PCR reaction solution was used as a template to synthesize double-stranded RNA (dsRNA) represented by SEQ ID NO. 5 in vitro.
The interference primer designed according to the open reading frame of the ILPR gene shown in the sequence of SEQ ID NO. 1 is a forward upstream primer sequence shown in SEQ ID NO. 2, and a reverse downstream primer sequence shown in SEQ ID NO. 3.
The dsRNA injection dose is 5 mu g/g.
The invention has the following beneficial effects:
the invention obtains dsRNA through the ILPR gene of the macrobrachium nipponense, and the dsRNA is injected into the pericardial cavity of the macrobrachium nipponense in a microinjection mode, and the result shows that: interference with the ILPR gene results in prolonged molting cycle and slow growth of Macrobrachium nipponense. The expression quantity change of the gene under the stimulation of dsRNA and the influence of the gene on the growth of the macrobrachium nipponensis prove that the ILPR gene can regulate the growth and development of the macrobrachium nipponensis, and the research result provides a new growth target gene for the fine breed breeding of the macrobrachium nipponensis.
Drawings
FIG. 1 is a graph showing the interference efficiency of male Macrobrachium nipponensis injected with dsRNA-ILPR according to an embodiment of the present invention; wherein 7d, 14d, 21d and 28d refer to 7 th, 14 th, 21 st and 28 th day after injection, respectively. P <0.01, the interfering group was injected with dsRNA-ILPR, 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 injected with dsRNA-ILPR according to an embodiment of the present invention; wherein 7d, 14d, 21d and 28d refer to 7 th, 14 th, 21 st and 28 th day after injection, respectively. P <0.01, the interfering group was injected with dsRNA-ILPR, and the control group was injected with the same dose of DEPC water.
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
The invention clones and obtains ILPR gene from the muscle of macrobrachium nipponense; an RNA interference primer is designed according to an open reading frame of the ILPR gene, and double-stranded RNA (dsRNA) is injected into macrobrachium nipponense bodies, so that the expression level of the ILPR gene can be effectively reduced; meanwhile, the growth of the macrobrachium nipponensis is obviously slowed down. The invention clones the mRNA sequence of the ILPR gene in the crustacean for the first time, defines that the ILPR gene has the function of regulating and controlling the growth and development of the macrobrachium nipponense for the first time, and provides an important economic character candidate gene for the fine breed breeding and the development and the utilization of the production performance of the macrobrachium nipponense.
Example 1: obtaining of the full-Length cDNA of the ILPR Gene of Macrobrachium nipponense
Total RNA extraction: selecting about 3g of Macrobrachium nipponensis (two each of male and female), taking the muscle, placing into a precooled mortar containing liquid nitrogen, and rapidly 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 Penaeus japonicus ILPR: designing two groups of primers according to the middle segment of the muscle transcriptome of the macrobrachium nipponensis to verify the middle segment (the amplified segments of the two groups of primers have an overlapping part so as to be convenient for splicing): forward primer MF1(SEQ ID NO:6) and reverse primer MR1(SEQ ID NO:7), forward primer MF2(SEQ ID NO:8) and reverse primer MR2(SEQ ID NO: 9). The PCR amplification reaction system is as follows:
Figure GDA0003346790220000031
Figure GDA0003346790220000041
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 glue 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 alpha, 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 Shanghai biological corporation for sequencing analysis.
Designing specific forward primers 3F (SEQ ID NO:10) and 5R (SEQ ID NO:11) according to the ILPR gene cDNA middle fragment sequence for respectively carrying out 3 'and 5' rapid amplification, and the operation steps are as follows
Figure GDA0003346790220000042
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 ILPR 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 of the shrimp ILPR gene of the macrobrachium nipponense, which is shown as the base sequence of SEQ ID NO. 1.
Example 2: synthesis of dsRNA of ILPR gene of macrobrachium nipponense
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 was designed within the open reading frame of the ILPR gene using online dsRNA primer design software (http:// www.flyrnai.org/cgi-bin/RNAi _ find _ primers. pl), and the following PCR reaction was carried out using total muscle RNA of Macrobrachium nipponense as a template according to the following reaction system to obtain a sequence shown in SEQ ID NO: 4.
Figure GDA0003346790220000043
Then, in vitro Transcription was performed to synthesize dsRNA according to the instructions of the Transcript AidTM T7 High Yield Transcription kit (Fermentas, Inc., USA), and in vitro synthesis of double-stranded RNA (dsRNA) using the PCR reaction solution obtained from SEQ ID NO. 4 as a template is 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 was dissolved in DEPC water for use.
Example 3: effect of dsRNA-ILPR injection on growth and development of Macrobrachium nipponense
1. Selection of experimental shrimps
Firstly, 80 adult male and female macrobrachium nipponensis with strong activity, uniform individual and weight of about 2g are selected in one-time experiment, and are averagely divided into two groups (40 per group), wherein one group is a dsRNA-ILPR injection group, and the other group is a DEPC water group control group. 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
The dsRNA solution was injected into the pericardial cavity from the basal part of the cephalic turbinates of Macrobrachium nipponensis at an injection dose of 5. mu.g/g, and the control group was injected with DEPC water. 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 the sample is extracted and inverted into cDNA. The interference efficiency was calculated by detecting the change in expression level of ILPR relative to the control group using a fluorescent quantitative PCR technique. The body weights of the control group and the experimental group were measured at the end of the experiment, and the data were analyzed.
3. Results and analysis of the experiments
As shown in FIG. 1, the expression level of the male Macrobrachium nipponensis ILPR was reduced to 88.03%, 89.59%, 86.51% and 87.01% at 7d, 14d, 21d and 28d after the interference, respectively, as compared with the expression level of the muscle of the control Macrobrachium nipponensis.
As shown in fig. 2, the expression level of female macrobrachium nipponensis ILPR decreased to 88.14%, 86.96%, 87.21% and 81.19% at 7d, 14d, 21d and 28d after the interference, respectively, as compared to the expression level of the muscle of the control macrobrachium nipponensis.
The above results indicate that injection of dsRNA effectively reduced the expression level of ILPR.
TABLE 1 changes in molting individuals and mean body weight in Macrobrachium nipponensis RNAi experiments
Figure GDA0003346790220000061
Note: capital letters indicate significant differences between the two sets of data.
As shown in table 1, the numbers of molts of male and female shrimps in the RNAi-interfered group were 12 and 10, respectively, while the numbers of molts of male and female shrimps in the control group were 31 and 29, respectively. Compared with the control group, the average body weight of male and female shrimps in the RNAi interference group is reduced by 0.66g and 0.36g respectively, and the difference between the control group and the interference group is obvious.
Research results show that the interference of the ILPR gene can prolong the ecdysis period of the macrobrachium nipponense and slow the growth of the macrobrachium nipponense, and the ILPR gene plays an important regulation and control function in the growth and development process of the macrobrachium nipponense. The invention ensures the function of the ILPR gene and provides a new growth target gene for the fine breed breeding of macrobrachium nipponensis. In addition, the invention also lays a foundation for clarifying the molecular mechanism of the growth and development of the crustacean.
Sequence listing
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aatgagaata gtattcattt gtacataagg aagtatcccc aagaacgagc tgaacgcaac 2940
atgactcagt atttgcagga tttaaaacaa aagaaatact tcataataat tggtgacttt 3000
tatgaagatg ttgctcgatc tgttatatgt gatgcttata atatgaagat gactggtgaa 3060
cagcgatatt tgtggtttct gcctcactgg ttctctgcgc attggtacga cactgataga 3120
ctgcgagaat ctgagggacc caatagaaat tatcaggatt cactcaggga tccagtagtt 3180
acttgtacaa ctgaacaaat gaggctcgct cttcaaggac atatgtcatt atcttacaaa 3240
tattttgctg aaaactcttc agtcatgcaa gaaaacaaaa ctgtcgaaga ttggcggaaa 3300
gaatactcta agactgtgaa gagtgttcaa gggcttgcta ctgagtctaa ctatgcaggc 3360
tttacatatg atgctgtgtg gacttatgcc ttggcactgg atgccctctt caaggaagat 3420
cagtcgtatg cagctgacct aagggcaccc aatgcaacaa aggcatacat cagtaagatt 3480
aaatcagttg cttttgatgg cgtctctggc ccaattaatt ttacatcagg ctcaagagta 3540
actgatatta ttgtgtggca attcaaaggc aactcgtatg aagaggttgg aatttttcat 3600
cctggactca cgagaaatga cactggaaaa ctgaatatca gattggagaa actcttctgg 3660
ccatctaatg agaaacctga tgatgggagt gacaaatgtg atatagaggg gtttaggaag 3720
ctgctcaatg tagagtgccg cacagcaatc atcatattgt gtgctgtttg ctttggtggc 3780
cttgcaacag tactcatagc atgctttgtt atctttaaaa gaaggtatga aaaaagactg 3840
gaacaaatcc aggaactgtg gaggggtcga ccgttgtttg aaatttttga cggatgggaa 3900
attcctcgag ataaagtggt aataaatcgt aaaattggtg aaggagcctt tggcacagtg 3960
tatggaggag aatgccagtt tgatacaaaa ggatgggaag ctgatagggt tgctgtggca 4020
gtgaagactc tgaagattgg atcaactatt tctgagaagt tggacttcct ttctgaagca 4080
gagatgatga aaaattttga gcatgaaaat attgttcaac taataggggt ctgcaccaag 4140
agtgagccaa tatatactgt tatggaattc atgctgtatg ctgacttgaa gacatatctt 4200
ctggctcgaa ggcatctcgt aaatgaaaaa agccgtaatg acgatgatga agtcagcaac 4260
aagaggctga cttcaatggc acttgatatt gcacgtgggc ttgcttattt agcagaaatg 4320
aaatatgtgc acagggatgt tgctagtcgt aactgtcttg tgaatgccaa cagaactgtc 4380
aaacttgcag attttggaat gaccaggcct atgtatgaga ataactacta caaatttaat 4440
cgtaaaggta tgctaccagt acgatggatg gcgccagaat cactaacaga gggagttttc 4500
actaccatga gtgatgtctg gtcatatggt gtgttgctgt atgaaattgt cacatttgga 4560
gcattcccgt ttcagggaat gtctaatgat caagtgttag agcatgtcaa agctggtcat 4620
actatagcca taccaaaagg agtgaaaccg caaatggaga ttttactgag gagttgctgg 4680
catcatgttc ccacaagaag gattcagatt ttacaaataa ttgactattt aacaaattac 4740
ccgcgtttga tatcgccttg tctagatgga ccacagtcat cagtgcagat tgaagacact 4800
gtctcacttg agttgagaat acctgataag acaaggaaac tgtcactatc aataaataat 4860
cgcttacctc aagtggtgtc gagtcgaaag cgatcaatga gtggaaatat ggtgatgaat 4920
gttcctcctc ttacctcgag tctaagcgag gatggaatga tatcggctca ttccaactta 4980
gatgctctca atctaaatca tgtgacttta gaggaaaatg aaatgggaga ggaccctttg 5040
ttaccccctg ctcaatatgt ctcctctgga tacatgtcta taccacacaa agacctaaaa 5100
gaaaaggaaa gtttagtaag tagacagcag ggagattatt gcgctacaga tctacccggt 5160
gacttgtgga ctaatgttac tcctgtatag ggaaacgcct ccttaggcaa atttaagtga 5220
ccaaagtagt gttctagtat tttacatata gatctaatat atatatatat gtatgtatgt 5280
gtatatatat atata 5295
<210> 2
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
taatacgact cactataggg ctgcgagaat ctgagggacc 40
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
taatacgact cactataggg cagtgccaag gcataagtcc 40
<210> 4
<211> 279
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctgcgagaat ctgagggacc caatagaaat tatcaggatt cactcaggga tccagtagtt 60
acttgtacaa ctgaacaaat gaggctcgct cttcaaggac atatgtcatt atcttacaaa 120
tattttgctg aaaactcttc agtcatgcaa gaaaacaaaa ctgtcgaaga ttggcggaaa 180
gaatactcta agactgtgaa gagtgttcaa gggcttgcta ctgagtctaa ctatgcaggc 240
tttacatatg atgctgtgtg gacttatgcc ttggcactg 279
<210> 5
<211> 279
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cugcgagaau cugagggacc caauagaaau uaucaggauu cacucaggga uccaguaguu 60
acuuguacaa cugaacaaau gaggcucgcu cuucaaggac auaugucauu aucuuacaaa 120
uauuuugcug aaaacucuuc agucaugcaa gaaaacaaaa cugucgaaga uuggcggaaa 180
gaauacucua agacugugaa gaguguucaa gggcuugcua cugagucuaa cuaugcaggc 240
uuuacauaug augcugugug gacuuaugcc uuggcacug 279
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gcgtgggtgt tgagaaacag cc 22
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acgaaagaag tatgggtaat cc 22
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cctgctcaga tactgttgaa cc 22
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ttccctgaaa cgggaatgct cc 22
<210> 10
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cagttgaaca ggagcattag gcctacg 27
<210> 11
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gttttcacta ccatgagtga tgtctgg 27

Claims (4)

1.一种调控日本沼虾生长发育的类胰岛素受体ILPR基因，其特征在于：所述类胰岛素受体ILPR基因的碱基序列如SEQ ID NO:1所示。1. An insulin-like receptor ILPR gene for regulating the growth and development of Macrobrachium japonicum, wherein the base sequence of the insulin-like receptor ILPR gene is as shown in SEQ ID NO: 1. 2.如权利要求1所述的类胰岛素受体ILPR基因在调控日本沼虾生长发育中的应用，其特征在于：利用RNA干扰技术根据SEQ ID NO:1所示的类胰岛素受体ILPR基因的开放阅读框设计干扰引物，以日本沼虾的肌肉总RNA为模板进行PCR反应，得到的序列如SEQ ID NO:4所示；再以所得PCR反应液为模板进行体外合成如SEQ ID NO:5所示的双链RNA（dsRNA），得到双链RNA（dsRNA）注入日本沼虾围心腔内，延缓日本沼虾的蜕皮周期和生长。2. the application of the insulin-like receptor ILPR gene as claimed in claim 1 in regulating the growth and development of Macrobrachium japonicum, it is characterized in that: utilize RNA interference technology according to the insulin-like receptor ILPR gene shown in SEQ ID NO:1. The open reading frame was designed to interfere with primers, and the total muscle RNA of Macrobrachium japonicum was used as a template to carry out PCR reaction, and the obtained sequence was shown in SEQ ID NO: 4; and then the obtained PCR reaction solution was used as a template for in vitro synthesis as shown in SEQ ID NO: 5 The double-stranded RNA (dsRNA) shown, the obtained double-stranded RNA (dsRNA) was injected into the pericardial cavity of Macrobrachium japonicus to delay the molting cycle and growth of Macrobrachium japonicus. 3.根据权利要求2所述的应用，其特征在于：所述根据SEQ ID NO:1所示的类胰岛素受体ILPR基因的开放阅读框设计干扰引物的正向上游引物序列如SEQ ID NO:2所示，反向下游引物序列如SEQ ID NO:3所示。3. application according to claim 2, is characterized in that: described according to the open reading frame of the insulin-like receptor ILPR gene shown in SEQ ID NO:1 The forward upstream primer sequence of interference primer is designed as SEQ ID NO: 2, the reverse downstream primer sequence is shown in SEQ ID NO:3. 4.根据权利要求2所述的应用，其特征在于：所述dsRNA的注射剂量为5 μg/g 。4. application according to claim 2 is characterized in that: the injection dose of described dsRNA is 5 μg/g.
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