CN118592405B - Application of ion receptor co-receptor gene in German cockroach behavior regulation - Google Patents
Application of ion receptor co-receptor gene in German cockroach behavior regulation Download PDFInfo
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Abstract
本发明属于生物技术领域，公开了离子型受体共受体基因在德国小蠊行为调控中的应用，具体公开了离子型受体共受体基因IR25a抑制剂和/或离子型受体共受体基因IR76b抑制剂在弱化蜚蠊目昆虫对性信息素的反应中的应用。本发明首次公开了可用于调控德国小蠊繁殖相关行为的离子型受体共受体基因IR25a和IR76b，通过离子型受体共受体基因IR25a抑制剂和/或离子型受体共受体基因IR76b抑制剂靶向下调IR25a和/或IR76b、降解IR25a和/或IR76b，降低雄虫对接触性信息素标准品的电生理反应，并导致雄虫个体对雌虫的求偶行为削弱或消失，达到干扰和遏制雌雄交配和繁殖的目的。The present invention belongs to the field of biotechnology, and discloses the application of ionotropic receptor co-receptor genes in the behavior regulation of German cockroaches, and specifically discloses the application of ionotropic receptor co-receptor gene IR25a inhibitor and/or ionotropic receptor co-receptor gene IR76b inhibitor in weakening the response of Blattaria insects to sex pheromones. The present invention discloses for the first time ionotropic receptor co-receptor genes IR25a and IR76b that can be used to regulate the reproduction-related behaviors of German cockroaches, and IR25a and/or IR76b are targeted and down-regulated, and IR25a and/or IR76b are degraded by ionotropic receptor co-receptor gene IR25a inhibitor and/or ionotropic receptor co-receptor gene IR76b inhibitor, so as to reduce the electrophysiological response of male insects to contact with sex pheromone standards, and cause the male insects' individual courtship behavior to female insects to be weakened or disappear, so as to achieve the purpose of interfering with and curbing the mating and reproduction of males and females.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of an ionic receptor co-receptor gene in German cockroach behavior regulation.
Background
Cockroaches are commonly called cockroaches, and are one of the oldest and most successful insect groups in the world. Presently recorded cockroaches are more than 5000, with less than 1% of species being associated with human life. Cockroaches are two most common species, namely cockroaches which are the most widely distributed important pests in blattaria, and are distributed in tropical, subtropical, temperate and frigid areas all over the world, wherein the cockroaches are distributed in a more widely-distributed, damp-loving, dark and warm indoor environment, are easily contacted with various pathogenic microorganisms, cause food pollution, are main allergens causing asthma or other allergic diseases, and seriously threaten human health. Currently, traditional chemical pesticides remain the primary means of controlling cockroach populations. Abuse of traditional pesticides results in cockroaches becoming resistant to the pesticide and even negatively impacting the ecological environment. Therefore, it is urgent to explore and develop an eco-friendly cockroach control strategy.
Sex pheromone is used as the most common chemical signal in an insect amphiprotic communication system, is a high-efficiency behavior regulator, and is used for leading coupling and mating behaviors and guaranteeing amphiprotic reproduction. In german cockroaches, taste sense is one of the main means of closely identifying female sex signals by males, and plays a vital role in initiating coupling actions and completing subsequent mating processes. The function of taste sensory neurons is mainly determined by taste receptors expressed therein, so that it is feasible to screen german cockroach coupling behavior modulators with taste receptors as targets. As a genetic manipulation means with high efficiency and high specificity, the potential of RNA interference technology in pest green control strategies has been attracting attention in recent years. In particular, insects of the order Blatta have a high sensitivity to RNA interference, making it possible to manipulate the expression of key genes for coupled reproductive behavior by molecular and genetic techniques. The key to realize the breakthrough and the prospect is to develop a molecular target for efficiently and exclusively controlling the propagation behavior of the German cockroaches. However, no key genes for regulating reproductive behaviors such as the mating of German cockroaches have been reported at present, which greatly hinders the development of cockroach control technologies.
Disclosure of Invention
The object of the first aspect of the present invention is to provide the use of an inhibitor of the ionotropic receptor co-receptor gene IR25a and/or an inhibitor of the ionotropic receptor co-receptor gene IR76 b.
The object of the second aspect of the present invention is to provide a dsRNA.
The object of the third aspect of the present invention is to provide a biomaterial associated with the dsRNA of the second aspect of the present invention.
The fourth aspect of the present invention is directed to a reagent.
The object of the fifth aspect of the present invention is to provide the use of the dsRNA of the second aspect of the present invention, the biomaterial of the third aspect of the present invention and/or the agent of the fourth aspect of the present invention.
The object of the sixth aspect of the invention is to provide a method.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention there is provided the use of an inhibitor of the ionotropic receptor co-receptor gene IR25a and/or an inhibitor of the ionotropic receptor co-receptor gene IR76b in any of (1) to (8):
(1) Weakening the response of the blattaria insects to sex pheromones;
(2) Preparing a product for weakening the reaction of the blattaria insects to sex pheromone;
(3) Interfering/suppressing male and female mating of cockroaches;
(4) Preparing a product which interferes/inhibits male and female mating of the cockroaches;
(5) Prolonging the coupling incubation period of the cockroach insects;
(6) Preparing a product for prolonging the coupling incubation period of the cockroach insects;
(7) Preventing and treating blattaria insects;
(8) The product for preventing and treating the blattaria insect is prepared.
In some embodiments of the present invention, the inhibitor of the ionotropic receptor co-receptor gene IR25a is at least one of a substance that inhibits the activity of the ionotropic receptor co-receptor gene IR25a, a substance that degrades the ionotropic receptor co-receptor gene IR25a, or a substance that reduces the expression level of the ionotropic receptor co-receptor gene IR25a, and further a substance that reduces the expression level of the ionotropic receptor co-receptor IR25 a.
In some embodiments of the invention, the inhibitor of the ionotropic receptor co-receptor gene IR76b is at least one of a substance that inhibits the activity of the ionotropic receptor co-receptor gene IR76b, a substance that degrades the ionotropic receptor co-receptor gene IR76b, or a substance that reduces the expression level of the ionotropic receptor co-receptor gene IR76b, and further a substance that reduces the expression level of the ionotropic receptor co-receptor gene IR76 b.
In some embodiments of the invention, the substance that reduces the expression level of the ionotropic receptor co-receptor gene IR25a or the ionotropic receptor co-receptor gene IR76b is at least one of (9) - (11):
(9) siRNA, dsRNA, miRNA, ribozymes, or shRNA targeting the ionotropic receptor co-receptor IR25a or the ionotropic receptor co-receptor gene IR76 b;
(10) A nucleic acid molecule encoding siRNA, dsRNA, miRNA, ribozyme, or shRNA of the targeted ionotropic receptor co-receptor gene IR25a or ionotropic receptor co-receptor IR76b of (9);
(11) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of (10).
In some embodiments of the invention, the inhibitor of the ionotropic receptor co-receptor gene IR25a or the inhibitor of the ionotropic receptor co-receptor gene IR76b is at least one of (12) to (14):
(12) dsRNA targeting the ionotropic receptor co-receptor gene IR25a or the ionotropic receptor co-receptor gene IR76 b;
(13) A nucleic acid molecule encoding the dsRNA targeting the ionotropic receptor co-receptor gene IR25a or the ionotropic receptor co-receptor gene IR76b of (12);
(14) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of (13).
In some embodiments of the invention, the dsRNA comprises double stranded RNA consisting of the nucleotide sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 14 and the nucleotide sequence set forth in the reverse complement thereof.
In some embodiments of the invention, the amino acid sequence of the ionic receptor co-receptor gene IR25a is shown in SEQ ID NO. 2, and the amino acid sequence of the ionic receptor co-receptor gene IR76b is shown in SEQ ID NO. 4.
In some embodiments of the invention, the sex pheromone comprises 3, 11-dimethyl-icosadecan-2-one.
In a second aspect of the invention, there is provided a dsRNA comprising a double stranded RNA consisting of the nucleotide sequence shown as SEQ ID NO.13 or SEQ ID NO. 14 and the nucleotide sequence shown as its reverse complement.
In a third aspect of the invention there is provided a biomaterial associated with the dsRNA of the second aspect of the invention, the biomaterial comprising any one of 1) to 12):
1) A nucleic acid molecule encoding a dsRNA of the second aspect of the invention;
2) An expression cassette comprising 1) the nucleic acid molecule;
3) A vector comprising 1) the nucleic acid molecule;
4) A vector comprising 2) the expression cassette;
5) A transgenic cell line comprising 1) said nucleic acid molecule;
6) A transgenic cell line comprising 2) said expression cassette;
7) A transgenic cell line comprising 3) the vector;
8) A transgenic cell line comprising 4) the vector;
9) A recombinant microorganism comprising 1) said nucleic acid molecule;
10A recombinant microorganism comprising 2) said expression cassette;
11A recombinant microorganism containing 3) the vector;
12A recombinant microorganism containing the vector of 4).
In some embodiments of the invention, the transgenic cell line does not comprise propagation material.
In a fourth aspect, the invention provides an agent comprising the dsRNA of the second aspect of the invention and/or the biomaterial of the third aspect of the invention.
In some embodiments of the invention, the agent is used in any one of a 1) to a 4):
a1 Weakening the response of the blattaria insect to sex pheromone;
a2 Interfering/suppressing male and female mating of cockroaches;
a3 Extension of the incubation period of cockroaches;
a4 Preventing and treating blattaria insects.
In some embodiments of the invention, the blattaria insect comprises at least one of periplaneta americana, periplaneta australis, periplaneta germanica, periplaneta japonica.
In some preferred embodiments of the invention, the blattaria insect is german cockroach.
In some embodiments of the invention, the sex pheromone comprises 3, 11-dimethyl-icosadecan-2-one.
In a fifth aspect of the invention there is provided the use of a dsRNA of the second aspect of the invention, a biomaterial of the third aspect of the invention and/or a reagent of the fourth aspect of the invention in any one of b 1) to b 8):
b1 Weakening the response of the blattaria insect to sex pheromone;
b2 Preparing a product for weakening the reaction of the blattaria insect to the sex pheromone;
b3 Interfering/suppressing male and female mating of cockroaches;
b4 Preparing a product which interferes/suppresses male and female mating of the blattaria insect;
b5 Extension of the incubation period of cockroaches;
b6 Preparing a product for prolonging the coupling incubation period of the cockroach-order insects;
b7 Preventing and treating blattaria insects;
b8 Preparing a product for preventing and treating the blattaria insects.
In some embodiments of the invention, the blattaria insect comprises at least one of periplaneta americana, periplaneta australis, periplaneta germanica, periplaneta japonica.
In some preferred embodiments of the invention, the blattaria insect is german cockroach.
In some embodiments of the invention, the sex pheromone comprises 3, 11-dimethyl-icosadecan-2-one.
In a sixth aspect, the present invention provides a method comprising the step of reducing the expression level and/or activity of an ionotropic receptor co-receptor gene IR25a and/or an ionotropic receptor co-receptor gene IR76b in an insect of the order Blatta, wherein the method is any one of c 1) to c 4):
c1 A method of interfering/suppressing male and female mating of an insect of the order blattaria;
c2 A method for prolonging the coupling incubation period of the cockroach insects;
c3 A method for controlling insects of the order blattaria;
c4 A method for weakening the response of the blattaria insect to the sex pheromone.
In some embodiments of the invention, the amino acid sequence of the ionic receptor co-receptor gene IR25a is shown in SEQ ID NO. 2, and the amino acid sequence of the ionic receptor co-receptor gene IR76b is shown in SEQ ID NO. 4.
In some embodiments of the invention, the step of reducing the expression level and/or activity of the ionotropic receptor co-receptor gene IR25a and/or the ionotropic receptor co-receptor gene IR76b in the blattaria insect is to introduce the dsRNA of the second aspect of the invention, the biomaterial of the third aspect of the invention and/or the agent of the fourth aspect of the invention into the blattaria insect.
In some embodiments of the invention, the means of introduction comprises oral feeding or injection.
In some embodiments of the invention, the oral feeding comprises administering, spraying, atomizing, or administering to the food or water a dsRNA of the second aspect of the invention, a biomaterial of the third aspect of the invention, and/or an agent of the fourth aspect of the invention.
In some embodiments of the invention, the injection is from injection into the haemolymph along the abdominal internode of an insect of the order blattaria.
In some embodiments of the invention, the blattaria insect comprises at least one of periplaneta americana, periplaneta australis, periplaneta germanica, periplaneta japonica.
In some preferred embodiments of the invention, the blattaria insect is german cockroach.
In some embodiments of the invention, the sex pheromone comprises 3, 11-dimethyl-icosadecan-2-one.
The beneficial effects of the invention are as follows:
The invention discloses an ionic receptor Co-receptor gene (Ionotropic Receptor Co-receptor, IR-Co) IR25a and IR76b for regulating and controlling the German cockroach reproduction related behaviors for the first time, wherein the ionic receptor Co-receptor gene IR25a inhibitor and/or the ionic receptor Co-receptor gene IR76b inhibitor is used for downwards regulating the ionic receptor Co-receptor gene IR25a and/or IR76b (inhibiting the expression of the ionic receptor Co-receptor gene IR25a and/or IR76 b), degrading the ionic receptor Co-receptor gene IR25a and/or IR76b and inhibiting the activity of the ionic receptor Co-receptor gene IR25a and/or IR76b, so that the electrophysiological reaction of male worms to a contact pheromone standard is reduced, the coupling behavior of male worms to female worms is weakened or disappeared, and the purposes of interference and female-male mating and reproduction inhibition are achieved.
The invention designs specific dsRNA which can effectively block the coupling behavior of male insects and prolong the coupling latency based on two genes of the ion type receptor co-receptor genes IR25a and IR76 b. The dsRNA corresponding to each gene is respectively injected into the bodies of the male worms, so that the individual of the partially treated group male worms can lose the coupling behavior to the wild female worms, and the coupling latency period of the individual of the male worms which can be coupled is obviously prolonged. In particular, dsIR a is injected into the German cockroach male insects, so that the electrophysiological reaction of the male insects to the contact pheromone standard can be obviously reduced, the activity of neurons in the male insects antenna thorn type sensor (subtype 2) is inhibited, the interference and the inhibition of mating and reproduction of male and female insects are achieved, and the final purpose of pest control is realized. The invention provides a potential molecular target for the prevention and control strategy of fundamentally controlling the population quantity of the German cockroach by interfering the mating and mating activities of the German cockroach. In addition, the gene editing technology in recent years realizes breakthrough in the German cockroaches, and the invention can provide a brand new molecular target for genetic control of the German cockroaches by utilizing the gene editing technology in the future.
Drawings
FIG. 1 shows the change in the expression level of IR25a gene (A) or IR76B gene (B) after dsRNA injection in example 3, wherein the reference gene is actin-5c, and P <0.001.
Fig. 2 shows the variation of the males' coupling behavior to wild females in example 4, wherein a is the rate of fin lifting (coupling rate) of males, P <0.05, P <0.001, and b is the coupling latency of males, P <0.05, and P <0.001.
Fig. 3 shows the electrophysiological response to sex pheromone standard after 2 dsIR a injections to male worms in example 5, wherein a is the response frequency (pulses per second) of male worm antenna thorn sensor to sex pheromone standard per second, P <0.001 is shown, b is a representative single sensor record, and black arrow represents the time point of initiation of sex pheromone stimulation.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 acquisition of coding sequences for the genes IR25a and IR76b of German cockroach
Based on German cockroach genome annotation information and tentacle difference transcriptome analysis data, firstly, an ionic acceptor Co-receptor (IR-Co) gene IR25a potential coding sequence (CDS) is regulated according to a gene ID C0J52_39515, and the potential coding sequence is further checked by utilizing a third generation genome and bioinformatics analysis means to obtain a complete CDS sequence with the length of 2796bp, wherein the nucleotide sequence is shown as SEQ ID N0:1. The gene codes 931 amino acid residues, and is shown as SEQ ID NO. 2. Based on German cockroach genome annotation information and tentacle difference transcriptome analysis data, firstly, a potential coding sequence (CDS) of an ionic acceptor co-acceptor gene IR76b is called according to a gene ID (code division multiple access) C0J52_38862, and the potential coding sequence is further checked by utilizing a third generation genome and bioinformatics analysis means to obtain a complete CDS sequence with the length of 1617bp, wherein the nucleotide sequence is shown as SEQ ID NO. 3. IR76b encodes 538 amino acid residues as shown in SEQ ID NO. 4. Both the IR25a and IR76b gene-encoded protein sequences have ligand binding and transmembrane domains that are conserved for the ionophore receptor.
ATGGCTCTAGAGAATCGCCTTCTTTTTCTTCTTCTAGCTATGTTCTTCGACTTCTCAGCATCACAAAAAATCAACATTATGATACTGAATGAAGAGCGTAACTGGGAAGCCTCAGACTCGTTCAGGGCGGCCCAGGAGTATTTCAAGCAGACTCCGACACTGGGCGTGGAGATCGGCAACCTCATGAACGTGACGGGGAACACCACGGACGCCAAGACATTCCTCGAAATGATGTGCACCGAGTACAACAACTCCATCGCCGAGAAGAACCCGCCTCATATTGTCTTGGATTTCACGATGAGCAGCGTGCCTTCGGAGACTGCCAAGTCCTTCACGGCCGCTCTTGGTCTGCCCACCATCAGCACATCCTTCGGCCAAGAAGGCGACCTCCGACAATGGCGCACCCTTGACGAAAGGGAAAAGAAGTACCTGATCCAGATAATGCCGCCAGCAGATATAATTCCGGAGGTGATTCGCAGCATCGTCGTGTCACAGAACATCACCAACGCTGGAATTCTCTTTGACGACTCGATTGTGATGGACCATAAGTACAAGTCATTACTGCAGAACATTCCAACTCGTCACATGATCGTTAAAGTGGACCAACAAGGTCCCAGAGCCCAACTGGAGAGAATGAGGTCGAGAGACATTTTCAACTACTTCATTGTCGGGCGCATGACAACCATTAAAAAAGTATTTGACGTAGCCTACAAGAAGAAATACTTTATGCGGCAATTTGCATGGCATGGGATTACCTCTGACAGTGGCAACGTTTATTGTGACTGCAAAAATGCAACAGTGTTTTATGTAAAGCCAAGACCAAATCAAGAGTACACAGATCGCTATGATTTGCTCAAGACCAAGTTTGGACTCAGTGGGACACCAGAGATTACCGTCTCTTTTTATTTTGACATTGCCGTCAGGACACTTATGGCAGCCAAAACAATTATGAAAGGGAAACCATACTGGGAAAACTATGTCACTTGCAATGACTATGATGAGAAATCACGCCCTGTTCCTGATGTCAATCTCATGAAAGCATTCACAGAGGTCACAGAGAAGCCATCATACGGACCTTTTGTTTTGAAATCCAATGGACAGAGCATGATGAAGTTCAACATGGACATGACATTGGTCACAATTAACAACAAGATACCCGTCAAGTCGGTGGAGGTGGCTACATGGGAGGCAGATCTTGTCAAAGGCCTTGATATAAAAAATGGAGACAAAATGGTAGAAAACTCTGCAGTAACTGTGTATAGGATAGTGACAGTGGTTCAAAAACCATTTGTGATGTATGATGGAGTTGACGACAAGAACAGAACCAAATTCAAGGGGTACTGCATTGACTTGATTGATGAAATTAGAAACATAACCAAGTTTGATTATGACATATTTGAGGCACCTGATGGGAAATTTGGCAACATGGATGAGAATGGAGAGTGGAATGGCATGATCAAGGAACTTATTCTTAAGAATGCAGACATTGCACTGGGCTCCTTGTCAGTGATGGCAGAGCGTGAAAATGTAGTGGACTTCACAGTGCCATATTATGACTTGGTCGGCATCACTATCCTCATGAAGAAACCTAAAGCTGCCACTTCGCTCTTCAAATTTTTAACTGTGCTTGAGAATGATGTGTGGCTGTGCATCCTCGCAGCATACTTCTTCACTAGTTTTCTGATGTGGGTGTTTGACCGCTGGAGTCCATACAGTTATCAGAATAACAGAGAAAAATACAAGGATGATGAAGAAAAGCGAGAATTCAATCTGAAGGAGTGCCTCTGGTTTTGCATGACCTCCCTCACACCTCAGGGTGGAGGTGAAGCCCCCAAGAACTTATCTGGAAGGCTCGTGGCTGCCACATGGTGGTTATTTGGCTTCATCATCATCGCCTCATATACTGCCAACTTAGCTGCTTTCTTGACAGTGTCTCGACTTGACACACCAGTTGAGTCTTTGGATGATCTCGCCAAACAATATAAGATTCAGTATGCACCACTTGCAAACTCATCTGCACAGACCTATTTCCAGAGGATGGCAGACATCGAGAGCAGGTTTTATGAAATCTGGAAAGACATGAGCCTCAATGACAGTTTAACAGACGTAGAAAGAGCTAAACTGGCCGTGTGGGACTACCCTGTAAGTGACAAGTACACCAAGATATGGCAGGCTATGAAAGAGGCAAAGTTTCCTGACTCTCTTGAAGCAGCAGTCGCCAGAGTTATGGAGTCCAAGTCTTCAAGTGAAGGCTTTGCATTCATTGGTGATGCCACAGACATTCGATATCTGGTGCTGACGAGTTGTGACTTGCAAATGGTTGGGGAAGAATTCTCGAGGAAGCCCTATGCCATAGCCACACAGCAGGGTTCACCTTTAAAGGATCAGTTCAACAATGCAATTCTGCAGCTCTTGAACAAAAGAAAGCTGGAGAAGCTGAAGGAACAGTGGTGGAACCAGAACCCAGAGAAGAAGAGGAACTGTGAGAAACAAGATGACCAAACAGATGGTATCAGCATTCAAAACATTGGTGGTGTATTCATTGTGATCTTTGTGGGCATTGGCTTGGCATGCATCACGCTGGCATTTGAGTATTGGTGGTACAGGTTCAAGAGAAACCCACAGGTCGTGGACACAGGGAATGTTGTGGTGCAACCTCGTACAATCCCTACGGCTGGAGGTGGCAAGATGGATCCTCTCACAATGCCAGGCTTCAGACCCCGCAACACTGGGTTCTCAGAGAGACCCTTTACCCAGAGAAATGCAGCATTGGCCGCTGTAAACAACCCCTGGTGA(SEQ ID NO:1).
MALENRLLFLLLAMFFDFSASQKINIMILNEERNWEASDSFRAAQEYFKQTPTLGVEIGNLMNVTGNTTDAKTFLEMMCTEYNNSIAEKNPPHIVLDFTMSSVPSETAKSFTAALGLPTISTSFGQEGDLRQWRTLDEREKKYLIQIMPPADIIPEVIRSIVVSQNITNAGILFDDSIVMDHKYKSLLQNIPTRHMIVKVDQQGPRAQLERMRSRDIFNYFIVGRMTTIKKVFDVAYKKKYFMRQFAWHGITSDSGNVYCDCKNATVFYVKPRPNQEYTDRYDLLKTKFGLSGTPEITVSFYFDIAVRTLMAAKTIMKGKPYWENYVTCNDYDEKSRPVPDVNLMKAFTEVTEKPSYGPFVLKSNGQSMMKFNMDMTLVTINNKIPVKSVEVATWEADLVKGLDIKNGDKMVENSAVTVYRIVTVVQKPFVMYDGVDDKNRTKFKGYCIDLIDEIRNITKFDYDIFEAPDGKFGNMDENGEWNGMIKELILKNADIALGSLSVMAERENVVDFTVPYYDLVGITILMKKPKAATSLFKFLTVLENDVWLCILAAYFFTSFLMWVFDRWSPYSYQNNREKYKDDEEKREFNLKECLWFCMTSLTPQGGGEAPKNLSGRLVAATWWLFGFIIIASYTANLAAFLTVSRLDTPVESLDDLAKQYKIQYAPLANSSAQTYFQRMADIESRFYEIWKDMSLNDSLTDVERAKLAVWDYPVSDKYTKIWQAMKEAKFPDSLEAAVARVMESKSSSEGFAFIGDATDIRYLVLTSCDLQMVGEEFSRKPYAIATQQGSPLKDQFNNAILQLLNKRKLEKLKEQWWNQNPEKKRNCEKQDDQTDGISIQNIGGVFIVIFVGIGLACITLAFEYWWYRFKRNPQVVDTGNVVVQPRTIPTAGGGKMDPLTMPGFRPRNTGFSERPFTQRNAALAAVNNPW(SEQ ID NO:2).
ATGGGCATCGGAGCAAAGCTAGTTGGAGCTCTTCTAGCCCATGTCTGTGTCAACCATGACGTGATAATAAAAAACGGCACAAATCTTCAAGGAGAGCATATTCCTTGCGACTTATGGACACCAAAGGCCCTCCATGGACAACATTTCCTGATAGCAGCACTGGACCAACCACCCCTGAGCAAAAGAGTAGGCGATAAGAACTATACTGGGATAGTGTTCGACTTCATCGACATTTTGAAGGAAAAGTATGGGTTCACTTACGACGTGACGTTCCCGCCCTCGGCTGAGGAAAACATCATGGGAGATGCAGACAGCGGAATTATCGGGCGCGTCTATAAACATGAAGTGGACATGGCTGCTGCATTCTTGCCTGTATTCCACGACTTGGACAAACTGGTCAACTTCTCGACGACTCTGGACGAATCGAACTGGGTGGTGCTGATGAAGCGTCCTTCCGAGTCAGCAACTGGCTCTGGATTGCTGGCGCCCTTCGACGAGACGGTTTGGCTTCTAATTCTGATATCTTTGATAGCCATTGGTCCTACCATCTACGCCATCGTCTTCATAAGAGCTATTGTGTGCAAGAACGACGAGGTTCTGGCGACTGTGGTGCCTCTAGACAACTGCATCTGGTTCGTGTATGGTGCCCTCATGAAGCAAGGCTCTACCCTCATGCCTGTCGCTGATTCGACCCGTATACTGTTTGCAACTTGGTGGATCTTCATAACCATCCTGACTTCATTCTACACGGCCAACTTGACTGCTTTCCTGACATTGTCGAAATTCACTTTGCCAATCGAAGGTCCAAAAGATCTGGCCAAGAATAGAGCAGGATGGATTTCACACAAAGGGACTTCTCTGGAATATCAGGTCGAGAACAACAAAGATTACGAGTACCTCAATAAAACTGTGCGAGATGGACGAGGCCGATTCCTCATCGAAAAGGACGAGGACATGTTGTTGATGGTAAAGAACAAGAAGTTTTCGCTGCTGAGAGAGAGGCGTGCTGTGGAGTACTTCATGTTCCGTGACTATCTCACGAAGGCAGAAAACAATGTCCCCGAGAATGAACGCTGTACCTATGTCGTCACACCAAAATCATTCATGGCCCATGGCATTGCTTTTGCTTATCCCAAGAACAGTACCTTGGCAAAACTATTTGACCCGATATTCCAGGCATTGGTGGAAACAGGAATTGTGAAACATCTGCTGAAGAAAGATCTGCCTCCCACTGAGATCTGTCCTCTGAACTTGAAGAGTACAGAACGACAGCTGAGGAACGGTGATCTGTTCACGACCTACATGGTGGTAGTTATTGGCTTCATTGCTGGAATTGCCGCTTTCATTGGAGAGGTGATGTACACCACTGTCAAGAGATGCGCTGCTGGAACTAAGATAGAGGTACCAGACACCGATTGGACAGGAAATGTCGGATACAAGAAATCGCAGCTATTCCCTCCGCCTTATTCTGTGTTTATGCAGCAACAACCGCCACCTTTTGGAAAACACCAAACTATCAACGGAAGAGATTATCTGGTCATTAACAACAAAACTGGCGATCCGCAACTGATTCCTGTTAGATCACCATCAGCTTTCCTTTTCCAATATTCTGCTTAA(SEQ ID NO:3).
MGIGAKLVGALLAHVCVNHDVIIKNGTNLQGEHIPCDLWTPKALHGQHFLIAALDQPPLSKRVGDKNYTGIVFDFIDILKEKYGFTYDVTFPPSAEENIMGDADSGIIGRVYKHEVDMAAAFLPVFHDLDKLVNFSTTLDESNWVVLMKRPSESATGSGLLAPFDETVWLLILISLIAIGPTIYAIVFIRAIVCKNDEVLATVVPLDNCIWFVYGALMKQGSTLMPVADSTRILFATWWIFITILTSFYTANLTAFLTLSKFTLPIEGPKDLAKNRAGWISHKGTSLEYQVENNKDYEYLNKTVRDGRGRFLIEKDEDMLLMVKNKKFSLLRERRAVEYFMFRDYLTKAENNVPENERCTYVVTPKSFMAHGIAFAYPKNSTLAKLFDPIFQALVETGIVKHLLKKDLPPTEICPLNLKSTERQLRNGDLFTTYMVVVIGFIAGIAAFIGEVMYTTVKRCAAGTKIEVPDTDWTGNVGYKKSQLFPPPYSVFMQQQPPPFGKHQTINGRDYLVINNKTGDPQLIPVRSPSAFLFQYSA(SEQ ID NO:4).
EXAMPLE 2 preparation of dsRNA fragments of German cockroach IR25a and IR76b genes
Based on the sequences shown in SEQ ID NO. 1 and SEQ ID NO. 3, specific Primer pairs for amplifying the IR25a and IR76b gene fragments were designed using NCBI on-line Primer design tool Primer-BLAST, respectively. Primer sequences for amplifying the IR25a gene fragment are shown below as 5'-TTGAGGCACCTGATGGGAAA-3' (SEQ ID NO: 5) and 5'-GTGTGAGGGAGGTCATGCAA-3' (SEQ ID NO: 6). Primer sequences for amplifying the IR76b gene fragment are shown below as 5'-ATGTCCCCGAGAATGAACGC-3' (SEQ ID NO: 7) and 5'-TGTTTTCCAAAAGGTGGCGG-3' (SEQ ID NO: 8).
For the acquisition of the DNA template required for the synthesis of the dsRNA of the IR25a gene, PCR amplification was performed with the use of a recombinant plasmid containing the sequence shown in SEQ ID NO. 1 as template, with the use of primer pairs SEQ ID NO. 5 and SEQ ID NO. 9 (5'-taatacgactcactataggGTGTGAGGGAGGTCATGCAA-3', wherein the lower case letter part is the T7 promoter sequence), and with the use of primer pairs SEQ ID NO. 6 and SEQ ID NO. 10 (5'-taatacgactcactatagg TTGAGGCACCTGATGGGAAA-3', wherein the lower case letter part is the T7 promoter sequence), respectively.
For the acquisition of the DNA template required for the synthesis of the dsRNA of the IR76b gene, PCR amplification was performed with the use of a recombinant plasmid containing the sequence shown in SEQ ID NO. 3 as template, with the use of primer pairs SEQ ID NO. 7 and SEQ ID NO. 11 (5'-taatacgactcactataggTGTTTTCCAAAAGGTGGCGG-3', wherein the lower case letter part is the T7 promoter sequence), respectively, and with the use of primer pairs SEQ ID NO. 8 and SEQ ID NO. 12 (5'-taatacgactcactatagg ATGTCCCCGAGAATGAACGC-3', wherein the lower case letter part is the T7 promoter sequence).
The PCR amplification procedure for the 2 gene fragments was 94℃for 2min, followed by 34 cycles of 94℃30s, 55℃30s and 72℃30s, and finally 72℃for 5min. The DNA polymerase used for PCR was Ex Taq enzyme from TAKARA. Further, dsRNA was synthesized and purified using T7 RiboMAXTM Express RNAI SYSTEM (Beijing Promega) in vitro transcription kit, designated dsIR a (SEQ ID NO: 13) and dsIR76b (SEQ ID NO: 14), respectively. The concentration was adjusted to 3. Mu.g/. Mu.L with RNase-FREE WATER water under the monitoring of a NanoDrop One ultraviolet spectrophotometer and stored in a-80℃refrigerator for use.
UUGAGGCACCUGAUGGGAAAUUUGGCAACAUGGAUGAGAAUGGAGAGUGGAAUGGCAUGAUCAAGGAACUUAUUCUUAAGAAUGCAGACAUUGCACUGGGCUCCUUGUCAGUGAUGGCAGAGCGUGAAAAUGUAGUGGACUUCACAGUGCCAUAUUAUGACUUGGUCGGCAUCACUAUCCUCAUGAAGAAACCUAAAGCUGCCACUUCGCUCUUCAAAUUUUUAACUGUGCUUGAGAAUGAUGUGUGGCUGUGCAUCCUCGCAGCAUACUUCUUCACUAGUUUUCUGAUGUGGGUGUUUGACCGCUGGAGUCCAUACAGUUAUCAGAAUAACAGAGAAAAAUACAAGGAUGAUGAAGAAAAGCGAGAAUUCAAUCUGAAGGAGUGCCUCUGGUUUUGCAUGACCUCCCUCACAC(SEQ IDNO:13).
AUGUCCCCGAGAAUGAACGCUGUACCUAUGUCGUCACACCAAAAUCAUUCAUGGCCCAUGGCAUUGCUUUUGCUUAUCCCAAGAACAGUACCUUGGCAAAACUAUUUGACCCGAUAUUCCAGGCAUUGGUGGAAACAGGAAUUGUGAAACAUCUGCUGAAGAAAGAUCUGCCUCCCACUGAGAUCUGUCCUCUGAACUUGAAGAGUACAGAACGACAGCUGAGGAACGGUGAUCUGUUCACGACCUACAUGGUGGUAGUUAUUGGCUUCAUUGCUGGAAUUGCCGCUUUCAUUGGAGAGGUGAUGUACACCACUGUCAAGAGAUGCGCUGCUGGAACUAAGAUAGAGGUACCAGACACCGAUUGGACAGGAAAUGUCGGAUACAAGAAAUCGCAGCUAUUCCCUCCGCCUUAUUCUGUGUUUAUGCAGCAACAACCGCCACCUUUUGGAAAACA(SEQ ID NO:14).
Example 3 efficiency detection of in vivo injection and RNA interference of dsIR a and dsIR76b
Male worms on the eclosion day are selected and randomly divided into 3 groups, wherein one group is used for injecting dsRNA (the sequence of which is shown as SEQ ID NO: 15) corresponding to a mouse lymphotoxin protein (Muslta) gene and is used as a control group, one group is used for injecting dsIR a or dsIR76b and is used as a dsIR a experimental group, and the other group is used for injecting dsIR76b and is used as a dsIR76b experimental group. After transient carbon dioxide anaesthesia of the males, dsRNA was injected from the 7/8 th internode of the abdomen to haemolymph with a microinjection instrument (Alcott Biotechnology) under microscopic magnification. The injection volume was 2. Mu.L and the dsRNA concentration was 3. Mu.g/. Mu.L. The injected cockroaches were placed into clean bioassay containers. Waiting for 10-20 min, allowing them to recover from the influence of carbon dioxide. The containers were labeled with date of injection, type and dose of dsRNA, and age of German cockroach. On the 3 rd day of emergence, the male worms were injected twice, with the same dose as the first time.
And on the 5 th day after eclosion, collecting 12-15 pairs of male worm antennae for each sample, and extracting total RNA by using a Trizol kit method. A first strand cDNA was then obtained by reverse transcription with 2. Mu.g total RNA. The qPCR primers were designed using NCBI on-line tool Primer Blast on-line tool, IR25a gene qPCR Primer sequences were 5'-ACCTCGTACAATCCCTACG-3' (SEQ ID NO: 16) and 5'-TTCTCTGGGTAAAGGGTCTC-3' (SEQ ID NO: 17), respectively, IR76b gene qPCR Primer sequences were 5'-GCAGGATGGATTTCACACAA-3' (SEQ ID NO: 18) and 5'-CCTTTTCGATGAGGAATCGG-3' (SEQ ID NO: 19), respectively, and the German cockroach actin gene (actin-5 c) was used as an internal reference gene for qPCR detection, with 4 biological replicates for each of the control and experimental groups.
The qPCR detection results are shown in FIG. 1, and it can be seen from the graph that the expression level of IR25a or IR76b in the tentacle of the male worms is remarkably inhibited after two injections of dsRNA. Wherein, the RNA interference efficiency of IR25a was about 94% (A in FIG. 1), and the RNA interference efficiency of IR76B was about 96% (B in FIG. 1).
CACCCUCUCCACGAAUUGCUCGGCCGUUCACUGGAACUCCUGGGCCUGACCCAGCUCCCUGCUAGUCCCUGCGGCCCACAGUUCCCCGGACCCGACUCCCUUUCCCAGAACGCAGUAGUCUAAGCCCUUAGCCUGCGGUUCUCUCCUAGGCCCCAGCCUUUCCUGCCUUCGACUGAAACAGCAGCAUCUUCUA(SEQ ID NO:15).
Example 4 injection dsIR a or dsIR b significantly inhibited the coupling rate of german cockroach males to wild females
The dsRNA living injection protocol as described in example 3 was used for RNA interference of IR25a or IR76b genes in the antenna of german cockroach males to analyze the variation of males' coupling behavior under the premise of effectively reducing the expression level of IR25a or IR76 b. And carrying out coupling behavioral detection by using a video recording means on the 5 th day after male insect emergence. The specific process comprises loading male single into single-port transparent glass tube (7 cm long and 2cm diameter), introducing 1 wild female on day 3 after eclosion, pairing male and female at 1:1, and blocking the other end of the glass tube with cotton ball. Recording the mating behavior of the German cockroach by using a Lumix GH4 high-definition camera for 15min. The acquired behavioural source video is analyzed, and 2 main behavioural parameters are analyzed, namely 1) the rate of lifting wings of the male insects (the marked behavioural characteristics of the coupling stage) and 2) the incubation period of lifting wings of the male insects (the time interval from the contact of male insects with antennae of the male insects to the occurrence of lifting wings of the male insects).
As a result, as shown in fig. 2, it can be seen that the males in the injection dsIR a or dsIR b were significantly reduced in the coupling rate compared to the control group, about 41% of the male individuals in the treatment group injected with dsIR a were no longer coupled to the wild-type females, and about 87% of the male individuals in the treatment group injected with dsIR b were no longer coupled to the wild-type females (fig. 2 a). In the remaining males that can be pupled, their puppet latency is significantly prolonged (B in fig. 2).
Example 5 injection dsIR a significantly inhibited the electrophysiological response of german cockroach males to sex pheromones
A standard of the German cockroach contact pheromone component 3, 11-dimethyl icosaxalan-2-one was prepared into an n-hexane solution with a concentration of 50 ng/. Mu.L. Taking a certain volume of normal hexane solution of the standard substance, naturally airing in a chromatographic bottle, adding 10mM KCl solution (containing 0.1% Triton X-100) with the same volume as normal hexane to prepare a single sensor recording stimulation liquid, and standing by vortex for standby. Control solution recorded as single sensor with 10mM KCl solution. dsRNA microinjection was performed on Blatta germanica males as described in example 3. Electrophysiological responses of males to sex pheromones were detected on day 6 after eclosion using a single sensor recorder (Synthch, germany) Taste Probe. Fixing the insect body and the antenna by using low-melting-point paraffin, inserting a silver wire of a reference electrode into the abdomen of the insect body, sleeving a capillary glass tube filled with a stimulating solution into the silver wire at the other end of the capillary glass tube to serve as a recording electrode. Under a microscope, the glass tip of the recording electrode is slowly sleeved into the top end of the feeler-pin-shaped sensor (subtype 2), so that the stimulating liquid contacts with the top hole of the sensor, and the recording electrode filled with the control solution is replaced to record the same sensor after the recording is successful. The number of pulses in response to the stimulus within 1s of the tentacles of the control and treatment groups, respectively, was counted using Autospike software.
As shown in fig. 3, it can be seen that the response to sex pheromone standard was weaker in the male worms after injection dsIR a compared to the control group, showing a significant decrease in the number of pulses per second of response.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (9)
1. Use of an inhibitor of the ionotropic receptor co-receptor gene IR25a and/or an inhibitor of the ionotropic receptor co-receptor gene IR76b in any of (1) to (8):
(1) Weakening the response of the blattaria insects to sex pheromones;
(2) Preparing a product for weakening the reaction of the blattaria insects to sex pheromone;
(3) Interfering/suppressing male and female mating of cockroaches;
(4) Preparing a product which interferes/inhibits male and female mating of the cockroaches;
(5) Prolonging the coupling incubation period of the cockroach insects;
(6) Preparing a product for prolonging the coupling incubation period of the cockroach insects;
(7) Preventing and treating blattaria insects;
(8) Preparing a product for preventing and treating the blattaria insects;
the ionic receptor co-receptor gene IR25a inhibitor or the ionic receptor co-receptor gene IR76b inhibitor is at least one of the following components (9) - (11):
(9) dsRNA targeting the ionotropic receptor co-receptor gene IR25a or the ionotropic receptor co-receptor gene IR76 b;
(10) A nucleic acid molecule encoding the dsRNA targeting the ionotropic receptor co-receptor gene IR25a or the ionotropic receptor co-receptor gene IR76b of (9);
(11) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of (10);
The dsRNA is double-stranded RNA composed of a nucleotide sequence shown as SEQ ID NO. 13 or SEQ ID NO. 14 and a nucleotide sequence shown as a reverse complementary sequence thereof;
The blattaria insect is Blatta germanica.
2. The use according to claim 1, wherein the amino acid sequence of the ionotropic receptor co-receptor gene IR25a is shown in SEQ ID NO. 2 and the amino acid sequence of the ionotropic receptor co-receptor gene IR76b is shown in SEQ ID NO. 4.
3. A dsRNA comprising a nucleotide sequence represented by SEQ ID NO. 13 or SEQ ID NO. 14 and a nucleotide sequence represented by the reverse complement thereof.
4. A biomaterial associated with the dsRNA of claim 3, the biomaterial comprising any one of 1) to 12):
1) A nucleic acid molecule encoding the dsRNA of claim 3;
2) An expression cassette comprising 1) the nucleic acid molecule;
3) A vector comprising 1) the nucleic acid molecule;
4) A vector comprising 2) the expression cassette;
5) A transgenic cell line comprising 1) said nucleic acid molecule;
6) A transgenic cell line comprising 2) said expression cassette;
7) A transgenic cell line comprising 3) the vector;
8) A transgenic cell line comprising 4) the vector;
9) A recombinant microorganism comprising 1) said nucleic acid molecule;
10A recombinant microorganism comprising 2) said expression cassette;
11A recombinant microorganism containing 3) the vector;
12A recombinant microorganism containing the vector of 4).
5. An agent comprising the dsRNA of claim 3 and/or the biological material of claim 4.
6. The reagent according to claim 5, wherein,
The reagent is used for any one of a 1) to a 4):
a1 Weakening the response of the blattaria insect to sex pheromone;
a2 Interfering/suppressing male and female mating of cockroaches;
a3 Extension of the incubation period of cockroaches;
a4 Preventing and treating blattaria insects.
7. Use of the dsRNA of claim 3, the biomaterial of claim 4 and/or the agent of claim 5 or 6 in any one of b 1) to b 8):
b1 Weakening the response of the blattaria insect to sex pheromone;
b2 Preparing a product for weakening the reaction of the blattaria insect to the sex pheromone;
b3 Interfering/suppressing male and female mating of cockroaches;
b4 Preparing a product which interferes/suppresses male and female mating of the blattaria insect;
b5 Extension of the incubation period of cockroaches;
b6 Preparing a product for prolonging the coupling incubation period of the cockroach-order insects;
b7 Preventing and treating blattaria insects;
b8 Preparing a product for preventing and treating the blattaria insects;
The blattaria insect is Blatta germanica.
8. A method comprising the step of reducing the expression level and/or activity of an ionic receptor co-receptor gene IR25a and/or an ionic receptor co-receptor gene IR76b in an insect of the order Blatta, wherein the method is any one of c 1) to c 4):
c1 A method of interfering/suppressing male and female mating of an insect of the order blattaria;
c2 A method for prolonging the coupling incubation period of the cockroach insects;
c3 A method for controlling insects of the order blattaria;
c4 A method for weakening the response of the blattaria insect to the sex pheromone;
The blattaria insect is German cockroach;
The step of reducing the expression level and/or activity of the ion receptor co-receptor gene IR25a and/or the ion receptor co-receptor gene IR76b in the blattaria insect is to introduce the dsRNA of claim 3, the biomaterial of claim 4, the agent of claim 5 or 6 into the blattaria insect.
9. The method according to claim 8, wherein the amino acid sequence of the ionotropic receptor co-receptor gene IR25a is shown in SEQ ID NO. 2 and the amino acid sequence of the ionotropic receptor co-receptor gene IR76b is shown in SEQ ID NO. 4.
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