Crassostrea hongkongensis gene related to adductor muscle tension and application thereof

Abstract

The application belongs to the field of marine biotechnology and particularly relates to a Crassostrea hongkongensis adductor muscle tension related gene and application thereof. The nucleotide sequence of the Crassostrea hongkongensis adductor muscle tension related gene ManIIa is shown as SEQ ID NO:1. The application injects a dsRNA interference sequence of the gene into the adductor muscle of Crassostrea hongkongensis, significantly reduces the adductor muscle tension of Crassostrea hongkongensis, and causes the mortality of Crassostrea hongkongensis under high temperature stimulation to be significantly higher than that of a control group. The application provides a new specific molecular target for RNA interference based biofouling prevention and removal and provides a new way for marine corrosion prevention.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of marine biotechnology, and particularly relates to a Crassostrea ariakensis adductor muscle tension-related gene and application thereof. BACKGROUND

[0002] Oyster is a typical dominant species in intertidal zone and estuary, and is highly adaptable and easy to attach to substrate structures such as coastal zone reefs and facility platforms, and is one of the most common marine fouling organisms. The coasts of China have serious corrosion problems due to the attachment of hard fouling organisms such as oysters on wharfs, dikes, ship bottoms and the like. Compared with other macrofouling organisms such as barnacles, the attachment of oysters can cause significant localized corrosion, and the formation of concentration cells between the surface of the metal and the like and the oysters can cause corrosion and damage; in addition, the reductive bacteria formed by the metabolic products or excretions of oysters can also indirectly cause corrosion, and the degree of harm is much greater than that of other species, so oysters are the main object of marine corrosion prevention and removal.

[0003] At present, the research on oyster attachment corrosion at home and abroad is still in its infancy. Common oyster fouling prevention measures include three categories: electrolytic antifouling method and other chemical methods, cathodic protection method and other physical methods, and biomimetic treatment method and other biological methods. The existing methods all change the external environment to form environmental stress on oysters to achieve the prevention effect, but do not modify the oysters themselves.

[0004] Crassostrea ariakensis is a major group in estuary, especially in the coastal areas of southern China. Compared with the main economic species of the same ecological niche, Crassostrea hongkongensis, it has strong adaptability, wider distribution and relatively lower economic value, and is the main oyster species to be prevented in the corrosion prevention engineering of wharfs, ships and the like in the coastal areas of southern China. Shell closure is an important high-adaptability defense mechanism of oysters and other bivalves, which is mainly controlled by the adductor muscle. With the realization of genome mapping and genetic manipulation in Crassostrea ariakensis, the key gene controlling shell closure, alpha-mannosidase-2 (ManIIa), has been identified. This gene has been proved to be an effector gene regulating muscle development in model species. However, there is no report on the effector gene regulating muscle development in Crassostrea ariakensis. SUMMARY

[0005] The application aims to provide a Crassostrea ariakensis adductor muscle tension-related gene and application thereof.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0007] The nucleotide sequence of a Crassostrea hongkongensis adductor muscle tension-related gene, Crassostrea hongkongensis adductor muscle tension-related gene ManIIa is shown as SEQ ID NO: 1.

[0008] The application of a Crassostrea hongkongensis adductor muscle tension-related gene, the application of Crassostrea hongkongensis adductor muscle tension-related gene ManIIa in regulating the muscle development of Crassostrea hongkongensis.

[0009] An interference sequence interfering with a Crassostrea hongkongensis adductor muscle tension-related gene, the interference sequence is a double-stranded sequence of 19 bp, and the ends contain a dTdT structure. Preferably, the 3' end of the dsRNA contains a dTdT structure.

[0010] The interference sequence is obtained by a pair of upstream and downstream specific primers, and the upstream and downstream specific primers are as follows:

[0011] The upstream primer ds ManIIa-F is 5'-GCUCUGUUCUACUGUUCAATT-3';

[0012] The downstream primer ds ManIIa-R is 5'-UUGAACAGUAGAACAGAGCTT-3'.

[0013] The application of an interference sequence interfering with a Crassostrea hongkongensis adductor muscle tension-related gene, the application of the interference sequence in preventing marine fouling organisms.

[0014] A method for preventing the corrosion of Crassostrea hongkongensis, in which an interference sequence dsRNA is synthesized in vitro, and then injected into Crassostrea hongkongensis to regulate the ManIIa gene in the adductor muscle of Crassostrea hongkongensis, inhibit the tension of the adductor muscle of Crassostrea hongkongensis, and further prevent the corrosion of Crassostrea hongkongensis to the environment.

[0015] Further, the step of injecting the dsRNA interference sequence of the Crassostrea hongkongensis adductor muscle tension-related gene ManIIa into the adductor muscle. The interference sequence is a double-stranded RNA molecule, and the dsRNA sequence of the sequence is synthesized in vitro by using specific primers. The Crassostrea hongkongensis is temporarily opened by anesthesia, and then the dsRNA sequence is injected. The results show that the sequence can significantly inhibit the expression of alpha-mannosidase-2 (ManIIa) gene, and the tension of the adductor muscle is significantly reduced. Further study on the influence of ManIIa gene on the high temperature tolerance of Crassostrea hongkongensis shows that the inhibition of the expression of ManIIa gene significantly reduces the survival rate of Crassostrea hongkongensis under acute high temperature stimulation.

[0016] A kit for interfering with the tension of the adductor muscle of Crassostrea hongkongensis, the kit containing the interference sequence.

[0017] A kit for interfering with the tension of the adductor muscle of Crassostrea hongkongensis, the kit containing a pair of upstream and downstream specific primers of the interference sequence.

[0018] Advantages of the present application:

[0019] The present application first discovers that alpha-mannosidase-2 (ManIIa) gene is involved in the process of shell closure of Crassostrea ariakensis, and inhibiting the expression of ManIIa gene significantly reduces the adductor muscle tension of Crassostrea ariakensis, and the mortality rate under high temperature. The gene can be used for developing related products for preventing and removing the corrosion of Crassostrea ariakensis.

[0020] The present application provides an interference sequence of alpha-mannosidase-2 (ManIIa) gene of Crassostrea ariakensis, which can significantly knock down the expression of ManIIa gene, reduce the adductor muscle tension, and increase the mortality rate of Crassostrea ariakensis under high temperature. The interference sequence can be used for developing specific, efficient, green and environmentally friendly anti-corrosion products to achieve the purpose of preventing and removing Crassostrea ariakensis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 4 shows the effect of injecting dsRNA of ManIIa gene on the expression of ManIIa gene of Crassostrea ariakensis (different letters represent significant difference, p<0.05); wherein, the experimental group is ds-ManIIa, and the control group is ds-GFP and SW.

[0022] Figure 2 Figure 5 shows the effect of injecting dsRNA of ManIIa gene on the adductor muscle tension of Crassostrea ariakensis (different letters represent significant difference, p<0.05); wherein, the experimental group is ds-ManIIa, and the control group is ds-GFP and SW.

[0023] Figure 3 Figure 6 shows the effect of injecting dsRNA of ManIIa gene on the high temperature tolerance of Crassostrea ariakensis; wherein, the experimental group is ds-ManIIa, and the control group is ds-GFP and SW. DETAILED DESCRIPTION

[0024] The specific embodiments of the present application are further described in conjunction with examples, and it should be pointed out that the specific embodiments described herein are only for the purpose of illustrating and explaining the present application, and are not limited to the present application.

[0025] Example 1

[0026] According to the previous study (Li A, Dai H, Guo X, Zhang Z, Zhang K, Wang C, Wang X, Wang W, Chen H, Li X, et al: Genome of the estuarine oyster provides insights into climate impact and adaptive plasticity. Communications Biology 2021, 4: 1287.), a high-continuity, chromosome-level genome assembly database of C. fluminea was obtained by using third-generation Nanopore, second-generation HiC and illumina sequencing methods, and the information of third-generation full-length transcriptome, gene prediction and genome annotation was integrated. By using bioinformatics methods, the full-length of ManIIa gene located on the fragment of 38914720-38926094 on chromosome 5 of C. fluminea with a length of 11375 bp (SEQ ID NO. 1) and the CDS sequence with a length of 480 bp (SEQ ID NO. 3) were obtained.

[0027] SEQ ID NO. 1

[0028] ttgaaatacaattactattgaaactagagcaaagctcgttgcaaagcaacgagtag gtcttccgttgaagctgcgaattaaaatgatgttaatgtacatggaaaggattatctt ca tggtaatgctttagtagctatactaaacgactaatataaacgtacaatttatcttgg acttttatttgtccttgtacatgtagaaatgtgtaacaggtattgtttgcaaataaaat tgggattgctattggaaatttaaaaacaaaatctgacgctttcaaattaaataataaa ttgaagtcaaattttttattaattgaaaataattaagatgagcatggatgaatgcttg tttacgttcaaataatttgtatgtttctaatcctgtgatctctgtacattcacatatag ttatatagcttgatacgtgcgtgaaacctgaaacatttatcagacatgttgtaaacttg acggatatagcgaattaagcttataaagaagtaaaattgcccgtccctggaccttcc ttataggcgtgttttactgtataaggcagatttatggtatagataaaaatattttcgg ataatttagagttatcaaacattgctaaggatcgaagatcataagtagggaatctgc ggtaaacactggttaatgatccacaataaacactggtaaatgataaacactggtaaat gataaacactggtgaatgataatcacgggtaaatgataaacactcagtctggtaaatga taaacactggtaaatgataaacactggtgaatgataatcacgggtaaatgataaacac tcagtctggtaaatgataaacactggtaaatgataaacactggtgaatgataatcacc ggtaaatgataaacactcagtctggtaaatgataaacactggtaaatgataaacactg gtgaatgataatcacgggtaaatgataaacactcagtctggtaaatgataaacactgg taaatgataaacactggtgaatgataatcacgggtaaatgataaacactcagtctgg taaatgataaacactggtaaatgataaacactggtgaatgataatcacgggtaaatga taaacactcagtctggtaaatgataaacactggtaaatgataaacactggtgaatgat aatcacgggtaaatgataaacactcagtctggtaaatgataaacactggtaaatgata aacactggtgaatgataatcacgggtaaatgataaacactcagtctggtaaatgataa acactggtaaatgataaacactggtgaatgataatcacgggtaaatgataaacactca gtctggtaaatgataaacactggtaaatgataaacactggtgaatgataatcacgggt aaatgataaacactcagtctggtaaatgataaacactggtaaatgataaacactggtg aatgataatcacgggtaaatgataaacactcagtctggtaaatgataaacactggtaa atgataaacactggtgaatgataatcacgggtaaatgataaacactcagtctggtaaa t900 actggtaaat gataaacac tggttaatga tccacagtaa acactggtaa atgataaacac 960 tggtaaatga taaacactgg taatatgata aacactggtt tatgatccac aggaacactg gttaatgat 1020 taacactggt aaatgacaaac actggttaat gat aaacactgg tttatgatcc acagtacctg aagtaggcc 1080 agtggagccg gagaatttcg cc tccaggaaa gagttgtcta ttgtgaattc tgaagactcc ttcttctct 1140 actgaaaact tagacctgta gaaaacaaga acagtatgtc agtatcaata tgtaaatatg agagacagac 1260 agacagacag acagacagac agacaggtag gcaggcaggg acacagtcag tatcaatatg taagtactgt 1320 aacctacttt tatttgcga caactttatt tcataatta aacaacttaa gatgaactgt ttcacgatga 1380 ctttttcgtg attttcgttt gagatgtca ttacaccaaa aagaaaagac tggttagggt gagaaatatt 1440 cggaattcaa gactctaac aaacctcgca aatatttctc acacgtacat aaaagttgtt tacagagaga 1500 tagaaagaga gagagagaga gagagagaga gagacaaaga gagagagaggg agacagaggg agaggaagag 1560 aaagggaaag agagattttc aaaagagaga ggagaaagga aaagagagag ggaaaaagag agggggaaag 1620 agaggggtgg ggaaaggggg aaagagagat ggggtatatt gagcgtaaga gatggaagaa aaaaagggaac 1680 agaggaaatg tcagttacac caggtaaatat gagagaaaat tttcttctta tttcttctta tttcttctt 1740 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 1800 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 1860 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 1920 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 1980 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 2040 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 2100 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 2160 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 2220 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 2280 atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt atttcttctt 2340 atttcttctt1800 aacagacaga ataagcacag ataatgcaaa atttctacct gcaccattga agaaagagag 1860 atggagggtg ggaaatgata atttaaagag agaaaggtaa atgtagaaaa aactaag 1920 agagtaagaag agggaatcag atggaaacag agtatgacgg tatgaatcta tacataata 1980 aagagatttt aaaaaactac acctgataca tggtaatata aggggtaagc tatttgtctt 2040 tgtgataata atcatactac agactcaaga aacgtatctc agtatcaata tctaaatatg 2100 agagagagag agagagagag agagagagag agagagagag agagagagag agggaggtag 2160 catgagagag gggggagggagggtgaaagagagagggggtgcaagaggaagagggggtg 2220 caagg aagagataag gtgagagagaaagtgagataaaaagaaatagggtgagaaaaaacaat 2280 agagagaaag gt aagaaaaag acacagataag ctagaagtat ttgtgataac attgtgact 2340 ggggtgatat ttgtgataac aatcagacta cagactcact gagctgaggg tccgtctcca 2400 tggtagtatg tgactgtggc cagactgttg ctggagctgt cctttctctg tgatatctga 2460 tatctgacaa ttcccagggc gggcagctcg gtcacaaaag acagctgtaa aacatacaac 2520 acaggatgtaaatcaatg actctactgg cagcttacag gcagcttaat cacaagggac ag 2580 ctttaaacag ataagtcaaa ggcttgaatg gtccaactgc catcttattt atttatttga 2640 aatcacaggt ttgagggcct tactgtcatat tactgattag tttttttaag tcattggct 2700 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 2760 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 2820 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 2880 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 2940 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 3000 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 3060 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 3120 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 3180 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 3240 gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg gttggtggtg 3300 gtt2700 tgaaaggctatgctggcatcttcctaattttttatcggaaatcaaaggcttgaaaggcct 2760 cactgccatcattaaacatttgaaatttagcatgcagtttatggagggaatcatgcaact 2820 ctgggtctgtcctgatctcttaagtgaatattttaattttcaaatcagctgaaattggta 2880 aattagtaaaatcacattcctgcgtgaagtgcttaatctggaacattataaaggaggatt 2940 ggtagtcaacaaattaaccatcagatctaactaaaattttaagatatagtttgtttagct 3000 ctaaactttgatttaataaagaaaaaatctgtaaaatttaccttttaaatttgggtattt 3060 ttctataatcttatatagagctattttgctaaaggagatcaacacattttaaaaatggcc 3120 atgcattggccattgaaccctcttaatttatcaatatcaaaacacagtccctgaaacatt 3180 cttctattccattttccatgtgttcaccgtacactcacagtgcattcaccatttgtgctc 3240 tccgctctctgttcacaaagtgctcacaaatcgttcaacttgcactcactgttcgttcag 3300 gatgcacttattgttcagtcagataatattatatttagactgataacaatacatagttac 3360 atgtagcttatttttttattttttcctgattaccgacaaaacacaatataaggataactc 3420 ttaaggcactcggaataaatcgttctgtcatcttgcgttcacatagcgtttactgtttac 3480 tttgcgcttgtgtttgtgctccgtttgcgctctgcgttcgctcacagttcactgttcata 3540aacactcaccaaattctgttcagcgttagctcacccttcactcagagtgcactcattgtt 3600 cttcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctc3600 cgttcagtgt gcgctcaccg ctcacttact gttcaagtgg gaaagtagaa tgtttcagga 3660 actgtatgtg aacttctgta atatatatct actttaacaa actcatatat ttatatata 3720 tttatcaac cacacttgca tcataaagcg tgtttttatg atgtagaact cacagtta 3780 tatattgcgt caaatgcgtt ttctagagtt taagtgattg tgatgtaata ttgaaatttc 3840 aatgttcttc aatcgctcct tttaggtgca cgatatatat gtacgaagcg aactggat 3900 ccgtcgccct taatactacc aaggtttaat cttcaccaat gtttcctaag taataactga 3960 gatattgata aacaaaaagc cttaeettaat ctctcctgag acatttcatt ggaatcct 4020 tccagaaggg gtcaatctga gaggttatga ccttgccctg agcgtccttg acctcaatat 4080 ttgggtcact gacatgaagg gtgactactt cctgtctggg gtgggtgagg gagttgaaca 4140 gtagaacaga gctgtaaaat aaggcaactt atacaaccct atatttatgt tatgtaaatg 4200 gttatagtt aacagattcc agtaaaataa ggcaactat acaaccctat atttatgtta 4260 tgtaaatgtt ttatagtgaa cagactcctg taaaataagg caactataca gccttatatt 4320 tattatata tgtaaatgtg ttatagtgaa cagaatcctg taaaataagg caactataca 4380 ccctatattt atgttatgta aatgggtta 4440 tagtgaacag actcctgtaa aataaggcaa ttatacaacc ctatatttat gtatatgtaa 4500 atgggttaat agtgaacaga ctccagtaaa ataa4500 ggcaactatac aactctatat ttatgttatg taaatgggtt atagtgaaca gactccagt 4560 aaaataaggc aactatacaa ccctatattt atgttatgta aatgggttat agtgaacata 4620 ctcctgtaaa ataggcaact atacaactct atatttatgt tatgtaaatt tgttatagc 4680 agacagagac ctgttcaata aagcctctat aaaactttgt aatgtagatt tctaatcaaa 4740 cgcgaggaat atctgcgtaa aatcacaaga agaatatctc atggaatttt aaaattgggc 4800 ttttattttt ctgatagatg taactaaatt aaactattaat gataaaaatt tggtgttag 4860 cgattttaaa attctcgcga tttaattcaa aattgcaaaa ttatttgttg gattgtaaaa 4920 ttaagtactc aagttataaa atagaaatct acagtattta tattatgtta tttgagat 4980 agcaaaacag actactgttt aataaattat aaagcctagt ctataaaact ttgtactgta 5040 gatttcaact atttccctat tttttgtttc ttctttttac tcttggttag ttcctgagaa 5100 catcatgtga tcgtaaaact ttaattttaa gaagtaaatag cagattttgc tattaata 5160 cagaaggcat tttgatgata tatataaatt aaagtatctt taattttccc aatttgacaa 5220 aaaatgtcga taattttccc gatggaaagg gacccggtcc ctgtaccaaa aggtgctgtg 5280 acagccctgt ttacttacct tggtttatcg ctgacctgaa tcacattctt tgttatcaga 5340 gaatcgtggt tctttctaat ttcatcctta aaaataaaag ttgatctaat taagtataag 54005400 gaatcattcttaatatgtatcctcacctgattatttgatcaaatccaatacacctctaaa 5460 ggcttatgataaatttaatcacgcccaaccatatcacggccactaaacaagatgaacttc 5520 atgcaaaatgatataccaaatgcattttgagactgaatcactgcgaaaaatatttgtgaa 5580 gcttgcacaaattttgtatgtgaataaaagttggtatacagtaaaacattaagagcaggg 5640 taaagttttaaaaactcatactaaattagttagaagtaaaattaacagataaaacactat 5700 tttttcacagaatataaaaaaataaacatataaatgaggctgagtggtcaagaaatgaac 5760 catcagacctacctaaaattttaggatatgatgtgtttacctataaactattatcaacga 5820 aataaataatctaaatttttaatttgagtatcttcctatattttaatatagatctctttt 5880 cttaaggagatcaaacactgtgacgaccatcgagcttcttaacccagacttacaatgtca 5940 aaaagatgttttgtttataatcactcttggttgccatggtgaggaaagacagagcctcag 6000 taatgatgatattacagttgttctcagctttcagtagcctgtaacaaacagaacaagagg 6060 acggctgtcagtagcctgtaacaaacagaacaagaggtaagttgtctgtagcctgtaaca 6120 aaaagaacaaaaagaagactgtcagtagcctgtaacaaacagaacaagaggaaggctgtc 6180 ggtagcctgtaacaaacagaccaagagaaagatcgtcagtagcctgtaacaaacagaaaa 6240 agaggacggctgttagtagcctgtaacaaacaaaacaagaggaaggttgtcagtagcctg 6300 taacaaacagaccaagagaaagatcgtcagtagcctgtaacaaacagaaaaagaggacgg6300taacaaacagaacaagaggaaggttgtcagtagcctgtaacaaacagaacaaaaggaagg 6360ttgtcagtagcctgtgacaaaaagaacaagagaaaagttgtcaaagtgacagtaaatata 6420ataaacgatagaaaagctgtcaaagtgaaagcaatcctataaacgatagaacagttgtca 6480aagtgacagcaaacaaaagctgtcaatgtgacagcaaacaaaagctgtcaatgtgacagc6540aaaccaaatctgtcaatgtgacattaaaccaaaactgtcaatgtaggagcagaccaaatt 6600tgccacaaataaaagctgtcactgtgacagcaaaccaagtgcttcaatgtgacagcaaac 6660caaagctgtcaaaatcacagcaaattcaagctgtcagtgtgacagcaaaccaaagctgtc 6720tatgtgacagaaaatctaaaaaaacaatagattagttgccaatgggacagcaaacctaat 6780aaaccgtagaatagctgtcaaagtgacagcgaacccaataacaatagaaaagttgtcaat 6840gtgacagcaaaccttataaacaatacaaaagttgtcattgtgagagcaaaccttataaac 6900aatagaaatgtttcaatgtgacagcaaaccagagctgtcaaagtgtcagcaaactaaaaa 6960tataaaaaaagcaaaccaaagatgtcaatatgacagcaaaacaggttttcttttgggtaa 7020acagtttcaataatccctttgggatctctgaattgataaacactatctattcaaagaaat 7080gtagtaccctatatgcaatattctgcaagaaaatgaaagtttaacctttttctttattta 7140tgagagtatatttaccaataaggtaaatattagtattaaggattcgacagtttcaatata7200 ctgagcctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctgctgctg ctgctgctga gctg8100 taacgtaaat tcaatataca tgtagttgat tataatgata taataatgtt cttaaaggt 8160 ttaggtttag aagcagcatt ttctaacaaa gacagaagaa tgattagaat taagcatgat 8220 accacttgac aatcatccag ctcaatccat tgttaaaact tgaagtgttt ttaacaacac 8280 ttcaaattat gacaatatat gaaagtaatg aaagttgttc ataatctttat aaaagacg 8340 aacggagaag agaggaaaac gatgaaaagg gaggaaatcg tcaataagga aagaacaat 8400 tgaacatgtc aataagggtg ctggtttttt gacataaaaa cgaaacctct ttacagttt 8460 gaccttacag ttgttattac ttcttacagt tgtacgcgtt atacattgta tata 8520 tgtgtaatat gtatcttaga gagcacaata tctctaccaa tgacaattgt taatataaca 8580 tgtatcagtg ttaacaggag agagagacaa agctagacag atagagagac agagatagac 8640 agagatagac agagatagac agagacagag acagagagat agaaagagag aaaaaacaga 8700 gagagataag agagttaatt atttagaccat tgggcgcacg gcgggtgtga ccggtcagc 8760 agaggatgct tatactcctc gcagcaatgt taaggcgtcc cgatgctaaa atacggctgg 8820 aaaacgatat ttatttgaa tcatcgcaaa aatactttga ccgagagtat ttcttacaat ct 8880 atgttacatt tattgacaac gatgttaaac attaaatttg atgcattttt gtgacgtcat 8940 atgtactttg taatcacgtg acgggcgaat cctaatattg caaatgatct atttaaatcg 90009000 catcggcgca agtgattaat gacatcaaat cataaatatt tttaagaaaa atccttggtt 9060 atgtcatata cttcgaagtt cttgcttggg cttgaaatag atatttcgtt tatggaagat 9120 attgttgaaa cattctacaa aatcatcaaa ataatgcccc ttttactaat caaaagcga 9180 tttacaaaac attggggtga atacgtaggt ttcccaagca cgattcacat tggcacttat 9240 attctctacca attttacgta aaatattcta aaattgtgtt gatctttcac ctgcgccaag 9300 gctggttttt acatgcattt aaaattcttc attcagttgt ttaaacgtag cagggagagt 9360 ctccaaacca ctagtttata aatagtattt tacttaaaac aaagctttaa aactgccgat 9420 tatttgatac tggtttttaa tttgaatgaa ttctgtacgt ctagcattaa aggcagcatc 9480 tatgtgaagg aaacatgcgg tttatactgg ttgatataat tcacttttaa cgttaaga 9540 tacattccca aagaactatc gactggaatg cagatcgtga cgtctaagtt aattatgagt 9600 tcatattgtat gaagtacaga caagtgactt actacacatc tctgtaaaat caatcggga 9660 agccttaaca tgcccgcgct ccatggcacc tgatcctacc tctatcttgg aggtccgtgt 9720 tgctctgctt tgaattgtat ttcgttttat ggatttttga gatggttgac ggtttgtta 9780 ttgtcatttt ttcatcgtgc aatagatagg caaatgtaaa tggagataca gacttcaata 9840 gagaaagatg ctataccaga gggcttacgt cacatgtttg atatttcgtt tatatattta 9900 aatgaaagaa aatgctataa ccagagggct tacaatcaca tcttctgtaa aatcaatcgg9900 ttttaataac ctttaaaatg aatattgcta aatcgttgta tat taaagggtac gtttcc 9960 tataaaaagg aagttcaatt gaagctgacg attttaatag ttgtcacaga gaaaataact 10020 aagagtacaa gcaaaggact aattgccagt tgtttttgtt tgcagaaagt cggatcagtc 10080 tgatactcaa acataatact aatatattaat caagaatatg tgtaaaattt catactgtg 10140 taacccagtg ataaatctaa cggtttgtag caacataatt agtattaggt ttatttaat 10200 caaaatataa ttacaaattc a aactcttttt ttaatcaaaa tttcattgca aatcaaaat 10260 aaatgttaaa tcaaaataca atattacaaa ttcaaatcca atgtttaatc aacatataat 10320 tacaaatcca aacctatgtt taatcaaaat ataactacaa ac caaatctat gtttactc 10380 atattaatga aatacgtatt ctttaaattat ttcaatccgt gataaataca tt tatgttt 10440 tcaattcata atgtatagct aattgtcaag taaagaagta catcctgacc tggttttat 10500 tccatctaac attgcgatgc atcagattac ggccgatat gatgtatttt atgatgtgaac 10560 gtttaccttg taaaacccca ctctgttaag acaggtaccat gacacatgga cac 10620 ctgttacact tctcatttaa atatgtaaca aaaacagctc catgttgtcc ttttagctaa 10680 aactgtgttg gggaaaccca gactttcaaa acaataaacag tgacacaagt cacatttta 10740 gtaaaaactg ttaaagaaca aaaactttaa aaacattaag ttttaagata atggtgtat10800 ctttataggat attttaggtt gttttttctt atttattcat ttgaaacctt tattttgt 10860 tcagaacttg cctctaaatt agaacttgct tatgaaaagg tataaaaaga tgagtg 10920 tctaatgttt caaatttgtataaaagag atcacattgt gtgcacattc acgagacaca ga 10980 cagagacaca gacagcgaca cagacatatat agatggaggg agaatcacat tagttttttt 11040 aataccttag gttttgaaca acatccaccc gactacatct gctataaaaa caaccacctt 11100 caacaacaga catgtcctta tcttaacatt ttttttaaaa ttaaaaaata catttattta 11160 gatctgaaga attgcactgc cttgcctgaa ttaaaaaaaa cattgtattc tagaatctgc 11220 tctgaatcat tggtgtggag ccagtctggt gtataaataa tttaaaacac gcaggtgaca 11280 aatcaagcgt cattttgata agctcgttcg catttataat aatcatgaat cattggccgc 11340 atgcatacca acaatcgtac ctttctttca atcat 11375

[0029] SEQ ID NO. 2

[0030] 1 Let Ile Glu Arg Lys Asp Glu Ile Arg Lys Asn His Asp Ser Leu Ile Thr Lys Asn Val

[0031] 21 Ile Gin Val Ser Asp Lys Pro Ser Ser Val Leu Leu Phe Asn Ser Leu Thr His Pro Arg

[0032] 41 Gin Glu Val Val Thr Leu His Val Ser Asp Pro Asn Ile Glu Val Lys Asp Ala Gin Gly

[0033] 61 Lys Val lie Thr Ser Gin Thr Asp Pro Phe Trp Lys Asp Ser Asn Glu Met Ser Gin Glu

[0034] 81 Arg Phe Lys Leu Ser Phe Val Thr Glu Leu Pro Ala Leu Gly lie Val Arg Tyr Gin lie

[0035] 101 Ser Gin Arg Lys Asp Ser Ser Ser Asn Ser Leu Ala Thr Val Thr Tyr Tyr His Gly Asp

[0036] 121 Gly Pro Ser Ala Gin Ser Lys Phe Ser Val Glu Lys Lys Glu Ser Ser Glu Phe Thr lie

[0037] 141 Asp Asn Ser Phe Leu Glu Ala Lys Phe Ser Gly Ser Thr Gly Leu Leu Gin Phe Arg Ala

[0038] 161 Trp

[0039] SEQ ID NO. 3

[0040] atgattgaaagaaaggatgaaattagaaagaaccacgattctctgataacaaagaatgtg 60 attcaggtcagcgataaaccaagctctgttctactgttcaactccctcacccaccccaga 120 caggaagtagtcacccttcatgtcagtgacccaaatattgaggtcaaggacgctcagggc 180 aaggtcataacctctcagactgaccccttctggaaggattccaatgaaatgtctcaggag 240 agatttaagctgtcttttgtgaccgagctgcccgccctgggaattgtcagatatcagata 300 tcacagagaaaggacagctccagcaacagtctggccacagtcacatactaccatggagac 360 ggaccctcagctcagtctaagttttcagtagagaagaaggagtcttcagaattcacaata 420 gacaactctttcctggaggcgaaattctccggctccactggcctacttcagtttcgggct 480 tggtag 486

[0041] Example 2

[0042] According to the above-mentioned Example 1, the sequence of the CDS region of Man IIa gene is shown as SEQ ID NO. 3, the specific dsRNA sequence (ds-Man IIa) primer is designed, and the specific primer with the highest knockdown efficiency is finally determined in combination with the pre-experiment (Table 1). Then, the primer sequence is handed over to Suzhou Jimabio Gene Co., Ltd. for in vitro artificial chemical synthesis.

[0043] In addition, the dsRNA of green fluorescent protein (ds-GFP) is used as a universal meaningless control sequence, and natural seawater (SW) is set as a negative control. The ds-GFP control sequence is handed over to Suzhou Jimabio Gene Co., Ltd. for in vitro artificial chemical synthesis (Table 1).

[0044] Table 1 dsRNA sequence designed in the application

[0045]

[0046] Example 3

[0047] The wild adult Japanese oysters are selected for in vivo RNAi experiment:

[0048] The specific operation is as follows: the wild near Jiang oysters are brushed clean, anesthetized in pre-prepared MgCl2 sea water, and after the oysters are opened, the dsRNA or sea water synthesized in the above examples is injected into the adductor muscle of the near Jiang oyster by a microsyringe (100 μL), wherein the dose of the dsRNA and the sea water is 10 μg per oyster. After 48 hours of injection, the adductor muscle tension is measured and the adductor muscle is sampled to measure the expression level of ManIIa gene.

[0049] The adductor muscle tension is measured according to an improved bivalve adductor muscle tension measuring device (CN 211746263 U; 2020.10.27), the opening height is set to 10 mm to ensure that the oysters are not physically damaged, and the distance from the adductor muscle to the shell edge measuring point is measured when sampling to correct the adductor muscle tension.

[0050] According to the CDS sequence of the ManIIa gene shown in SEQ ID NO. 3, the primer is designed by using Primer Premier5 software (Table 2). The total RNA of the adductor muscle of the above-mentioned near Jiang oyster is extracted, and after reverse transcription, the expression levels of ManIIa gene and housekeeping gene Ef1a are detected by real-time quantitative PCR (qRT-PCR) (see Figure 1 and 2 ). The qRT-PCR reaction system is 20 μL, each reaction includes 3 μL of cDNA, 5.8 μL of DEPC water, 10 μL of SYBR Green mixture, 0.4 μL of upper and lower primers, and 0.4 μL of ROX dye II. The amplification process includes: 30 seconds of DNA polymerase activation time at 95℃; 40 cycles of 5 seconds at 95℃ and 30 seconds at 60℃ for melting, extension reaction; the melting curve program is: 15 seconds at 95℃, 1 minute at 60℃, 30 seconds at 95℃ and 15 seconds at 60℃.

[0051] Table 2 Primers for qRT-PCR detection

[0052]

[0053] Example 4

[0054] In order to verify whether the ManIIa gene affects the resistance of oysters, a heat tolerance experiment is carried out. That is, 50 wild near Jiang oysters are injected with ds-ManIIa (i.e., dsRNA obtained by specific primers), ds-GFP and natural sea water respectively after being anesthetized, and then placed in preheated sea water at 42℃ for acute heat shock for 1 hour, and then placed in normal sea water for 10 days, fed with spirulina powder every day and changed water once a day, and the number of deaths is recorded every day, and the death standard is determined as follows: the bivalve shell opened by external force cannot be closed.

[0055] Six individuals in each of the experimental and control groups were selected for qRT-PCR verification and determination of the adductor muscle tension according to the method described in Example 3 above (see Figure 3 ).

[0056] Compared with the control groups, the ManIIa gene expression of the individuals in the experimental groups was significantly knocked down, about 0.47-0.55( Figure 1 ) of the control groups, and there was no significant difference between the two control groups, indicating that the dsRNA of the application can successfully interfere with the ManIIa gene in Crassostrea hongkongensis; the adductor muscle tension of the oysters in the experimental groups was significantly smaller than that in the control groups( Figure 2 ), indicating that knocking down the ManIIa gene weakened the adductor muscle tension of the oysters.

[0057] Further detection of the effect of the gene on the survival rate of Crassostrea hongkongensis under acute high temperature stress found that the survival rate of the experimental group was significantly decreased( Figure 3 ), indicating that knocking down the ManIIa gene reduced the high temperature tolerance of the oysters.

Claims

1. A dsRNA that interferes with the expression of a muscle tension-related gene of Crassostrea hongkongensis, characterized in that: dsRNA is a 19bp double strand, which contains dTdT structure at the end; the sequence of the dsRNA is ds ManIIa-F: 5'-GCUCUGUUCUACUGUUCAATT-3'; ds ManIIa-R: 5'-UUGAACAGUAGAACAGAGCTT-3'.

2. The use of the dsRNA interfering with the gene encoding the adductor muscle tension-related protein of Crassostrea hongkongensis according to claim 1, characterized in that: The dsRNA inhibits the adductor muscle tension of Crassostrea rivularis and is used for preventing and removing Crassostrea rivularis.

3. A method of preventing corrosion of Crassostrea rivularis, characterized by: The dsRNA of claim 1 is injected into Crassostrea hongkongensis to regulate the expression of the gene in the adductor muscle of Crassostrea hongkongensis, so as to inhibit the tension of the adductor muscle of Crassostrea hongkongensis and prevent the corrosion of Crassostrea hongkongensis to the environment. ManIIa The dsRNA of claim 1 is injected into Crassostrea hongkongensis to regulate the expression of the gene in the adductor muscle of Crassostrea hongkongensis, so as to inhibit the tension of the adductor muscle of Crassostrea hongkongensis and prevent the 4. A kit for interfering with the closing muscle force of Crassostrea gigas, characterized in that it comprises: The kit contains the dsRNA of claim 1.

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