Carpovirus Hsc70_1 and Hsc70_2 genes and their applications

Abstract

The present application relates to the technical field of molecular biology, in particular to a Bactrocera cucurbitae Hsc70_1 and Hsc70_2 gene and application thereof.The technical problem to be solved by the present application is to provide a new option for green and sustainable control of pest sterilization technology.The technical scheme of the present application is a Bactrocera cucurbitae Hsc70_1 gene and / or Hsc70_2 gene, the nucleotide sequence of the Hsc70_1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the Hsc70_2 gene is shown as SEQ ID No.2.By reducing the expression of the two genes, the fertility of Bactrocera cucurbitae male insects can be reduced, and potential targets are provided for green and sustainable control of pest sterilization technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a Bactrocera cucurbitae Hsc70_1 and Hsc70_2 gene and application thereof. BACKGROUND

[0002] Bactrocera cucurbitae (Coquillett) is an agricultural quarantine pest that is extremely harmful to fruit and vegetable production and is widely distributed in tropical and subtropical regions. Bactrocera cucurbitae lays eggs in fruits, and after the eggs hatch, the larvae bore into the fruits, which can introduce a series of plant pathogens, causing the infected fruits to rot and deform. At present, chemical methods are mainly used to control Bactrocera cucurbitae in agricultural production, and the control effect is relatively significant, but the problem of drug resistance cannot be ignored. Moreover, the abuse of chemical pesticides can cause irreparable harm to the environment, and Bactrocera cucurbitae mainly lays eggs in the interior of host plants, so the effect of using chemical pesticides is very limited.

[0003] Sterile insect techniques (SIT) refers to the release of treated male insects that are infertile and have competitiveness with wild type males into the field, so that they can mate with wild type females, reduce the mating quantity of wild type males, and thus reduce the number of offspring. This method has been successfully applied in many insects in many places and has achieved good results. More and more researches are trying to elucidate the genes and molecular processes that control insect spermatogenesis in order to identify key genes that can be used as potential SIT targets. Further research to identify key genes essential for spermatogenesis is also necessary and provides an opportunity to further improve male sterile techniques. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a new choice for green and sustainable insect control by sterile techniques.

[0005] The technical solution of the present application is a coding protein of a Bactrocera cucurbitae Hsc70_1 gene and / or a coding protein of a Hsc70_2 gene, the amino acid sequence of the coding protein of the Hsc70_1 gene is shown in SEQ ID No. 24, and the amino acid sequence of the coding protein of the Hsc70_2 gene is shown in SEQ ID No. 25.

[0006] The present application also provides a Bactrocera cucurbitae Hsc70_1 gene and / or a Hsc70_2 gene, the nucleotide sequence of the Hsc70_1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the Hsc70_2 gene is shown in SEQ ID No. 2.

[0007] The application also provides application of the protein encoded by the Hsc70_1 or / and Hsc70_2 gene of B. cucurbitae in regulating insect spermatogenesis or the reproductive capacity of male insects.

[0008] The application also provides application of the Hsc70_1 or / and Hsc70_2 gene of B. cucurbitae in regulating insect spermatogenesis or the reproductive capacity of male insects.

[0009] Specifically, the regulation is negative regulation.

[0010] The application also provides application of the protein encoded by the Hsc70_1 or / and Hsc70_2 gene of B. cucurbitae in insect control.

[0011] The application also provides application of the Hsc70_1 or / and Hsc70_2 gene of B. cucurbitae in insect control.

[0012] Further, the application also provides a method for controlling insects. The main step of the method is to reduce the expression of the Hsc70_1 or / and Hsc70_2 gene in the insects.

[0013] Specifically, the expression of the Hsc70_1 or / and Hsc70_2 gene in the insects is reduced by using RNAi technology.

[0014] Further, in the RNAi technology, the amplification primers of the dsRNA for the Hsc70_1 gene are shown as SEQ ID No. 11 and SEQ ID No. 12; and the amplification primers of the dsRNA for the Hsc70_2 gene are shown as SEQ ID No. 13 and SEQ ID No. 14.

[0015] Further, the insects are fed with the dsRNA for the Hsc70_1 or / and Hsc70_2 gene.

[0016] The insects are Tephritidae insects.

[0017] Further, the Tephritidae insects are B. cucurbitae, B. oleae, B. oleivora or B. tryoni.

[0018] The insects are male insects.

[0019] The application also provides a substance for reducing the expression of the Hsc70_1 or / and Hsc70_2 gene, which is a dsRNA for interfering gene expression by using RNAi.

[0020] Further, the amplification primers of the dsRNA for the Hsc70_1 gene are shown as SEQ ID No. 11 and SEQ ID No. 12; the amplification primers of the dsRNA for the Hsc70_2 gene are shown as SEQ ID No. 13 and SEQ ID No. 14.

[0021] The application further provides an application of the substance for reducing the expression of the Hsc70_1 or / and Hsc70_2 gene in insect control.

[0022] The application further provides an application of the substance for reducing the expression of the Hsc70_1 or / and Hsc70_2 gene in regulating spermatogenesis of insects.

[0023] Specifically, the regulation is negative regulation.

[0024] The application has the following beneficial effects: the developmental stage and tissue expression profile of the Hsc70_1 and Hsc70_2 genes of the B. cucurbitae show that both the genes are specifically highly expressed in the testis of the male insect, especially in the conversion zone and the maturation zone of the testis tissue of the adult B. cucurbitae, the conversion zone is where the morphological differentiation of the spermatocyte occurs and the spermatocyte is converted into a sperm with a tail, and participates in the regulation of the morphological differentiation of the spermatocyte; the maturation zone is where the spermatogonia start meiosis and are converted into spermatocytes, and participate in the regulation of the meiosis of the spermatogonia. The Hsc70_1 and Hsc70_2 genes play an important function in maintaining the male reproduction process of the B. cucurbitae, when the Hsc70_1 and Hsc70_2 genes of the male B. cucurbitae are inhibited, the number of mature sperms of the male B. cucurbitae is reduced, and when the treated male insect is mated with a normal female insect, the hatching rate of the eggs laid by the female insect is significantly reduced, resulting in the reduction of the fecundity of the male B. cucurbitae. The two genes are expected to become potential targets for SIT and be applied to pest control, and provide a new idea for the green control of the B. cucurbitae. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 . Expression patterns of Hsc70_1 (A) and Hsc70_2 (B) in different tissues of B. cucurbitae; the columnar chart represents the average value ± standard error (SE) of the gene expression amount, and different letters on the column in the chart represent significant difference (P<0.05, one-way ANOVA, LSD).

[0026] Figure 2 . Localization of Hsc70_1 and Hsc70_2 in the testis of B. cucurbitae. (A) Morphology of the testis, which is divided into the spermatogonium zone, the growth zone, the maturation zone and the conversion zone; (B) negative control FISH signal in the testis of B. cucurbitae; (C) fluorescence signal of Hsc70_1 in the testis sample; (D) fluorescence signal of Hsc70_2 in the testis sample. The scale bar is marked in the lower right corner.

[0027] Figure 3. dsRNA delivery by feeding, 9 days later, Hsc70_1 (A) and Hsc70_2 (B) gene silencing efficiency detection. The column chart represents the average value ± standard error (SE) of gene expression, and the asterisk indicates significant difference (**P <0.01; ***P <0.001).

[0028] Figure 4 . Statistics of the number of testis sperm cells and mature sperm after RNAi, the column chart represents the average value ± standard error (SE) of gene expression, and the asterisk indicates significant difference (*P <0.05; ***P <0.001).

[0029] Figure 5 . Sperm fluorescence signal of each treatment. (A) Sperm fluorescence signal of B. cucurbitae treated by dsGFP negative control. (B) Sperm fluorescence signal of B. cucurbitae treated by dsHsc70_1. (C) Sperm fluorescence signal of B. cucurbitae treated by dsHsc70_2. The scale bar is in the lower right corner.

[0030] Figure 6 . Effect of interfering Hsc70_1 and Hsc70_2 on the oviposition of B. cucurbitae, the column chart represents the average value ± standard error (SE) of gene expression.

[0031] Figure 7 . Effect of interfering Hsc70_1 and Hsc70_2 on the egg hatching rate of B. cucurbitae, the column chart represents the average value ± standard error (SE) of gene expression, and the asterisk indicates significant difference (***P <0.001). DETAILED DESCRIPTION

[0032] There are very few target sequences for genetic manipulation based on target genes to achieve the technology of controlling and preventing Dacus cucurbitae male sterility. The inventors have accumulated a lot of research over the years, and have cloned two genes Hsc70_1 and Hsc70_2 which are highly expressed in the testis of adult Dacus cucurbitae from the genome of Dacus cucurbitae. In order to further clarify whether Hsc70_1 and Hsc70_2 genes can be used for prevention and control based on male sterility technology, the inventors have also carried out a series of follow-up experiments.

[0033] The inventors analyzed the expression patterns of the two genes and found that Hsc70_1 and Hsc70_2 genes were highly expressed in the testis of adult Dacus cucurbitae, which indicated that the two genes might be involved in the regulation of the reproductive capacity of male insects. Further, in situ hybridization experiments confirmed the localization of the two genes in testis tissue. This indicates that the two genes have potential application value.

[0034] Furthermore, to determine the function of the genes, the inventors used RNAi technology to prepare dsRNA suitable for the genes. Feeding male insects with dsRNA effectively reduced the expression of the Hsc70_1 and Hsc70_2 genes, and significantly reduced the number of mature sperm cells in adults. After mating with normally oviparous females, the hatching rate of fertilized eggs was also significantly reduced. This demonstrates that the Hsc70_1 and Hsc70_2 genes can serve as targets for sterilization in insect control.

[0035] Example 1: Obtaining the open reading frame of the Hsc70 gene in the melon fly

[0036] Full-length primers for the *F. melongrassus* Hsc70_1 / 2 were designed using the online primer design website Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-BLAST). Using adult male *F. melongrassus* cDNA as a template, the full-length Hsc70_1 / 2 was cloned using the aforementioned specific primers. PCR amplification conditions were as follows: pre-denaturation: 98℃ for 3 min, followed by 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min, repeated 35 times, with a final extension at 72℃ for 10 min. A 25 μL reaction mixture contained 9.5 μL of nuclease-free water, 12.5 μL of 2×PrimeSTAR Max Premix (TaKaRa, Japan), 1 μL each of forward and reverse primers (10 μM), and 1 μL of adult *F. melongrassus* testis cDNA as a template.

[0037] After the PCR reaction was completed, the PCR products were detected by 1% agarose gel electrophoresis to check the length of the electrophoretic bands. Once confirmed to be correct, the target band was cut from the gel and recovered using a PCR product purification kit. The concentration of the recovered product was determined using a NanoDrop One nucleic acid and protein analyzer. The product was then compared with... The T-Easy vector (Promega) was ligated and transformed using heat-cold shock, then the recombinant vector was transferred into *E. coli* DH5α. The culture was expanded using LB liquid medium with ampicillin resistance at 37°C with shaking for 2 hours. Subsequently, the bacterial culture, IPTG, and X-gal were plated onto LB solid medium with ampicillin resistance and incubated overnight at 37°C. Finally, white spots were picked and transferred to LB liquid medium with ampicillin resistance and shaken at 37°C for 1 hour. PCR was performed to detect the bacterial culture, and positive monoclonal cultures were sent to BGI Genomics Co., Ltd. for sequencing.

[0038] Table 1. Primer pairs used for cloning, qRT-PCR, and dsRNA synthesis

[0039] C. Hsc70_1 open reading frame sequence, SEQ ID No. 1

[0040] ATGGCTAAAGCGCCCGCTGTCGGCATTGATCTTGGCACGACATACTCATGCGTCGGTGTGTTTCAACA

[0041] TGGCAAAGTGGATATTATCGCCAATGACCAGGGCAATCGGGTGACTCCATCCTATGTGGCCTTCACCG

[0042] ATACGGAGCGGCTAATTGGCGATGCGGCTAAAAATCAAGTTGCCATGAATCCGAACAATACTATCTTTG

[0043] ATGCAAAACGTTTGATCGGTCGTAAGTTCGATGATATCACTGTGCAGAGCGACATGAAACATTGGCCC

[0044] TTCGAGGTCTTCAACGATAATGGCAAGCCGAAAATGCGTATCGAATATAAGGGCGAAAAGAAAAGTT

[0045] TCTTTCCGGAAGAAATCTCTTCTATGGTTTTGACAAAGATGAAGGAGACGGCCGAAGCTTATCTCGGC

[0046] AAGTCTGTAACAGATGCTGTGGTCACTGTACCAGCTTACTTTAATGACTCACAACGTCAAGCCACCAA

[0047] AGATGCTGGCGCCATTGCCGGTTTAAATGTGCTGCGTATCATTAACGAACCGACGGCGGCGGCCATCG

[0048] CTTACGGTCTGGACAAGAAGGGCACCAGTGAACGTAATGTGCTCATTTTTGATCTTGGTGGCGGCACT

[0049] TTTGATGTTTCCATACTCACCATTGAAGATGGCATCTTTGAGGTAAAGTCCACAGCAGGCGATACGCAT

[0050] TTAGGCGGTGAAGATTTCGACAATCGCATGGTGAATCATTTTGTACAAGAGTTCCAACGCAAGTACAA

[0051] GAAGGACTTGGCGCAAAATAAACGCGCTTTAAGGCGACTGCGCACGGCTTGTGAACGCGCCAAGCGT

[0052] ACGCTATCCGCCTCTTCGCAGGCTAGCATTGAGATCGATTCGCTATACGAAGGTATAGACTTTTATACCT

[0053] CCATTACACGTGCACGCTTCGAGGAATTGAATGGTGACCTCTTCAGGGGTACTATGGAGCCGGTGGCG

[0054] AAGGCTTTACGTGACGCTAAAATGGATAAAGGACAGATACACGATATAGTTTTGGTTGGCGGTTCAAC

[0055] GCGTATACCGAAAGTACAAAAACTACTGCAGGACTTCTTCAATGGTAAAGAGCTCAACAAGTCTATTA

[0056] ATCCGGATGAGGCCGTCGCTTATGGTGCCGCTGTACAAGCCGCCATACTCTGTGGCGATAAGTCCGAA

[0057] GCGGTGCAAGATCTTTTACTGCTCGATGTAACACCACTATCGTTGGGTATCGAAACCGCCGGCGGTGT

[0058] TATGACGGTGCTGATCAAACGTAATACCACAATACCAACGAAGCAAACCCAAGTCTTCACCACCTACT

[0059] CAGACAATCAGCCGGGCGTGTTGATACAGGTATTCGAGGGTGAGCGTGCCATGACCAAAGACAACAA

[0060] TATACTCGGCAAATTTGAGCTGAGCGGCATACCGCCGGCGCCACGTGGTGTGCCACAAATCGAAGTC

[0061] ACCTTCGACATTGACGCGAATGGTATTTTAAATGTCACCGCGGTGGAAAAGTCGACAGGCAAAGAGA

[0062] ATAGAATCACTATAACCAACGACAAGGGTCGGCTGAGCAAGGACGATATCGAGCGCATGGTAAACGA

[0063] GGCAGAGCAATATCGCAACGAGGATGAGAAGCAGCGCGAACGCATTAATGCCAAGAATGCGCTTGAG

[0064] TCTTACTGCTTCCAAATGAAGTCCACCATGGATGATGAGAATATACGCGCTAAAATTTCGGACTCCGAT

[0065] CGTCAGCTGATATTGCAGAAATGTGACGAGACGATCAGCTGGTTGGATAGTAATCAACAGGCGGAGA

[0066] AGGATGAATTCGAGTATAGACAGAAAGAGTTGGAGAAAATTTGCAGTCCGATTATAACACGCTTGTAT

[0067] CAGGGTGGCGTGCCGCCACCACCGCCAAATACAGGTGGTCCGGGTGCTGGTGGCTCCGGCGGCGCA

[0068] GCTGGTGGTCCCACCATAGAAGAGGTGGATTAA

[0069] Coding protein of the Bactrocera dorsalis Hsc70_1 gene SEQ ID No. 24

[0070] MAKAPAVGIDLGTTYSCVGVFQHGKVDIIANDQGNRVTPSYVAFTDTERLIGDAAKNQVA

[0071] MNPNNTIFDAKRLIGRKFDDITVQSDMKHWPFEVFNDNGKPKMRIEYKGEKKSFFPEEISS

[0072] MVLTKMKETAEAYLGKSVTDAVVTVPAYFNDSQRQATKDAGAIAGLNVLRIINEPTAAAIA

[0073] YGLDKKGTSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFVQE

[0074] FQRKYKKDLAQNKRALRRLRTACERAKRTLSASSQASIEIDSLYEGIDFYTSITRARFEELNG

[0075] DLFRGTMEPVAKALRDAKMDKGQIHDIVLVGGSTRIPKVQKLLQDFFNGKELNKSINPDEA

[0076] VAYGAAVQAAILCGDKSEAVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQVFTT

[0077] YSDNQPGVLIQVFEGERAMTKDNNILGKFELSGIPPAPRGVPQIEVTFDIDANGILNVTAVEK

[0078] STGKENRITITNDKGRLSKDDIERMVNEAEQYRNEDEKQRERINAKNALESYCFQMKSTM

[0079] DDENIRAKISDSDRQLILQKCDETISWLDSNQQAEKDEFEYRQKELEKICSPIITRLYQGGVPPPPPNTGGPGAGGSGGAAGGPTIEEVD.

[0080] Culex quinquefasciatus Hsc70_2 open reading frame sequence, SEQ ID No. 2

[0081] ATGGTTAAGGCACCAGCAATCGGTATTGATTTGGGCACCACCTACTCCTGTGTGGGCGTTTGGCAGAA

[0082] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0083] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0084] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0085] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0086] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0087] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0088] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0089] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0090] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0091] GATACGGAACGTTTGATCGGCGATGCGGCCAAGAATCAGGTCGCTATGAACGCCAAAAATACAGTATT

[0092] GCACAAACGCGATTTGTCCAGCAATGTACGCGCTTTGCGACGTCTGCGCACCGCCTGTGAGCGCGCC

[0093] AAGCGTACGCTGTCCAGCAGCACAGAGGCTTCTATTGAAATCGATGCCTTGCATGAGGGTATTGACTT

[0094] CTATTCGAAGATCAGTCGCGCACGTTTCGAAGAGCTGAATATGGACTTGTTCCGTTCGACTCTGCAAC

[0095] CAGTTGAGCGTGCGTTGAATGACGCCAAAATGGACAAGGCGGCCATACACGATGTCGTACTGGTGGG

[0096] CGGTTCTACACGCATTCCGAAAATACAAAAGATGTTGCAGGACTTCTTTGGCGGCAAACAGCTGAAC

[0097] TTGTCCATCAATCCCGATGAGGCTGTGGCTTATGGTGCCGCCGTGCAAGCTGCCATTCTCACCGGTGT

[0098] GGGTAGTTCACAAATTCAGGATGTGCTACTGGTTGATGTCACCCCACTTTCGTTGGGTATCGAAACTG

[0099] CCGGTGGTGTGATGACCAAACTAATTGAACGCAATGCACGCATTCCATGCAAACAACAACAAACCTT

[0100] CACAACATACAGTGACAACCAGAATGCTGTCACCATTCAAGTGTACGAAGGTGAACGCGCCATGACC

[0101] AAGGACAACAATCTTTTGGGTACTTTCAATTTGACTGGCATACCACCAGCACCACGTGGTGTACCCAA

[0102] AATCGAGGTCACCTTTGATCTGAATGCCGATGGCATATTGCATGTATCCGCTAAGGATAACAGCACCGG

[0103] TAAATCTGAGAAGATTACCATCACCAATGATAAGGGACGTCTATCCAAGGCCGAAATCGATCGTATGTT

[0104] ATCGGACGCCGAACGTTATAAGGATGAGGATGAGAAACAGAAGGAGCGCATACAGTCGAGGAATGCT

[0105] TTGGAGAGTTACATCTTCAGCTGCAAGCAGGCCATTGAAGATGCACCCGCTGGACGTCTGACAGACG

[0106] CCGACAAGACAACGGTACGTGATAAATGCACTTCGGAAATGTCTTGGCTGGATGCCAATACACTCGCC

[0107] GAAAAGGACGAGTTCGACGATCATCTCAAGGAGTGTCAACGCGTTTGTGGTCCAGTAATGGCGAAGA

[0108] TGCATGGAGGTGCTGGCAGTGGTGATGCAGGTGCGGCAAAAGGAGCACAAGGCGGACGCGCCGGCG

[0109] GACCGACTGTCGAGGAAGTCGATTAA

[0110] The encoded protein of the Bactrocera dorsalis Hsc70_2 gene is SEQ ID No. 25

[0111] MVKAPAIGIDLGTTYSCVGVWQNNKVEIIANDQGNRTTPSYVAFNDTERLIGDAAKNQVAMNAKNTVFD

[0112] AKRLIGRRFDDTKIQEDMKHWPFKVVNDCGKPKIEIEFKGEVKRFAPEEVSSMVLTKMREIAEVYLGGKV

[0113] TDAVITVPAYFNDSQRQATKDAGSIAGLNVLRIINEPTAAALAYGLDKNLKGEKNVLIFDLGGGTFDVSILS

[0114] IDEGSLFEVKSTAGDTHLGGEDFDNRLVNHFVEEFKRKHKRDLSSNVRALRRLRTACERAKRTLSSSTEAS

[0115] IEIDALHEGIDFYSKISRARFEELNMDLFRSTLQPVERALNDAKMDKAAIHDVVLVGGSTRIPKIQKMLQD

[0116] FFGGKQLNLSINPDEAVAYGAAVQAAILTGVGSSQIQDVLLVDVTPLSLGIETAGGVMTKLIERNARIPCKQ

[0117] QQTFTTYSDNQNAVTIQVYEGERAMTKDNNLLGTFNLTGIPPAPRGVPKIEVTFDLNADGILHVSAKDNS

[0118] TGKSEKITITNDKGRLSKAEIDRMLSDAERYKDEDEKQKERIQSRNALESYIFSCKQAIEDAPAGRLTDAD

[0119] KTTVRDKCTSEMSWLDANTLAEKDEFDDHLKECQRVCGPVMAKMHGGAGSGDAGAAKGAQGGRAGGPTVEEVD.

[0120] Example 2 Tissue expression profile of H. cucurbitae Hsc70

[0121] The relative expression levels of Hsc70_1 and Hsc70_2 genes in the tissues of adult male and female *F. melonfieldii* were detected using qRT-PCR, including the midgut, Malpighian tubules, fat body, and testis tissues. The qRT-PCR specific primer sequences are shown in Table 1. αTub and βTub1 were used as internal reference genes to evaluate the expression of Hsc70_1 and Hsc70_2 in different tissues. PCR amplification conditions were as follows: pre-denaturation: 95℃ for 2 min, followed by denaturation at 95℃ for 15 s, annealing and extension at 60℃ for 30 s, repeated 40 times. Finally, the primer amplification specificity was analyzed using a melting curve analysis at 60-95℃. The reaction system contained 5 μL of qPCR reagent (Shanghai Nearshore Protein), 3.5 μL of nuclease-free water, 1 μL (10 μM) of each downstream primer, and 0.5 μL of *F. melonfieldii* testis cDNA as template. Figure 1 It is the melon fly Hsc70_1 ( Figure 1 A) and Hsc70_2 Figure 1 B) Relative expression levels in different tissues (midgut, Malpighian tubules, fat body, and testis) of male and female worms. Error bars represent the standard error of the mean of three biological replicates; different letters on the columns indicate significant differences in expression levels (P < 0.05, one-way ANOVA, LSD). From Figure 1 It can be seen that the Hsc70_1 and Hsc70_2 genes of the melon fly are highly expressed in the testes of the adult fly.

[0122] Example 3: Localization of testicular tissue in the fruit fly Hsc70

[0123] Fluorescent probes were synthesized using the nucleic acid sequences shown in Table 2 for in situ hybridization analysis. Two-day-old male melon fly adults were dissected in 1×PBS solution to remove intact testicular tissue. The attached fat bodies were removed, and the testicular tissue was fixed overnight at 4°C in 4% paraformaldehyde solution. After fixation, the 4% paraformaldehyde solution was aspirated with a pipette, and the tissue was washed three times with 0.3% PBST solution (containing 1×PBS and 0.3% Triton X-100), 20 min each time. After washing, the 0.3% PBST solution was aspirated, and 1× proteinase K solution was added, followed by permeabilization at 37°C for 20 min. The 1× proteinase K solution was aspirated, and the tissue was washed three more times with 0.3% PBST solution, 5 min each time. After washing, prehybridization solution was added, and the tissue was incubated in a hybridization oven at 68°C for 60 min. The prehybridization solution was aspirated, and a probe diluted 500 times with the prehybridization solution was added, followed by incubation in a hybridization oven for 48 h. The control group received only the same volume of prehybridization solution. After incubation, the probe was aspirated and washed with 0.3% PBST solution. Wash three times with PBST solution, 5 min each time; after washing, aspirate the 0.3% PBST solution, dilute the DAPI stock solution 2000 times with 0.3% PBST solution, and add it to the sample for nucleation; after nucleation, aspirate the DAPI, wash three times with 0.3% PBST solution, 5 min each time, and finally add a fluorescence antiquenching agent to prepare the slide and observe it under a laser confocal microscope.

[0124] Figure 2 Display Hsc70_1( Figure 2 C) and Hsc70_2 Figure 2 D) Localization in the testis tissue of *F. melongrassus* showed that Hsc70_1 signaling appeared in the transformation region, while Hsc70_2 signaling appeared in the maturation region. It is speculated that the Hsc70_1 gene may exert its physiological function through participation in spermatocyte morphological transformation, while the Hsc702 gene may exert its physiological function through participation in spermatocyte meiosis.

[0125] Table 2. Hsc70_1 and Hsc70_2 in situ hybridization probe sequence information

[0126] Gene Sequence No. Probe sequence Hsc70_1 SEQ ID No. 21 ctataccttcgtatagcgaatcgatctcaatgctagcctgcgaag Hsc70_2 SEQ ID No. 22 cttgaatggtgacagcattctggttgtcactgtatgttgtgaagg

[0127] Example 4: Preparation of dsRNA from ZcHsc70_1 and ZcHsc70_2 genes

[0128] Based on the open reading frame sequences of Hsc70_1 and Hsc70_2, dsRNA primers were designed using the NCBI Primer BLAST online website (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast), and a T7 promoter sequence (SEQ ID No. 23: TAATACGACTCACTATAGGG) was added to the 5' end of the primer sequence. The sequence information is shown in Table 1.

[0129] Using the 5-day-old testis tissue cDNA of the Mediterranean fruit fly as a template, the partial sequences of ZcHsc70_1 and ZcHsc70_2 genes were amplified by ordinary PCR using dsRNA-specific primers. The positive clones were sent to Huada Gene for sequencing. After sequencing, PCR amplification was performed using the respective bacterial liquid as a template and the dsRNA primers, and the reaction system and conditions were the same as those in the molecular cloning. The PCR product was recovered and purified, and a higher concentration of the PCR product was used as a template to synthesize and purify dsRNA according to the instructions of the Transcript Aid T7 High Yield Transcription Kit (Thermo, USA). The purity and integrity of the dsRNA were detected by 1.2% agarose gel electrophoresis, and its concentration was measured at 260 nm using a UV spectrophotometer (Thermo, USA). Then it was stored at -80°C for standby use.

[0130] Example 5 Inhibition of the male reproductive capacity of the Mediterranean fruit fly by feeding dsHsc70_1 and dsHsc70_2 synthesized from the Hsc70_1 and Hsc70_2 gene fragments, respectively

[0131] (1) Feeding of dsHsc70_1 and dsHsc70_2 synthesized from the Hsc70s gene fragments

[0132] Adult Mediterranean fruit flies that emerged on the same day were collected and normally reared to the second day, and then male and female adult insects were separated and reared separately. Ten male insects were reared in one small insect cage, 100 μL of adult liquid feed containing brilliant blue dye and 20 μg of dsRNA were added in the morning every day, and the adult feed was supplemented normally in the afternoon, and the adult feed was removed at night. Female insects were normally reared with adult feed. Continuous rearing was performed to the 9th day.

[0133] (2) Detection of the Hsc70 gene silencing efficiency

[0134] Five male adults of C. pipiens f. quinquefasciata were collected after 9 days of continuous feeding, and total RNA was extracted using TRIzol (Invitrogen, USA) reagent. The template cDNA was obtained by reverse transcription using RQ1 RNase-Free DNase reagent (Promeg, USA) and PrimeScriptTM RT Reagent Kit (TaKaRa, Japan). The relative expression of Hsc70_1 and Hsc70_2 genes was detected by qRT-PCR using the above-mentioned primers and methods to calculate the silencing efficiency of the target genes. Figure 3 The results showed that after 9 days of continuous feeding of dsRNA, the expression of Hsc70_1 and Hsc70_2 was significantly down-regulated by 81.30% and 57.12% compared with the control, respectively, indicating that the feeding method can effectively silence the expression of target genes.

[0135] (3) Changes in male reproductive ability were observed by changes in the number of sperm cells, mature sperm, and the number of eggs laid and egg hatching rate

[0136] The male adults fed with dsRNA for 8 days were collected, and the testes were dissected in HEPES buffer containing 10% bovine serum albumin. The testicular tissue was transferred to 50 μL of 0.25% trypsin solution, and the testicular epidermis was destroyed with forceps to release the testicular fluid. Each group had 3 pairs of testicular tissue. After 10 min of shaking, 5 μL of sperm suspension was stained with 1 μL of fluorescent dye SYBR-14 (50x) for 10 min, and the slides were prepared and counted under a laser confocal microscope. The treated males were mated with normal female adults of the same age, and the control was set as males fed with dsGFP mated with normal female adults. The females were fed separately after mating for 2 days, and fresh tender pumpkin was used to induce oviposition. The number of eggs laid was counted, and 100 eggs were randomly selected and placed in a water film to count the hatching rate within 48 h. The number of eggs laid and the hatching rate were counted continuously for 7 days, with 3 replicates.

[0137] Figure 4 and Figure 5 The number of sperm cells and mature sperm of C. pipiens f. quinquefasciata adults treated with dsHsc70_1, dsHsc70_2, and dsGFP was counted. The results showed that the number of sperm cells in males treated with dsHsc70_1 and dsHsc70_2 was significantly increased by 207.65% and decreased by 54.73% compared with the control group, respectively. The number of mature sperm cells was significantly reduced by 91.13% and 61.22% compared with the control group, respectively. Figure 6 The number of eggs laid by males treated with dsRNA for 9 days and normal female adults was counted for 7 consecutive days. The results showed that there was no significant difference in the number of eggs laid between the target gene dsRNA treatment group and the control group. Figure 7The male insects were fed with dsRNA for 9 days, and then mated with normal female insects. The egg hatching rate within 7 days was determined. The results showed that the egg hatching rate of the target gene dsRNA feeding group was significantly lower than that of the control group.

Claims

1. Use of the protein encoded by the Hsc70_1 gene and / or the protein encoded by the Hsc70_2 gene of the Mediterranean fruit fly in insect control; characterized in that: The amino acid sequence of the protein encoded by the Hsc70_1 gene is shown as SEQ ID No. 24, and the amino acid sequence of the protein encoded by the Hsc70_2 gene is shown as SEQ ID No. 25; the nucleotide sequence of the Hsc70_1 gene is shown as SEQ ID No. 1, and the nucleotide sequence of the Hsc70_2 gene is shown as SEQ ID No. 2; and the insect is a male fruit fly.

2. A method of controlling insects, characterized by: The expression of the Hsc70_1 or / and Hsc70_2 gene in an insect is reduced; the nucleotide sequence of the Hsc70_1 gene is shown as SEQ ID No. 1, and the nucleotide sequence of the Hsc70_2 gene is shown as SEQ ID No. 2; and the insect is a male fruit fly.

3. The method of claim 2, wherein: The expression of the Hsc70_1 or / and Hsc70_2 gene in an insect is reduced by using an RNAi technology.

4. The method of claim 3, wherein: In the RNAi technology, the amplification primers for the dsRNA against the Hsc70_1 gene are shown as SEQ ID No. 11 and SEQ ID No. 12; and the amplification primers for the dsRNA against the Hsc70_2 gene are shown as SEQ ID No. 13 and SEQ ID No.

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5. The method of claim 4, wherein: The insect is fed with the dsRNA against the Hsc70_1 or / and Hsc70_2 gene.

6. A substance which reduces the expression of the Hsc70_1 or / and Hsc70_2 gene, characterized in that: The nucleotide sequence of the Hsc70_1 gene is shown as SEQ ID No. 1, and the nucleotide sequence of the Hsc70_2 gene is shown as SEQ ID No. 2; the substance is a dsRNA for interfering gene expression by using an RNAi technology; further, the amplification primers for the dsRNA against the Hsc70_1 gene are shown as SEQ ID No. 11 and SEQ ID No. 12; and the amplification primers for the dsRNA against the Hsc70_2 gene are shown as SEQ ID No. 13 and SEQ ID No.

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7. Use of a substance as claimed in claim 6 for insect control, characterized in that: The insect is a male fruit fly.

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