CN115925856B - Brown planthopper heat shock transcription factor NlHsf, cDNA and application - Google Patents
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Abstract
The invention discloses a cDNA sequence, a coding protein sequence of brown planthopper transcription factor NlHsf and application thereof in pest control. The invention discovers that the transcription factor NlHsf1 is involved in reproductive regulation of brown planthoppers; the double-stranded dsRNA of the gene of the targeted interference brown planthopper NlHsf is also designed, and the double-stranded dsRNA can influence the ovary development of the brown planthopper, greatly reduce the spawning quantity, enable the eyespot on the egg to be inverted, greatly reduce the hatching rate, and reduce the killing of other organisms while preventing and controlling the brown planthopper. The transcription factor NlHsf gene found by the invention participates in the development of the female egg nest of brown planthoppers and the incubation of eggs, and is applied to the research on the reproduction of agricultural and forestry pests and the pest control, and the dsRNA is also utilized to interfere the NlHsf gene on the nucleotide level, so that the damage to natural enemies and other non-target organisms caused by the conservation of the amino acid sequence is avoided, the ecological control function is fully exerted while the control of brown planthoppers is hopeful to be realized, and the environmental pollution is prevented.
Description
Technical Field
The invention belongs to the technical field of animal genetic engineering, and relates to a pest transcription factor and application thereof, in particular to a brown planthopper transcription factor and application thereof in pest control.
Background
Brown planthopper (NILAPARVATA LUGENS)) Belongs to the plant hopper family (DELPHACIDAE) of the order Hemiptera (Hemiptera), and is the first pest on Asian rice. The brown planthoppers are single in selection of host plants, and only feed and reproduce offspring on rice and common wild rice. Brown planthoppers influence the normal growth and development of rice mainly through piercing and sucking host rice phloem juice, saliva sheath formed during feeding can block vascular bundles, and a large amount of honeydew is secreted after feeding, and the honeydew contains a large amount of amino acids and sugar substances, so that the rice can be infected with bacteria. When a large number of brown planthoppers burst, paralysis and lodging of the rice can be caused, the phenomenon of 'lice firing' of the rice is caused, and serious yield reduction and even harvest of the rice are finally caused. At present, brown planthoppers are largely protected from attack by using pesticides. However, with the massive unscientific use of pesticides, the drug resistance of brown planthoppers is increased, the reproductive capacity is stimulated, and the brown planthoppers burst out, so that the rice yield is seriously reduced.
The problems caused by brown planthoppers are closely related to their strong fertility. Thus, studies on reproductive mechanisms of brown planthoppers have been a hotspot. The fecundity of brown planthoppers is closely related to environmental factors, particularly the temperature, and has great influence on the development of the ovary and the incubation of eggs of brown planthoppers. In addition, many studies have shown that the rise in temperature caused by global climate change promotes reproduction of brown planthoppers. However, the molecular mechanism of how temperature affects insect reproduction is not clear and remains to be studied and addressed.
Disclosure of Invention
In order to solve the problems in the background technology, the invention aims to provide the brown planthopper heat shock transcription factor NlHsf and the application of the cDNA sequence thereof in pest control.
Based on the important role of NlHsf in brown planthopper reproduction regulation, the invention discloses a cDNA sequence, an amino acid sequence and application thereof in pest control.
The invention adopts the following technical scheme:
1. Brown planthopper heat shock transcription factor NlHsf1:
The amino acid sequence is shown in SEQ.NO. 2.
Substitution and/or deletion and/or insertion of amino acids on the basis of the amino acid sequence gives at least 90% homology with the SEQ.NO. 2.
2. CDNA of brown planthopper heat shock transcription factor NlHsf:
The gene sequence is shown in SEQ.NO. 1.
The double-stranded dsRNA synthesized by the cDNA is combined with mRNA of a transcription factor NlHsf to degrade the mRNA, so that the reproductive capacity of brown planthoppers is inhibited.
3. Brown planthopper heat shock transcription factor NlHsf1 or application of the cDNA in brown planthopper pest control.
In particular to application in dsRNA novel pesticides which take transcription factor NlHsf gene as target in brown planthopper control.
4. The application method of brown planthopper heat shock transcription factor NlHsf or cDNA in brown planthopper pest control adopts RNA interference mode to reduce fertility of brown planthopper.
5. A pesticide for preventing and controlling brown planthoppers, which takes the brown planthopper heat shock transcription factor NlHsf protein as a target.
The invention discovers that a transcription factor NlHsf which participates in reproductive regulation of brown planthoppers is a heat shock transcription factor, and the transcription factor activated during heat stress can regulate synthesis of heat shock proteins, so that tolerance of brown planthoppers organisms is improved.
According to the characteristic that the sequence coded by the transcription factor NlHsf is relatively conserved in amino acid sequence but the nucleotide sequence has lower homology with other organisms, the RNA interference is carried out on the target gene NlHsf1, the inhibition of the transcription accumulation of the NlHsf1 gene is realized on the mRNA level, and the fecundity of brown planthoppers is obviously reduced.
The invention also designs double-stranded dsRNA of the gene of the targeted interference brown planthopper NlHsf, and discovers that the double-stranded dsRNA can influence the ovary development of the brown planthopper, greatly reduce the spawning quantity, invert the eyespot on the egg and greatly reduce the hatching rate. This also suggests that NlHsf, a central regulator in this heat stress response, plays an important role in the reproduction of brown planthoppers.
The invention uses RNA interference fragments to specifically design gene sequences of different species, uses the gene as a target gene for biological control of brown planthoppers, and reduces killing of other organisms while controlling brown planthoppers.
The invention has the beneficial effects that:
The transcription factor NlHsf gene of the present invention is a part of brown planthopper gene, and its nucleotide sequence is different from that of other species. The gene NlHsf of the transcription factor is involved in the development of the egg nest of the female brown planthopper and the incubation of eggs.
The research of heat shock transcription factors in the aspect of reproduction of agricultural and forestry pests and the application of the heat shock transcription factors in pest control have not been reported. The method specifically comprises the following steps: reduced fecundity of brown planthoppers, reduced direct harm of pest feeding to rice crops, and the like.
Based on the characteristic that the nucleotide sequence of the target gene has low homology with other organisms, the dsRNA is utilized to interfere NlHsf a1 on the nucleotide level, so that the phenomenon that natural enemies and other non-target organisms are killed due to the conservation of the amino acid sequence is avoided, and the ecological management function is expected to be fully exerted while the brown planthopper prevention and control is realized.
The method and the application of the invention can effectively inhibit the propagation of brown planthoppers, prevent the environmental pollution, effectively control the brown planthoppers, and can be used for screening and finding new brown planthopper control targets, thereby having important practical significance.
Drawings
FIG. 1 shows the relative transcriptional accumulation of the brown planthopper NlHsf gene at different developmental stages. Wherein 1d, 3d, 5d, 7d represent days after emergence of the adults.
FIG. 2 shows the relative transcriptional accumulation of the brown planthopper NlHsf gene at different tissue sites.
FIG. 3 is the effect of RNA interference on the transcriptional accumulation of the brown planthopper NlHsf gene.
FIG. 4 is the effect of RNA interference on brown planthopper ovary morphology. Wherein the scale bar is 1mm.
FIG. 5 is the effect of RNA interference on the spawning amount of brown planthoppers.
FIG. 6 is the effect of RNA interference on the morphology of brown planthopper eggs. Wherein the arrow indicates the eye point, and the scale bar is 200 μm.
FIG. 7 is the effect of RNA interference on the hatchability of brown planthoppers.
Detailed Description
The invention is further described below with reference to the drawings and detailed description.
Embodiments of the invention are as follows:
1. Materials and methods
1.1, Brown planthopper to be tested
The test brown planthopper population was initially harvested in the Huajia pond school paddy field (N30 deg. 16'35.21 ", E120 deg. 11' 37.16") at Zhejiang university, hangzhou, china. Brown planthoppers are raised in a climatic chamber at 26+/-0.5 ℃ and 70% relative humidity, and the illumination is set to 16 hours of illumination and 8 hours of darkness. The rice variety for raising brown planthoppers is TN1.
1.2 Cloning of full-Length cDNA of brown planthopper NlHsf Gene
Trizol method was used to extract total RNA from brown planthopper. Sample selection: 5 male and female brown planthoppers respectively take 30-50 nymphs and about 500 eggs. RNA quality was measured using Nanodrop 2000 (Thermo) and samples were stored in a-80℃refrigerator. cDNA was synthesized by reverse transcription using 1. Mu.g of total RNA as a template and using reverse transcription kit (HiScript II 1st Strand cDNA Synthesis Kit) from Nanjinouzan corporation, and stored in a-20℃refrigerator.
According to the transcriptome sequencing sequence information of the laboratory, the partial sequence of the brown planthopper NlHsf gene is obtained and is identified by NCBI website sequence comparison. Primers NlHsf-F and NlHsf1-R were designed using PRIMER PREMIER 5.0.0 software and the identified sequences were verified by comparison.
Subsequently, polymerase Chain Reaction (PCR) was performed using a Kit (KOD One PCR MASTER Mix Kit), and PCR conditions were set as follows: 98 ℃ for 5 minutes; 98 ℃,10 seconds, 50 ℃,68 ℃,10 seconds, 35 cycles; 68℃for 5 minutes. The PCR amplified products were detected by 1% agarose gel electrophoresis, gel recovered using a gel recovery kit (Favorgen), followed by ligation to pMD18-T vector (Takara) and transformation into DH 5. Alpha. Competent cells. 1mL of LB liquid medium was shake-cultured at 37℃for 2 hours, 50. Mu.L of the bacterial liquid was spread on LB solid medium containing 1% of ampicillin, and cultured overnight at 37 ℃. The next day, about 10 single colonies were randomly picked up and shake-cultured in 1.5mL centrifuge tubes at 37℃for 6 hours in 1mL LB liquid medium containing 1% ampicillin, and 1. Mu.L of the bacterial liquid was used for bacterial liquid PCR identification of positive clone strains and sent to Hangzhou Shangya Biotechnology Co., ltd for sequencing. Sequencing results were verified by Geneious software and primordial sequence alignment.
According to RACE kit @RACE 5'/3'Kit User Manual) specification, outer primers NlHsf1-3F1 and NlHsf1-5R1 (Table 1) and inner primers NlHsf1-3F2 and NlHsf1-5R2 (Table 1) were designed, and nested PCR was performed to amplify, electrophorese, gel recover, ligate, transform, and sequence the 5 'and 3' ends of NlHsf 1. The sequencing results are aligned and spliced to obtain the full-length cDNA sequence NlHsf. The full-length verification primers NlHsf-FL-F and NlHsf-FL-R of NlHsf1 were then designed to verify the spliced full-length sequence based on the full-length cDNA sequence obtained.
1.4, Analysis of the spatial-temporal expression Pattern of the Brown planthopper NlHsf Gene
Collecting brown planthopper eggs (3 rd day after delivery), nymphs (1, 2,3, 4 and 5 years), female adults and male adults with different wings 1, 3,5 and 7 days after emergence, and respectively extracting RNA of brown planthoppers at different development stages; and respectively extracting RNA of each tissue from 50 heads of the male and female brown planthoppers after the anatomical eclosion according to the sequence of the heads, the feet, the wings, the midgut, the Markov tube, the epidermis, the fat body, the ovary and the testis. Subsequently, cDNA was synthesized by reverse transcription using a reverse transcription kit (REVERTRA ACE QPCR RT MASTER Mix with gDNA Remover kit).
The cDNA product is diluted by 6 times and then the kit is usedPremix Ex Taq TM kit) was subjected to a real-time fluorescent quantitative PCR (qRT-PCR) reaction. Specific primers for NlHsf1 were designed using PRIMER PREMIER 5.0.0 software as NlHsf-Q-F and NlHsf-Q-R (Table 1).
Brown planthoppers 18S were used as reference genes, and primers 18S rRNA-Q-F and 18S rRNA-Q-R were designed (table 1).
1.5 Synthesis of double-stranded RNA interference fragments
Primers dsNlHsf1-F and dsNlHsf-R for the synthesis of the dsRNA interference fragments were designed based on the full-length cDNA sequence and a T7 promoter sequence (TATATACGACTCACTATAGGG) was added before the 5' end of the specific primer. The dsRNA region does not overlap with the qPCR region, so that mRNA transcription accumulation detection after RNA interference is not affected.
1.6 Microinjection method for RNA interference
A capillary tube with a length of 75mm, an outer diameter of 1.0mm and an inner diameter of 0.6mm is selected for manufacturing the microinjection needle. The capillary was drawn into a glass needle suitable for microinjection using a P-97 capillary needle puller, with instrument parameters set at heat=750, pull=100, vel=150, time=40. The newly emerged female brown planthoppers were selected and injected using a TRANSFER MANNK model 2 microinjection apparatus, wherein the microinjection apparatus parameters were generally set to 1000hPa for the injection pressure, 0.5s for the pressure maintenance time, and 20hPa for the compensation pressure.
1.7 Observation of phenotype
Ovarian morphology observation: female adults of brown planthoppers 3 days and 6 days after dsGFP and dsNlHsf1 injections, respectively, were collected, placed on a glass slide, and 100. Mu.l of PBS buffer was added to dissect the ovaries of brown planthoppers. Subsequently, observations were made under a Zeiss stereo microscope (Zeiss Leica S8AP 0) and photographed. Each treatment was repeated biologically with 10 female adults of brown planthoppers.
Female worm spawning amount detection: single-head transfer of dsGFP and dsNlHsf1 injected female adults into glass tubes (35 cm. Times.5.5 cm) with one rice seedling (30 d seedling age), simultaneously each tube is respectively connected with 2 untreated male adults to mate with the female adults, sucking out female adults after 7 days, dissecting rice seedlings, and counting the number of eggs.
Detecting the nymph hatching rate: in the spawning test, the treated female adults and the untreated male adults are paired one by one, and the female adults and the male adults are sucked out after spawning for 7 d. Continuously culturing rice seedlings, recording the number of hatched nymphs on each rice seedling, dissecting the rice seedlings, recording the number of unhatched eggs, and calculating the hatching rate of the nymphs.
Morphology observation of eggs: the female adults were transferred to new glass tubes on the fourth day after injection as described in the oviposition test, and removed after 6 hours of oviposition. Subsequently, the morphology of the eggs was observed and photographed every day.
2. Data statistics and analysis
Independent sample t-test was performed using Data Processing System (DPS) 13.5 software and plotted. Data in the figures are presented as mean ± standard error, where "×" indicates that the inter-group differences are very significant (P < 0.01).
3. Results and analysis
3.1 CDNA full-length clone of brown planthopper NlHsf Gene and sequence analysis
The NlHsf gene has a total length of 2720bp and comprises a 220bp 5'-UTR and a 199bp 3' -UTR region. NlHsf 1A predicted to contain the 2301bp open reading frame (GenBank accession number: OK 500008) and encoding a 766 amino acid polypeptide. NlHsf1 protein molecular weight is 83182.59Da, theoretical isoelectric point is 4.88, subcellular localization is cell nucleus.
The open reading frame of the NlHsf gene was predicted by the on-line software https:// www.ncbi.nlm.nih.gov/orffinder. Isoelectric points and molecular weights were calculated using http:// web. Expasy. Org/computer_pi. Cell localization using http:// www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/predict NlHsf protein.
3.2, Analysis of the space-time expression Pattern of Brown planthopper NlHsf Gene
NlHsf1 genes are transcribed at different developmental stages of brown planthoppers, and have higher transcription accumulation in adults. Furthermore, the transcript accumulation of NlHsf1 genes in eggs was higher compared to the nymph stage (fig. 1). Tissue quantification results showed that NlHsf gene had the highest transcript accumulation in ovaries and spermary (FIG. 2). This result suggests that NlHsf1 may play a key role in the development of the reproductive system and incubation of eggs of brown planthoppers. Therefore, the inventors further explored the role of NlHsf1 in brown planthopper fertility.
3.3, NlHsf influence of knock-down of Gene expression level on reproduction of brown planthopper
Within 1-5 days after injection dsNlHsf a very significant reduction in the transcript accumulation of NlHsf1 occurred compared to the control group injected with dsGFP. This suggests that dsNlHsf1 successfully interfered with NlHsf1 gene expression at the mRNA level (fig. 3).
NlHsf1 interference results found: brown planthopper ovarian development was inhibited (fig. 4), and spawning amount was reduced (fig. 5); inversion of the eyespot occurred (fig. 6), and the egg hatching rate decreased (fig. 7).
TABLE 1 primers used in the present invention
While the foregoing is directed to the preferred embodiments of the present invention, it is evident that many alternatives and modifications will be apparent to those skilled in the art. Accordingly, all such improvements or modifications are intended to be within the scope of the present invention as set forth in the claims.
The sequence related to the invention is as follows:
SEQ ID No.1：
name: cap-F1 primer nucleotide sequence
Type of DNA: genomic DNA
Organism origin: brown planthopper (NILAPARVATA LUGENS))
ATGCATTCAATAACAGAATTCGAAGGTAGTATTCCAGCATTTCTTGCTAAACTATGGAATATGGTTGATGATCCCAAATTAGACGAACTTATTGCGTGGGGAGCGAATGGATTAAGCTTCGTCATAAATGACCAAATGCGGTTTGCCAAAGAATTGTTACCGATGTACTACAAGCACAACAATATGGCTAGTTTTATTAGACAGCTGAATATGTATGGCTTCCACAAGAACTTATCACTGGAATCTGGCTGCGCCAAAATGGAAGACGTTGTAATAGAGTTTTATCATCCATGTTTCCAGAAGAATAATCCCAATTTACTCCAGAATATCAAGCGAAAGATGAACGCACGCGGCGGAACGGCGGGCGGGGGAAGTGGTGGAGGAGCCGTGAGCGCTTCACCCGACCCGATGCTGGTGAGTCAGCTGTTGCTGGACGTGAAGCGCATGAAGTCCCGACAGGACTCGGTCGACTCGCGCTTCAATCAGCTCAAGATGGAGAACGAAGCCCTCTGGCGCGAGGTGGCCATCATGCGCCAGAAGCATATCAAACAACAGCAGATTGTCAACAAGTTGATCCAGTTCCTATTGACCGTGGTGCAGCATCCGTCGAATGGATTCAGCGTGAAGCGGCGAGGCCTGCCTTTGATGCTGCAAGATCACTCAGTTCCTAGCAATCAGTTGAATAAAAACAATCAGAGCCTGTCAGAAGTGTCTCGATCGCCGACTGGACCCGTGATACACGAGATCGATCCAAACGAGTACGCCGCCTCTTTGCTCTTGAACGATGTGCAATCCGCATCTCAACAGACACCCGACCAGCAAGTCAACGATCAGACGATAACTACTGACGCGACCGCGACCACAACAGCTGACGAATTGCTCGATTTGCCCAATCAGCTGTTGGCAGAGCAACTATCAGCTGATGGCGACGAGCAGACTGAACAGCTGTTTGCCAGACCCAAGCAGATTAGGCCCGGCAAACGACGCCCCCGGAAGCCGGCCGCAACTAAAAAGTTGCCGAGCAAAGTTGCCGATGTGTGTCCCGGTCAACCTGATATCGATGGGTTGGATTTGGACGACTTCCCTCTCATAATCAACGGCTCCCAGGCCCTGGAGCAACTCACCAATCTACCTACATCATCCAATGATGGCCTATTGCCTTCTGACAATGATGCCAACATTGAAGCTCTCACAACACCGATCACTAACCTAACAGCTGATTCATCCATTGCCAACCTTACAACGGACACGCCCATAATCAGTCTATCAGCTGATTCGCCTATTACCAACCTCACAGCTGATGTGCCCATCATCAGTCTATCAGCTGATTCGCCGATTACCAACCTCACAGCTGATGCACCCATAATCAGTCTAACAGCTGATTCGCCGGTTACCAACCTCACAGCTGATGCACCCATAATCAGTCTAACAGCTGATTCTTCACCAATTTCTAACCTAACAGCTGACTCGCCGGTACCAATAGTGATCCCTGCGTCGCCGTACAGTGCTGATTTGGCGGATATTGTGGACAACAGTCAACAGCCAATTGTCAATTGTCAACAGTCGATTGTGAACAGCACAGTGCCAATTGTGGCCGCCGACAAGTCTCGCTACAAGAAGATGGCGCCTAAACTTGTCATTCCCAGCCAGCAGTCTCAGATGAAAGTGATCAGGCCTCAGCGGAAACGGAAGACGGCGGTCGCTGCTCAGACGAAAAGTGGCGGGAAACGGTCGAAAGCGGATGGCGGGAAATTCAAAAGTGTGTCGGCCACGGTTGCTAGGCAACCAGAGACGACGCCCGCCAATCAGAACAGTGATAGGACTGTCGCCTGTATGACACCCACCAACTCACAACAGTCTATGAAGCAGGAAATCGACAATCATGTAGACACCATACAAAATGACCTGGACACACTGAAAGAGCTTCTAAAGGGCGAATCAATTAGTTTGGACGCAAATACATTGTTGGGGCTATTCAATTCAGAAGATCCTTTCTCATTTACAATGGATTTACCCAGCCAGACGACTGAGGAAAAATTGAAAGAACCTGATAACAACCAGCTGGTATCGTACAATTCGGGGTCCCAACAGGGGGGGCAGGGAGACCTGATGGGAGATCTGTTCGACGACTGGATCCTGTCGCCCCCTGCGGGAGGTGGGGAGGGGACAGAGGTGGGGGCGAACGATTTTCCCCTCCAGCCAGATGACGAGGATGACGTCATCAACACACCCGACATACTCTTGACACCGGAGCCACTGCTCATGTCCAATGATGGAGGAGGGAAGAGGAGGAAGAAGTGA
SEQ ID No.2：
Name: brown planthopper heat shock transcription factor NlHsf amino acid sequence
Organism origin: brown planthopper (NILAPARVATA LUGENS))
MHSITEFEGSIPAFLAKLWNMVDDPKLDELIAWGANGLSFVINDQMRFAKELLPMYYKHNNMASFIRQLNMYGFHKNLSLESGCAKMEDVVIEFYHPCFQKNNPNLLQNIKRKMNARGGTAGGGSGGGAVSASPDPMLVSQLLLDVKRMKSRQDSVDSRFNQLKMENEALWREVAIMRQKHIKQQQIVNKLIQFLLTVVQHPSNGFSVKRRGLPLMLQDHSVPSNQLNKNNQSLSEVSRSPTGPVIHEIDPNEYAASLLLNDVQSASQQTPDQQVNDQTITTDATATTTADELLDLPNQLLAEQLSADGDEQTEQLFARPKQIRPGKRRPRKPAATKKLPSKVADVCPGQPDIDGLDLDDFPLIINGSQALEQLTNLPTSSNDGLLPSDNDANIEALTTPITNLTADSSIANLTTDTPIISLSADSPITNLTADVPIISLSADSPITNLTADAPIISLTADSPVTNLTADAPIISLTADSSPISNLTADSPVPIVIPASPYSADLADIVDNSQQPIVNCQQSIVNSTVPIVAADKSRYKKMAPKLVIPSQQSQMKVIRPQRKRKTAVAAQTKSGGKRSKADGGKFKSVSATVARQPETTPANQNSDRTVACMTPTNSQQSMKQEIDNHVDTIQNDLDTLKELLKGESISLDANTLLGLFNSEDPFSFTMDLPSQTTEEKLKEPDNNQLVSYNSGSQQGGQGDLMGDLFDDWILSPPAGGGEGTEVGANDFPLQPDDEDDVINTPDILLTPEPLLMSNDGGGKRRKK
SEQ ID No.3：
Name: primer 18S rRNA-Q-F nucleotide sequence
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
GTAACCCGCTGAACCTCC
SEQ ID No.4：
Name: primer 18S rRNA-Q-R nucleotide sequence
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
GTCCGAAGACCTCACTAAATCA
SEQ ID No.5：
Name: primer NlHsf nucleotide sequence of Q-F
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
TCGCTGCTCAGACGAAAAGT
SEQ ID No.6：
Name: primer NlHsf nucleotide sequence of Q-R
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
GAGTTGGTGGGTGTCATACA
SEQ ID No.7：
Name: primer dsGFP-F nucleotide sequence
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
TAATACGACTCACTATAGGGAAGTTCAGCGTGTCCGGCGA
SEQ ID No.8：
Name: primer dsGFP-R nucleotide sequence
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
TAATACGACTCACTATAGGGCACCTTGATGCCGTTCTTCT
SEQ ID No.9：
Name: primer dsNlHsf nucleotide sequence 1-F
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
TAATACGACTCACTATAGGGGCCAAAATGGAAGACGTTGT
SEQ ID No.10：
Name: primer dsNlHsf nucleotide sequence 1-R
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
TAATACGACTCACTATAGGGTCGTCTGATCGTTGACTTGC
SEQ ID No.11：
Name: primer NlHsf nucleotide sequence 1-F
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
GCGCCAAAATGGAAGACGTT
SEQ ID No.12：
Name: primer NlHsf nucleotide sequence 1-R
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
GGCGACAGTCCTATCACTGT
SEQ ID No.13：
Name: primer NlHsf nucleotide sequence of 1-3F1
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
GTGTGTCGGCCACGGTTGCTAGGCAACC
SEQ ID No.14：
Name: primer NlHsf nucleotide sequence of 1-3F2
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
AGGCAACCAGAGACGACGCCCGCCAA
SEQ ID No.15：
Name: primer NlHsf nucleotide sequence of 1-5R1
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
CTGACTCACCAGCATCGGGTCGGGT
SEQ ID No.16：
Name: primer NlHsf nucleotide sequence of 1-5R2
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
ACCAGCATCGGGTCGGGTGAAGCGCT
SEQ ID No.17：
Name: primer NlHsf nucleotide sequence of FL-F
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
ATGCATTCAATAACAGAATTCGAAGGTAG
SEQ ID No.18：
Name: primer NlHsf nucleotide sequence FL-R
Type of DNA: genomic DNA
Organism origin: artificial sequence (ARTIFICIAL SEQUENCE)
TCACTTCTTCCTCCTCTTCCCTCCTCCAT。
Claims (3)
1. A brown planthopper heat shock transcription factor NlHsf, characterized in that: the amino acid sequence is shown in SEQ.NO. 2.
2. A cDNA of brown planthopper heat shock transcription factor NlHsf, characterized in that: the gene sequence is shown in SEQ.NO. 1.
3. A method for controlling brown planthopper pests, which is characterized in that the method adopts an RNA interference mode to reduce the expression of the brown planthopper heat shock transcription factor NlHsf as set forth in claim 1.
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| CN202210851021.9A CN115925856B (en) | 2022-07-19 | 2022-07-19 | Brown planthopper heat shock transcription factor NlHsf, cDNA and application |
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| CN202210851021.9A CN115925856B (en) | 2022-07-19 | 2022-07-19 | Brown planthopper heat shock transcription factor NlHsf, cDNA and application |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861054A (en) * | 2015-04-14 | 2015-08-26 | 中国水稻研究所 | Brown planthopper protein translation elongation factor NlEF1gamma, and encoded protein and application thereof |
| CN109666675A (en) * | 2018-11-21 | 2019-04-23 | 中国计量大学 | Brown paddy plant hopper NlAtg3 gene, coding albumen and its application |
| CN112195180A (en) * | 2020-10-19 | 2021-01-08 | 扬州大学 | dsRNA sequence for regulating and controlling male brown planthopper seminal fluid Selenoprotein F and application |
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2022
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861054A (en) * | 2015-04-14 | 2015-08-26 | 中国水稻研究所 | Brown planthopper protein translation elongation factor NlEF1gamma, and encoded protein and application thereof |
| CN109666675A (en) * | 2018-11-21 | 2019-04-23 | 中国计量大学 | Brown paddy plant hopper NlAtg3 gene, coding albumen and its application |
| CN112195180A (en) * | 2020-10-19 | 2021-01-08 | 扬州大学 | dsRNA sequence for regulating and controlling male brown planthopper seminal fluid Selenoprotein F and application |
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| CN115925856A (en) | 2023-04-07 |
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