Application of brown planthopper anchoring protein gene NlGPI as a target in controlling brown planthopper

Abstract

The present invention discloses the application of brown planthopper anchor protein gene NlGPI as a target in the prevention and control of brown planthopper. Based on the transcriptome data of brown planthopper, the present invention clones the full-length cDNA sequence of NlGPI by PCR technology; analyzes its nucleic acid and protein sequence characteristics by bioinformatics means; detects its spatiotemporal expression law by qRT-PCR technology; and explores the biological function of NlGPI by RNAi technology. The present invention finds that NlGPI is closely related to the process of YLS release from brown planthopper to hemolymph, and plays an important role in the growth, development and reproduction of brown planthopper, and can be used as a potential target for the prevention and control of brown planthopper.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the application of a brown planthopper anchor protein gene N1GPI as a target in preventing and controlling the brown planthopper. Background Art

[0002] Brown planthopper (Nilaparvata lugens ) belongs to the family Delphacidae in the order Hemiptera and is a major pest that harms rice in my country. Brown planthoppers not only cause mechanical damage by sucking rice phloem sap and laying eggs in the center of rice leaf sheaths, but also spread a variety of viral diseases, causing large-scale grain failure and production reduction in my country. How to safely and effectively prevent and control brown planthoppers is a bottleneck problem that needs to be solved in the current prevention and control of brown planthoppers.

[0003] There are a large number of yeast-like symbionts (YLS) in the abdominal fat body of brown planthoppers, which participate in biological processes such as steroid metabolism, amino acid supply and nitrogen cycle of brown planthoppers, and play an important role in the growth, development, reproduction and pesticide resistance of brown planthoppers. Therefore, YLS is expected to become a new target for the control of brown planthoppers. In-depth research on the vertical transmission process of YLS in brown planthoppers is conducive to providing new ideas for the control of brown planthoppers. The previous study of the present invention showed that a small amount of YLS can be detected in the hemolymph of newly emerged female insects, while a large amount of YLS exists in the hemolymph of female insects 72 hours after eclosion. In order to explore the influencing factors and regulatory mechanisms of the process of YLS release from fat body to hemolymph, we conducted transcriptome sequencing analysis on the fat body of newly emerged female insects (the time point when YLS begins to release) and female insects 72 hours after eclosion (the period of large-scale release of YLS), and found that the expression of the anchoring protein gene NlGPI was significantly different during the YLS release process.

[0004] GPI-anchored proteins are proteins that are anchored to the membrane by covalently binding glycophosphatidylinositol (GPI) at the C-terminus. They were first discovered in Bacillus cereus. Ferguson et al. successfully isolated and identified the first GPI molecule in Brucei variant. GPI is a complex glycolipid compound that is widely present in eukaryotes. To date, more than 50 GPI structures have been confirmed. The C-termini of membrane surface proteins and glycoproteins in many lower or higher eukaryotic organisms bind to GPI in the form of a covalent bond to form GPI-anchored proteins and anchor them to the cytoplasmic membrane. In essence, the appearance of GPI proteins is a post-translational modification of proteins. GPI-anchored proteins are mainly anchored to lipid rafts on the cytoplasmic membrane. Lipid rafts are a dynamic structure on the plasma membrane that is rich in microdomains of cholesterol and sphingomyelin. When GPI-anchored proteins are synthesized in large quantities and anchored to the plasma membrane, they will spontaneously form GPI-anchored protein clusters. In the past few decades, through studies on the surface of human immune T cells and epithelial cells, it has been found that the protein clusters formed by GPI on the surface of different types of cells are different. Therefore, it can be inferred that the size and number of GPI-anchored protein clusters are related to the type of cell and the external environment of the cell. To date, more than 250 eukaryotic membrane proteins have been shown to be connected to the plasma membrane through GPI anchoring. The functions of GPI-anchored proteins in mammals mainly include activating immune responses, promoting spermatogenesis and development, and participating in signal transduction between cells or between cells and the environment. However, the role of GPI-anchored proteins in insects such as brown planthoppers has not yet been clarified. Summary of the invention

[0005] In order to explore the function of GPI-anchored protein in brown planthoppers, especially the important role it plays in the process of YLS release from fat body to hemolymph, the present invention cloned the brown planthopper GPI-anchored protein gene (NlGPI), and performed biological analysis and spatiotemporal expression pattern analysis on its cDNA sequence, and then conducted a preliminary functional exploration of NlGPI through RNAi technology, and explored the role of NlGPI in the release process of YLS of brown planthoppers, providing a new direction and theoretical basis for the control of brown planthoppers.

[0006] In order to achieve the above invention objectives, the present invention provides the use of brown planthopper anchor protein gene NlGPI as a target in controlling brown planthoppers, and the nucleotide sequence of the NlGPI gene is shown in SEQ ID NO.1.

[0007] Specifically, the present invention provides the use of the brown planthopper anchor protein gene NlGPI as a target in the preparation of a drug for controlling brown planthoppers.

[0008] The invention provides a dsRNA of a brown planthopper anchoring protein gene N1GPI. The dsRNA consists of two complementary nucleotide sequences, the nucleotide sequence of the sense chain is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.3.

[0009] The present invention also provides the use of the dsRNA of the brown planthopper anchor protein gene N1GPI in preventing and controlling brown planthopper pests.

[0010] Preferably, the application is: using the dsRNA to prepare a drug for controlling brown planthopper.

[0011] The present invention also provides a recombinant expression vector, a recombinant microorganism or a transgenic cell line comprising the dsRNA.

[0012] The present invention also provides a method for controlling brown planthoppers, which comprises introducing dsRNA of the brown planthopper anchor protein gene N1GPI into the body of the brown planthopper.

[0013] Preferably, the brown planthopper is a nymph.

[0014] Preferably, the introduction is by feeding or injection.

[0015] The experimental results of the present invention show that the NlGPI gene has obvious spatiotemporal specificity. NlGPI is basically not expressed from the egg stage to the first feather. The expression level begins to increase significantly 24 hours after eclosion. The gene expression level 72 hours after eclosion is significantly higher than that of other age stages. There is no significant difference in the expression level of NlGPI in the chest, abdomen, ovary and intestine, but they are all significantly higher than the expression in the head. The results of RNAi analysis show that after NlGPI interference, the number of YLS in the hemolymph of brown planthoppers decreased significantly, the mortality rate of brown planthoppers increased significantly, and the egg laying amount and hatching rate decreased significantly. Therefore, NlGPI is closely related to the process of releasing YLS from the body of brown planthoppers into the hemolymph, and plays an important role in the growth, development and reproduction of brown planthoppers, and can be used as a potential target for the prevention and control of brown planthoppers. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is a schematic diagram of the cDNA sequence of NlGPI and its encoded amino acid sequence;

[0017] Figure 2 is the expression level of NlGPI in different developmental stages of brown planthopper;

[0018] Figure 3 is the expression level of NlGPI in different tissues of brown planthopper;

[0019] Figure 4 is the expression level of NlGPI gene in brown planthopper after RNA interference;

[0020] Figure 5 is the number of YLS in brown planthopper at different stages after RNA interference;

[0021] Figure 6 is the growth and survival curve of brown planthopper after RNA interference;

[0022] Figure 7 is the total amount of eggs laid by the sample after RNA interference;

[0023] Figure 8 is the number of unhatched eggs after RNA interference. DETAILED DESCRIPTION

[0024] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0025] Brown planthoppers were collected from the artificial climate chamber of the Zhejiang Provincial Key Laboratory of Biometrics and Inspection and Quarantine Technology and were continuously raised with TN1 sensitive rice seedlings. The rearing conditions were: temperature 26±1℃, humidity: 70%~80%, and photoperiod 16L:8D.

[0026] RNA extraction kit MiniBEST Universal RNAExtraction Kit, first-strand cDNA synthesis kit PrimeScriptTM 1st Strand cDNA Synthesis Kit, Taq enzyme, gel recovery kit MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0, fluorescent quantitative PCR reagents Premix ExTaqTMII was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; dsRNA (double-stranded RNA) synthesis kit RNAi Kit was purchased from Invitrogen, USA; primer synthesis and sequencing were completed by Hangzhou Youkang Biotechnology Co., Ltd.

[0027] Data analysis used the brown planthopper Actin gene as an internal reference and used 2 -ΔΔCt The relative expression of NlGPI was calculated by the method. The experimental results were expressed as mean ± standard error, and data processing and analysis were performed using dps 7.05 software. One-way ANOVA was used for significant difference test, and the graphs were drawn using GraphPad Prism Software 8.0.

[0028] Example 1 Gene cloning and biological analysis of N1GPI

[0029] Based on the existing transcriptome data of brown planthoppers in our laboratory and combined with the whole genome information of brown planthoppers, the full-length sequence of NlGPI cDNA was obtained (NCBI accession number: XM_022342741.2). To verify the cloning of NlGPI, the total RNA of brown planthopper adults was first extracted using an RNA extraction kit. After the quality and concentration were tested by the micro-spectrophotometer NanoDrop ND-2000 (ThermoScientific, USA), the first-strand cDNA was synthesized using a reverse transcription kit, and then PCR amplification was performed using this as a template. The PCR amplification primer sequences are shown in Table 1. PCR reaction system: 25 μL of Ex Taq enzyme, 1 μL of upstream and downstream primers (10 μmol / L) each (NlGPI-F and NlGPI-R in Table 1), 1 μL of cDNA template, and ddH 2 O to make the volume 50μL. PCR amplification program: pre-denaturation 95℃, 5min; denaturation 95℃, 30s; annealing 55℃, 30s; extension 72℃, 90s; 30 cycles; 72℃, 10min. The obtained PCR product was detected by 1.5% agarose gel electrophoresis, and the gel block with the target band was cut out and the product was purified and recovered using a gel recovery kit. The recovered DNA sample was sent to the company for sequencing analysis.

[0030] By BLAST comparison of the whole genome sequence information of brown planthopper, the cDNA sequence of the NlGPI gene cloned in this experiment is completely consistent with the sequence with accession number XM_022342741.2 in GenBank. The ORF of the NlGPI gene has a total of 813 nucleotides, encoding 270 amino acids, with a predicted molecular weight of 26989.57 and a predicted isoelectric point of 4.84. The gene has a signal peptide (Sec / SPI), and the cleavage site of the signal peptide is located between the 19th and 20th amino acids, and contains a domain with a GltG protein. There are 44 phosphorylation modification sites, including 28 serine modification sites (S), 8 threonine modification sites (T), 8 tyrosine modification sites (Y), and no N-glycosylation (N) modification site ( Figure 1 ). Figure 1 The numbers on the left indicate the positions of nucleotides and amino acids. The bold black fonts in the figure are predicted phosphorylation modification sites of serine (S), threonine (T), and tyrosine (Y). The black underline indicates the conserved domain of the gene.

[0031] * indicates a stop codon.

[0032] Example 2 Analysis of spatiotemporal expression patterns of N1GPI

[0033] Eggs, nymphs of 1-5 instars, and females 1-5 days after first eclosion were collected as samples of brown planthoppers at different developmental stages, and head, thorax, abdomen, ovary and intestinal tissues of female brown planthoppers 72 hours after eclosion were collected as samples of different brown planthopper tissues. Total RNA was extracted and reverse transcribed to synthesize cDNA as a template for qRT-PCR reaction. qRT-PCR reaction system: cDNA 2μL, Premix Ex TaqTMII 10 μL, upstream and downstream primers (10 μmol / L) 1 μL each (qNlGPI-F and qNlGPI-R in Table 1), and ddH 2 O to make the volume 20 μL. Amplification program: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s; annealing and extension at 60°C for 30 s, 40 cycles, with 3 replicates for each group.

[0034] The spatiotemporal expression pattern of NlGPI was studied by qRT-PCR. Figure 2 and 3 As shown in the figure, the results showed that NlGPI was basically not expressed from the egg stage to the initial eclosion stage, and the expression level began to increase significantly after 1 day of eclosion, and there were significant differences in the gene expression levels of NlGPI at 1d, 2d and 3d after eclosion. The gene expression level reached the highest 3d after eclosion, and then decreased significantly with the extension of the developmental period, but there was no significant difference in the expression level at 4d and 5d after eclosion. The study of NlGPI expression in different tissues of brown planthoppers at 3d after eclosion showed that its expression levels in the chest, abdomen, ovary and intestine were not significantly different, but were significantly higher than those in the head. Figure 2 In the gene expression levels of brown planthoppers at different developmental stages, the horizontal axis 0 represents the egg stage of brown planthoppers, N1-N5 represents the 1st to 5th instar nymphs of brown planthoppers, and A0-A5 represents the females of brown planthoppers from the initial eclosion state to 5 days after eclosion. In the gene expression levels of different tissues of brown planthoppers, the collected samples are tissues of females with normal growth and development to 3 days after eclosion. Different letters in the figure represent significant differences (P < 0.05), the same below.

[0035] Example 3 Effects of RNA interference NlGPI on the release of YLS and growth, development and reproduction of brown planthopper

[0036] Based on the cDNA sequences of NlGPI and green fluorescent protein GFP (control) genes, dsRNA synthesis primers (dsNlGPI-F and dsNlGPI-R in Table 1) were designed. The RNAi Kit instructions were used to synthesize dsNlGPI and dsGFP. After the dsRNA was tested by NanoDrop ND-2000 and 1.5% agarose gel electrophoresis, ddH 2O was diluted to 5000ng / μL for subsequent injection. The newly emerged female insects were placed under a microscope, and 50nL dsRNA was slowly injected into the area between the second and third legs of the thorax of the brown planthopper using a microinjector. After injection, the brown planthoppers were transferred to a warm net room for breeding. The brown planthoppers injected with dsNlGPI were used as the experimental group, and the injection of dsGFP was used as the control group. Three parallel experiments were set up, with 50 test insects in each group. The disturbed brown planthopper samples were raised separately, and samples were collected 24h, 48h, and 72h after injection. The expression level of NlGPI in the body, the number of YLS in the hemolymph, the survival rate of the brown planthoppers, the egg laying amount, and the hatching rate were detected respectively.

[0037] Table 1 Primers used in the present invention

[0038]

[0039] The results of the interference efficiency test of NlGPI in brown planthoppers are as follows Figure 4 As shown, there were extremely significant differences in NlGPI gene expression between the experimental group and the control group at different times after treatment (P < 0.01). The NlGPI gene expression in the experimental group was significantly lower than that in the control group, and decreased by 82.74%, 78.14%, and 68.60% respectively after 24h, 48h, and 72h of injection compared with the control group.

[0040] After interfering with NlGPI, the number of YLS per unit mass of hemolymph in the samples at 24h, 48h, and 72h was counted. The results are as follows: Figure 5 As the treatment time prolonged, the amount of YLS in the hemolymph of the control group increased significantly (P < 0.05), reaching the highest level at 72h after injection. However, the amount of YLS in the hemolymph of the experimental group decreased significantly compared with the control group (P < 0.05), decreasing by 72.4%, 68.2%, and 72.3% at 24h, 48h, and 72h after injection, respectively.

[0041] like Figure 6 As shown in the figure, the survival rate of brown planthoppers in the control group decreased with a smaller slope, the survival curve trend was relatively gentle, and the survival rate was 0 on the 16th day after injection, that is, all the samples died. The survival rate of brown planthoppers in the experimental group decreased rapidly, the survival curve trend was steeper, and all the samples died on the 10th day after injection, indicating that interfering with NlGPI is not conducive to the normal growth and development of brown planthoppers.

[0042] like Figure 7 and 8As shown in the data, after RNA interference, the egg-laying capacity of brown planthoppers in the control group was as high as 300 and as low as 80, the number of unhatched eggs was less than 18, and the average hatching rate was greater than 90%; while the egg-laying capacity of brown planthoppers in the treatment group was as high as 56, the number of unhatched eggs was greater than 15, and the average hatching rate was less than 20%, which was significantly different from the control group (P < 0.05), indicating that RNA interference had a significant negative impact on the physiological processes of brown planthoppers such as egg-laying and hatching.

[0043] The above results show that the expression of NlGPI in brown planthoppers has obvious spatiotemporal specificity. The expression level of NlGPI shows obvious differences in the different developmental stages of brown planthoppers: NlGPI is basically not expressed from the egg stage to the first feather, and the expression level begins to increase significantly 24 hours after eclosion. The gene expression level reaches the highest level 72 hours after eclosion, and then the expression level decreases significantly with the extension of the developmental period. Previous observations on the number of YLS in the hemolymph of brown planthoppers found that the number of YLS in the hemolymph of the first eclosion females is almost 0, and NlGPI is basically not expressed at this time; the number of YLS in the hemolymph of females reaches the maximum 72 hours after eclosion, and the expression level of NlGPI reaches the highest at this time. It is speculated that NlGPI is very likely to play an important role in the release of YLS.

[0044] The present invention shows that the release of YLS is affected by NlGPI. Through the expression of RNAi NlGPI, the amount of YLS in the hemolymph of brown planthoppers is significantly reduced, and the survival rate, egg-laying rate and hatching rate are also significantly reduced. It is inferred that after NlGPI is silenced, the amount of YLS in the hemolymph is significantly reduced, that is, the process of YLS being released from the fat body to the hemolymph is affected, thereby causing the amount of YLS in the oocyte to be reduced, resulting in a significant reduction in the amount of egg-laying and hatching rate of brown planthoppers. Since the process of YLS being released to the hemolymph is affected, the vertical transmission of YLS in the body of brown planthoppers is affected. The necessary physiological processes in the growth and development of brown planthoppers are affected, so brown planthoppers cannot grow and develop normally, and the phenomenon of increased mortality occurs. Therefore, NlGPI is closely related to the process of YLS being released from the fat body to the hemolymph, which may affect the exocytosis process of YLS being released to the hemolymph in the body of brown planthoppers by changing the structure of the plasma membrane, and affect the physiological processes such as the growth and reproduction of brown planthoppers. Therefore, NlGPI can be used as a potential target for the prevention and control of brown planthoppers.

[0045] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Brown planthopper anchor protein gene NlGPI As a target for the control of brown planthoppers, It is characterized in that The brown planthopper anchor protein gene NlGPI The nucleotide sequence is shown in SEQ ID NO.

1.

2. A brown planthopper anchoring protein gene NlGPI Application of dsRNA in controlling brown planthopper pests, It is characterized in that The dsRNA consists of two complementary nucleotide sequences, the nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

3.

3. The use according to claim 2, Features The application is: using the dsRNA to prepare medicine for preventing and controlling brown planthoppers.

4. A method for controlling brown planthopper, It is characterized in that The method comprises the steps of: NlGPI The dsRNA is introduced into the brown planthopper, wherein the dsRNA consists of two complementary nucleotide sequences, the nucleotide sequence of the sense chain is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.

3.

5. The method according to claim 4, It is characterized in that The brown planthopper is a nymph.

6. The method according to claim 4 or 5, It is characterized in that The introduction method is feeding or injection.

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