CN116004638A - Application of brown planthopper anchoring protein gene NlGPI as target spot in brown planthopper control - Google Patents

Abstract

The invention discloses application of brown planthopper anchoring protein gene NlGPI as a target in brown planthopper control. The invention uses PCR technique to clone the full-length cDNA sequence of NlGPI based on the data of the brown planthopper transcriptome; analyzing the nucleic acid and protein sequence characteristics by using a bioinformatics means; detecting a time-space expression rule by a qRT-PCR technology; the biological function of NlGPI was ascertained using RNAi technology. The invention discovers that NlGPI is closely related to the process of releasing YLS to haemolymph in brown planthopper, plays an important role in the growth and development and propagation processes of brown planthopper, and can be used as a potential target for brown planthopper prevention and control.

Description

The application of the brown planthopper anchor protein gene NlGPI as a target in the control of brown planthopper

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of brown planthopper anchor protein gene NlGPI as a target in controlling brown planthopper.

Background technique

Brown planthopper (Nilaparvata lugens ) belongs to the family Hemiptera (Hemiptera: Delphacidae), and is an important pest that endangers rice in my country. The brown planthopper not only causes mechanical damage by sucking rice phloem juice and laying eggs in the center of rice leaf sheaths, but also spreads a variety of viral diseases, causing large-scale crop failure and production reduction in my country. How to safely and effectively control brown planthopper is a bottleneck problem to be solved urgently in the current control work of brown planthopper.

There are a large number of Yeast-like symbionts (YLS) in the abdominal fat body of N. lugens, which participate in biological processes such as sterol metabolism, amino acid supply, and nitrogen cycle of N. lugens. All play an important role, so YLS is expected to become a new target for the control of N. lugens. The in-depth study on the vertical transmission process of YLS in N. lugens is helpful to provide new ideas for the control of N. lugens. The preliminary research of the present invention shows that a small amount of YLS can be detected in the hemolymph of the primate female, while a large amount of YLS exists in the hemolymph of the 72-hour eclosion female. In order to find out the influencing factors and regulation mechanism of the process of YLS release from fat body to hemolymph, we carried out the fat body of first-feather females (YLS release time point) and eclosion 72h females (YLS massive release period). Transcriptome sequencing analysis revealed significant differences in the expression of the ankyrin gene NlGPI during the release of YLS.

GPI-anchored protein is a protein anchored on the membrane by covalently binding sugar phosphatidylinositol (GPI) at the C-terminus, and was first discovered in Bacillus cereus. Ferguson et al. successfully isolated and identified the first GPI molecule in Trypanosoma brucei (Brucei variant). GPI is a kind of glycolipid compound with complex structure, which widely exists in eukaryotes. Up to now, there are more than 50 confirmed GPI structures. In many lower or higher eukaryotic organisms, the c-terminus of membrane surface proteins and glycoproteins binds to GPI in the form of covalent bonds, forming GPI-anchored proteins and anchoring them on the plasma membrane. In essence, the appearance of GPI proteins is An act of post-translational modification of proteins. GPI-anchored proteins are mainly anchored on the lipid rafts of the plasma membrane. Lipid rafts are dynamic structures on the plasma membrane that are 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, research on the surface of cells such as human immune T cells and epithelial cells has 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 amount of GPI-anchored protein clusters are related to the type of cells and the external environment of cells. So far, more than 250 eukaryotic membrane proteins have been elucidated to be connected to the plasma membrane through GPI anchoring. The functions of GPI-anchored proteins in mammals mainly include activating immune response, 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 planthopper has not yet been clarified.

Contents of the invention

In order to ascertain the function of the GPI-anchored protein in N. lugens, especially the important role played by YLS in the process of YLS being released from fat body to hemolymph, the present invention cloned the N. lugens GPI-anchored protein gene (NlGPI), and The biological analysis of the cDNA sequence and the analysis of the spatiotemporal expression pattern were carried out, and then the function of NlGPI was initially explored by RNAi technology, and the role of NlGPI in the release of YLS from the brown planthopper was found out, which provided a new direction and theoretical basis for the control of brown planthopper.

In order to achieve the purpose of the above invention, the present invention provides the application of the brown planthopper anchor protein gene NlGPI as a target in the control of brown planthopper, the nucleotide sequence of the NlGPI gene is shown in SEQ ID NO.1.

Specifically, the present invention provides the use of the brown planthopper anchor protein gene NlGPI as a target in the preparation of drugs for controlling brown planthopper.

The present invention provides a kind of dsRNA of brown planthopper anchor protein gene NlGPI, said dsRNA is made up of two complementary nucleotide sequences, the nucleotide sequence of sense strand is shown in SEQ ID NO.2, the core of antisense strand The nucleotide sequence is shown in SEQ ID NO.3.

The invention also provides the application of the dsRNA of the brown planthopper anchor protein gene NlGPI in preventing and controlling brown planthopper pests.

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

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

The present invention also provides a method for controlling brown planthopper, the method comprising introducing the dsRNA according to claim 2 into the body of brown planthopper.

Preferably, the brown planthopper is a nymph.

Preferably, the way of introduction is feeding or injection.

The experimental results of the present invention show that the NlGPI gene has obvious time-space specificity, NlGPI is basically not expressed from the egg stage to the first feather, and the expression level begins to increase significantly after 24 hours of eclosion, and the gene expression level of 72 hours after eclosion is significantly higher than that of other ages. The expression levels of NlGPI in the thorax, abdomen, ovary and intestine were not significantly different, but were significantly higher than those in the head. The results of RNAi analysis showed that after NlGPI interference, the number of YLS in the hemolymph of N. lugens significantly decreased, the mortality of N. lugens significantly increased, and the number of eggs laid and hatching rate decreased significantly. Therefore, NlGPI is closely related to the release of YLS to the hemolymph in N. lugens, and plays an important role in the growth, development and reproduction of N. lugens. It can be used as a potential target for the control of N. lugens.

Description of drawings

Figure 1 is a schematic diagram of the cDNA sequence of NlGPI and its encoded amino acid sequence;

Figure 2 shows the expression levels of NlGPI at different developmental stages of the brown planthopper;

Fig. 3 is the expression level of NlGPI in different tissues of brown planthopper;

Figure 4 is the expression level of NlGPI gene in N. lugens planthopper after RNA interference;

Figure 5 shows the number of YLS in N. lugens at different periods after RNA interference;

Fig. 6 is the growth and survival curve of brown planthopper after RNA interference;

Figure 7 is the total amount of eggs laid by samples after RNA interference;

Figure 8 shows the number of unhatched eggs after RNA interference.

Detailed ways

The present invention will be further elaborated and described below in combination with specific embodiments. The embodiments are merely exemplary of the disclosure and do not delineate the scope of limitation. The technical features of the various implementations in the present invention can be combined accordingly on the premise that there is no conflict with each other.

The source of brown planthopper insects was collected from the artificial climate chamber of Zhejiang Key Laboratory of Biometrics and Inspection and Quarantine Technology, and the rice seedlings of TN1 sensitive strains were continuously raised. The feeding conditions were: temperature 26±1°C, humidity: 70%-80%, photoperiod 16L : 8D.

RNA Extraction Kit MiniBEST Universal RNA Extraction Kit, First Strand cDNA Synthesis Kit PrimeScriptTM 1st Strand cDNA Synthesis Kit, Taq Enzyme, Gel Recovery Kit MiniBEST Garose Gel DNA Extraction Kit Ver.4.0, Reagents Required for Fluorescence Quantitative PCR Premix ExTaqTM II was purchased from Bio-Technology (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.

For data analysis, the Actin gene of N. lugens was used as an internal reference, and the relative expression of NlGPI was calculated by the 2 ^-ΔΔCt method. The experimental results were expressed as mean ± standard error, and the data processing and analysis were performed by dps 7.05 software. One-way ANOVA was used to test the significance of difference, and the graph was drawn by GraphPad Prism Software 8.0.

The gene cloning and biological analysis of embodiment 1 NlGPI

Based on the existing N. lugens transcriptome data in our laboratory and combined with the whole genome information of N. lugens, 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 N. lugens adults was first extracted with an RNA extraction kit. After the quality and concentration were tested by a micro-spectrophotometer NanoDrop ND-2000 (ThermoScientific, USA), the first-strand cDNA was synthesized with a reverse transcription kit. , and then use this as a template for PCR amplification. The primer sequences for PCR amplification are listed in Table 1. PCR reaction system: 25 μL of Ex Taq enzyme, 1 μL of upstream and downstream primers (10 μmol/L) (NlGPI-F and N1GPI-R in Table 1), 1 μL of cDNA template, supplemented to 50 μL with ddH ₂ O. PCR amplification program: pre-denaturation at 95°C, 5min; denaturation at 95°C, 30s; annealing at 55°C, 30s; extension at 72°C, 90s; 30 cycles; 72°C, 10min. The obtained PCR products are detected by 1.5% agarose gel electrophoresis, and the gel pieces of the target bands are cut out, and the products are purified and recovered by using the gel recovery kit, and the recovered DNA samples are sent to the company for sequencing analysis.

By BLAST comparison of the whole genome sequence information of N. lugens, the cDNA sequence of the NlGPI gene cloned in this experiment was completely consistent with the sequence with the accession number XM_022342741.2 in GenBank. The ORF of NlGPI gene has a total of 813 nucleotides, encoding 270 amino acids, the predicted molecular weight is 26989.57, and the predicted isoelectric point is 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 GltG protein. A total of 44 phosphorylation modification sites, including 28 serine modification sites (S), 8 threonine modification sites (T), 8 tyrosine modification sites (Y), no N-glycosylation (N) Modification site (Fig. 1). The numbers on the left in Figure 1 indicate the positions of nucleotides and amino acids. The black bold font in the figure is the predicted phosphorylation site of serine (S), threonine (T) and tyrosine (Y), and the black underline Indicates the conserved domain of the gene, * indicates the stop codon.

The spatio-temporal expression pattern analysis of embodiment 2 NlGPI

Collect eggs, 1-5 instar nymphs, and females 1-5 days after first feathering as samples of N. lugens at different developmental stages, and the head, chest, abdomen, ovary and intestinal tissues of female N. lugens after eclosion for 72 hours as different N. lugens tissues. The total RNA was extracted from the sample, and cDNA was synthesized by reverse transcription as a template for qRT-PCR reaction. qRT-PCR reaction system: cDNA 2μL, Premix Ex TaqTM II 10 μL, upstream and downstream primers (10 μmol/L) 1 μL each (qNlGPI-F and qN1GPI-R in Table 1), supplement the volume with ddH ₂ O to 20 μL. Amplification program: pre-denaturation at 94°C, 30s; denaturation at 94°C, 5s; annealing and extension at 60°C, 30s, 40 cycles, with 3 replicates for each group.

The temporal and spatial expression patterns of NlGPI were studied by qRT-PCR technology, as shown in Figures 2 and 3. The results showed that: NlGPI was basically not expressed from the egg stage to the first feather stage, and the expression level began to increase significantly after 1d eclosion, and the expression level began to increase significantly after 1d and 2d eclosion. There was a significant difference in the gene expression levels of NlGPI and 3d. The gene expression level reached the highest level at 3d after eclosion, and then decreased significantly with the extension of developmental duration, but there was no significant difference between the expression levels of 4d and 5d after eclosion. The study of NlGPI expression in different tissues of eclosion 3d N. lugens showed that there was no significant difference in the expression levels in the thorax, abdomen, ovary and intestine, but they were all significantly higher than the expression in the head. Figure 2 In the gene expression levels of N. lugens at different developmental stages, 0 in the abscissa represents the egg stage of N. lugens, N1-N5 represents the 1st to 5th instar nymphs of N. lugens, and A0-A5 represents the nymphs of N. lugens from the initial eclosion state to 5 days after eclosion. female. In the gene expression levels of different tissues of N. lugens, the collected samples were female tissues from normal growth and development to 3 days after eclosion. Different letters in the figure represent significant differences (P<0.05), the same below.

Example 3 The effect of RNA interference NlGPI on the release of YLS and the growth, development and reproduction of brown planthopper

Based on the cDNA sequences of NlGPI and green fluorescent protein GFP (control) genes, design dsRNA synthetic primers (dsNlGPI-F and dsNlGPI-R in Table 1), refer to The RNAi Kit instructions were used to synthesize dsNlGPI and dsGFP. After the dsRNA is qualified by NanoDrop ND-2000 and 1.5% agarose gel electrophoresis, it is diluted with ddH ₂ O to 5000 ng/μL for subsequent injection. Place the first-feathered female under the microscope, and slowly inject 50 nL of dsRNA at the site between the second and third legs of the thorax of the brown planthopper using a microinjector. After the injection, the brown planthoppers were transferred to the greenhouse for rearing. The N. lugens injected with dsNlGPI was used as the experimental group, and the dsGFP was used as the control group. Three parallel experiments were set up, with 50 test insects in each group. The disturbed N. lugens samples were raised separately, and the samples were collected 24h, 48h, and 72h after injection, and the expression level of NlGPI in the body was detected, and the number of YLS in the hemolymph was observed microscopically. and the survival rate, egg laying rate and hatching rate of brown planthopper.

Table 1 primers used in the present invention

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

After interfering with NlGPI, the number of YLS in the hemolymph per unit mass of the sample at 24h, 48h, and 72h was counted, and the results are shown in Figure 5. With the prolongation of treatment time, the number of YLS in the hemolymph of the control group increased significantly (P<0.05), and the number reached the highest at 72 hours after injection. The number of YLS in the hemolymph of the experimental group was significantly lower than that of the control group (P<0.05), and decreased by 72.4%, 68.2%, and 72.3% after injection for 24h, 48h, and 72h, respectively.

As shown in Figure 6, the decline slope of the survival rate of N. lugens in the control group was relatively small, and the trend of the survival curve was relatively flat. The survival rate was 0 on day 16 after injection, that is, all the samples died. The survival rate of N. lugens in the experimental group decreased rapidly, the survival curve tended to be steeper, and all samples died 10 days after injection, which indicated that interference with NlGPI was not conducive to the normal growth and development of N. lugens.

As shown in Figures 7 and 8, after RNA interference, the maximum number of eggs laid by BPH in the control group was 300, the lowest was 80, the number of unhatched eggs was less than 18, and the average hatching rate was greater than 90%. The highest was 56, the number of unhatched eggs was more than 15, and the average hatching rate was less than 20%. Compared with the control group, the difference was significant (P<0.05), indicating that RNA interference had a greater negative impact on the physiological processes such as oviposition and hatching of N. lugens. To influence.

The above results indicated that the expression of NlGPI in N. lugens had obvious spatio-temporal specificity. The expression level of NlGPI showed obvious differences due to different developmental stages of N. lugens: NlGPI was basically not expressed from the egg stage to the first feather, and the expression level began to increase significantly after 24 hours after eclosion, and the gene expression level reached the highest level at 72 hours after eclosion, and then with the extension of developmental duration The expression level decreased significantly. Previous observations on the number of YLS in the hemolymph of N. lugens found that the number of YLS in the hemolymph of the first-feathered females was almost zero, and NlGPI was basically not expressed at this time; the number of YLS in the hemolymph of females reached the maximum 72 hours after eclosion, and NlGPI was expressed at this time. level up to the max. It is speculated that NlGPI may play an important role in the release of YLS.

The present invention shows that the release of YLS is affected by NlGPI. Through the expression of RNAi NlGPI, the number of YLS in the hemolymph of N. lugens was significantly reduced, and the survival rate, egg laying rate and hatching rate were also significantly reduced. It is inferred that after NlGPI is silenced, the number of YLS in the hemolymph decreases significantly, that is, the process of YLS release from the fat body to the hemolymph is affected, which leads to a decrease in the number of YLS in the oocyte, resulting in a significant decrease in the egg production and hatching rate of N. lugens. . Because the release of YLS to the hemolymph was affected, the vertical transmission of YLS in N. lugens was affected. The necessary physiological processes in the growth and development of the brown planthopper were affected, so the brown planthopper could not grow and develop normally, and the mortality rate increased. Therefore, the process of NlGPI and YLS release from fat body to hemolymph is closely related, which may affect the exocytosis process of YLS release to hemolymph in N. lugens by changing the structure of plasma membrane, and affect the physiological processes such as growth and reproduction of N. lugens. . Therefore, NlGPI can be used as a potential target for the control of N. lugens.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. For those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims (8)

1. the application of the brown planthopper anchor protein gene NlGPI as a target in the control of brown planthopper, characterized in that the nucleotide sequence of the NlGPI gene is as shown in SEQ ID NO.1. 2. A dsRNA of the brown planthopper anchor protein gene NlGPI, the dsRNA is composed of two complementary nucleotide sequences, the nucleotide sequence of the sense strand is as shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand The sequence is shown in SEQ ID NO.3. 3. the application of the dsRNA of the brown planthopper anchor protein gene NlGPI of claim 2 in the prevention and treatment of brown planthopper pests. 4. The application according to claim 3, characterized in that the application is: using the dsRNA to prepare a medicament for controlling brown planthopper. 5. A recombinant expression vector, a recombinant microorganism or a transgenic cell line comprising the dsRNA of claim 2. 6. A method for controlling brown planthopper, characterized in that the method comprises introducing the dsRNA according to claim 2 into the body of brown planthopper. 7. The method according to claim 6, wherein the brown planthopper is a nymph. 8. The method according to claim 6 or 7, characterized in that the way of introduction is feeding or injection.

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