The salivary gland specific gene LsSGSP of Laodelphax striatellus and its applications

Abstract

The present invention discloses a salivary gland-specific gene LsSGSP of Laodelphax striatellus and its application. The nucleic acid sequence of the salivary gland-specific gene LsSGSP of Laodelphax striatellus is shown as SEQ ID NO.1. After introducing the dsRNA of the LsSGSP gene into Laodelphax striatellus and silencing the LsSGSP gene, the feeding ability of Laodelphax striatellus can be significantly reduced, and its survival rate can be decreased. This indicates that the salivary gland-specific gene LsSGSP of Laodelphax striatellus plays a key role in the feeding process of Laodelphax striatellus, and at the same time has important value in the biological control of Laodelphax striatellus, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological control, specifically to the Laodelphax striatellus salivary gland specific gene LsSGSP, and also to the application of LsSGSP in the control of Laodelphax striatellus. Background Art

[0002] The small brown planthopper (Laodelphax striatellus) is an important pest in rice-growing areas in China and other parts of Asia. Since the 1960s, the small brown planthopper has frequently broken out and caused disasters in many rice-growing areas in China, seriously threatening China's food security. The small brown planthopper mainly damages plants by directly sucking plant sap, laying eggs, and transmitting rice viruses. In the field, adult and nymph small brown planthoppers often gather in rice clusters to suck the sap of stem and leaf tissues, consuming plant nutrients and making the grains not plump and reducing the 1000-grain weight. At present, the control of the small brown planthopper mainly relies on chemical pesticides. With the continuous increase in the resistance of the small brown planthopper to chemical pesticides, the dosage and frequency of chemical pesticides have been increasing, which also poses a serious threat to the farmland ecological environment and the quality and safety of rice. In view of the severity of its harm, in 2020, the Ministry of Agriculture and Rural Affairs included the rice planthopper including the small brown planthopper in the "List of First-Class Crop Pests and Diseases".

[0003] The salivary gland is an important secretory organ of the small brown planthopper. The colloidal saliva and watery saliva secreted during the feeding process of the small brown planthopper are mainly secreted by the salivary gland and injected into plant tissues through the salivary duct. The salivary gland plays an important role in the feeding, digestion, virus transmission, etc. of the small brown planthopper. It has been found that many genes related to the feeding of the small brown planthopper are specifically expressed in the salivary gland, and the functions of these genes and their uses in the control of the small brown planthopper remain to be further explored.

[0004] RNA interference technology has been a research hotspot in life sciences in recent years. It specifically degrades or inhibits the expression of target gene mRNA through small double-stranded RNA, thereby inhibiting or shutting down specific genes. RNA interference technology has the advantages of high specificity, high efficiency, and easy operation. At present, this technology has been widely applied to the control of various agricultural pests, that is, by introducing specific dsRNA into insects to achieve the purpose of controlling the pest population or reducing the virus transmission ability of pests. Summary of the Invention

[0005] In view of this, one of the purposes of the present invention is to provide a Laodelphax striatellus salivary gland specific gene LsSGSP; the second purpose of the present invention is to provide primers for amplifying the Laodelphax striatellus salivary gland specific gene LsSGSP; the third purpose of the present invention is to provide the application of a reagent for silencing the Laodelphax striatellus salivary gland specific gene LsSGSP in the preparation of a Laodelphax striatellus control agent. The present invention targets the Laodelphax striatellus salivary gland specific gene LsSGSP to achieve the purpose of inhibiting the secretion amount of honeydew, inhibiting the feeding of the small brown planthopper, reducing the survival rate of the small brown planthopper, and reducing the damage of the small brown planthopper to rice, providing a new control agent for the control of the small brown planthopper.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] 1. The Laodelphax striatellus salivary gland specific gene LsSGSP, and the amino acid sequence encoded by the Laodelphax striatellus salivary gland specific gene LsSGSP is as shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the Laodelphax striatellus LsSGSP gene of the present invention is as shown in SEQ ID NO.1.

[0009] 2. Primers for amplifying the Laodelphax striatellus salivary gland specific gene LsSGSP, and the primers are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] 3. dsRNA of the Laodelphax striatellus salivary gland specific gene LsSGSP.

[0011] Preferably, the nucleotide sequence of the dsRNA of the present invention is as shown in SEQ ID NO.5.

[0012] Preferably, the dsRNA of the present invention is prepared by the following method: using the DNA template shown in SEQ ID NO.5, with the T7 High Yield RNA Transcription Kit, in a reaction system of 2 μl 10×Reaction Buffer, 2 μl ATP solution, 2 μl UTP solution, 2 μl CTP solution, 2 μl GTP solution, 2 μl enzyme mix, 1 μg DNA template, and made up to 20 μl with RNase-free water. After the reaction system is prepared, mix well and react overnight at 37°C; then add TURBO DNase and react at 37°C for 15 min to eliminate the DNA in the reaction system, and denature at 65°C for 5 min to obtain.

[0013] 4. Application of a reagent for silencing the Laodelphax striatellus salivary gland specific gene LsSGSP in the preparation of a Laodelphax striatellus control agent.

[0014] Preferably, the reagent for silencing the Laodelphax striatellus salivary gland specific gene LsSGSP is the dsRNA of the Laodelphax striatellus salivary gland specific gene LsSGSP.

[0015] 5. Application of a reagent for silencing the Laodelphax striatellus salivary gland specific gene LsSGSP in reducing the honeydew secretion amount of Laodelphax striatellus.

[0016] 6. Method for controlling Laodelphax striatellus, silencing the expression of the Laodelphax striatellus salivary gland specific gene LsSGSP in Laodelphax striatellus, and the nucleotide sequence encoded by the Laodelphax striatellus salivary gland specific gene LsSGSP is as shown in SEQ ID NO.2.

[0017] The beneficial effects of the present invention are as follows: The present invention discloses the Laodelphax striatellus salivary gland specific gene LsSGSP. By introducing the dsRNA obtained from the LsSGSP gene into Laodelphax striatellus and silencing the LsSGSP gene, the feeding ability of Laodelphax striatellus can be significantly reduced, and its survival rate can be decreased. This indicates that the Laodelphax striatellus salivary gland specific gene LsSGSP plays a key role in the feeding process of Laodelphax striatellus, and at the same time has important value in the biological control of Laodelphax striatellus (such as transgenic rice, etc.), and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0019] Figure 1 Experimental results of PCR amplification of LsSGSP fragment (504bp);

[0020] Figure 2 Experimental results of dsRNA synthesis of LsSGSP gene;

[0021] Figure 3 Experimental results of the survival rate of introducing dsRNA of LsSGSP gene into Laodelphax striatellus;

[0022] Figure 4 Honeydew secretion amount of Laodelphax striatellus. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following further describes the present invention in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0024] Example 1. Cloning of Laodelphax striatellus LsSGSP gene fragment

[0025] Amplification of Laodelphax striatellus LsSGSP gene fragment

[0026] (1) Take Laodelphax striatellus nymphs or adults, place them on ice for anesthesia; add pre-cooled 1×PBS (37 mM NaCl, 2.68 mM KCl, 8.1 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4) on the glass slide, and immediately dissect the salivary glands of the planthoppers under a stereomicroscope;

[0027] (2) Add the dissected salivary glands to 1 ml of Trizol (Takara) and grind thoroughly; add 400 μl of chloroform, mix vigorously, and centrifuge at 12,000 rpm at 4 °C for 15 minutes. Carefully aspirate the upper aqueous phase liquid and transfer it to a new centrifuge tube; add an equal volume of isopropanol, mix well, and let it stand at room temperature; after 10 minutes, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, add 1 ml of 75% ethanol to the precipitate; centrifuge at 9,600 rpm for 5 minutes, discard the supernatant, and add an appropriate amount of RNase-free water to the precipitate; measure the RNA concentration using NanoDrop;

[0028] (3) Use the ReverTra Ace qPCR RT Master with gDNA remover kit to reverse transcribe the total RNA extracted from the salivary glands of Laodelphax striatellus: First, denature the total RNA at 65 °C for 5 min; take 1 μg of total RNA, add 2 μl of 4×DNMaster Mix, and make up to 8 μl with RNase-free water; mix well and incubate at 37 °C for 5 min to remove contaminating genomic DNA; then, add 2 μl of 5×RT Master Mix to the system; mix well and incubate at 37 °C for 1 h. Finally, denature at 98 °C for 5 min to inactivate the reverse transcriptase and finally obtain the cDNA of the salivary glands of Laodelphax striatellus;

[0029] (4) Design primers as shown in Table 1 according to the open reading frame (SEQ ID NO.1) of the LsSGSP gene, and the encoded amino acids are as shown in SEQ ID NO.2. The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd.;

[0030] Table 1. Primers for amplifying the LsSGSP gene

[0031]

[0032] (5) Using the cDNA of the salivary glands of Laodelphax striatellus as a template, use the primers shown in Table 1 to perform PCR amplification of the target gene. The specific amplification system is as follows: Max Buffer 25 μl, dNTP Mix (10 mM each) 1 μl, MaxSuper-Fidelity DNA Polymerase 1 μl, 1 μl each of the upstream and downstream primers, 1 μl of the cDNA template of the salivary glands of Laodelphax striatellus, and finally make up to 50 μl with ddH 2 O. The PCR amplification conditions are: 95 °C for 3 minutes; 95 °C for 15 seconds, 60 °C for 1 minute, 35 cycles; 72 °C for 10 minutes.

[0033] Obtaining monoclonal strains of the LsSGSP gene of Laodelphax striatellus:

[0034] (1) Separate the PCR amplification products by 1% agarose gel electrophoresis. The results are as Figure 1 shown. After separation, cut out the target fragment with a blade;

[0035] (2) Use a DNA agarose gel recovery kit (Sangon Biotech, Shanghai, SK8131) to recover the target DNA: Cut out the agarose containing the target fragment from the agarose gel and put it into a centrifuge tube; Add 5 times the volume of gel dissolution solution Buffer B2 and incubate in a 70°C water bath for 10 minutes; Add the dissolved gel solution to the adsorption column and centrifuge at 8,000 g for 30 seconds, discard the waste liquid; Add 500 μl of wash Solution and centrifuge at 9,000 g for 30 seconds, discard the waste liquid; Centrifuge the empty adsorption column at 9,000 g for 1 minute; Put the adsorption column into a new centrifuge tube and add 30 μl of ddH 2 O in the center of the adsorption column; Centrifuge at 10,000 g for 2 minutes to finally obtain the purified DNA fragment of the small brown planthopper LsSGSP gene;

[0036] (3) Use a blunt-end cloning kit (Beijing Tsingke Biotechnology Co., Ltd.) to ligate the DNA fragment of the small brown planthopper LsSGSP gene to the pClone007 vector: Add 1 μl of the DNA fragment of the small brown planthopper LsSGSP gene, 1 μl of pClone007 Blunt simple vector, and 1 μl of 10× buffer to a 0.2 ml centrifuge tube in sequence. Finally, make up to 10 μl with ddH 2 O. Mix the sample and react at 25°C for 5 minutes;

[0037] (4) Add the ligation product to the competent cells, gently flick the tube wall with your finger, and incubate on ice for 30 minutes; Transfer the centrifuge tube to a 42°C water bath for 90 seconds, quickly take it out and let it stand on ice for 2 minutes; Add 500 μl of antibiotic-free LB medium to the centrifuge tube and shake at 180 rpm for 1 hour; Take 100 - 200 μl of the bacterial solution and spread it on an LB plate containing ampicillin resistance;

[0038] (5) Pick a single colony into 1 ml of LB liquid medium containing ampicillin resistance and shake at 180 rpm for 6 hours. Perform colony PCR on the bacterial solution. The reaction system is: Max Buffer 12.5 μl, dNTP Mix (10 mM each) 0.5 μl, Max Super-Fidelity DNA Polymerase 0.5 μl, 0.5 μl of each upstream and downstream primer, 1 μl of the bacterial solution, and finally make up to 25 μl with ddH 2 O; The PCR amplification conditions are: Pre-denaturation at 95°C for 3 minutes; Denaturation at 95°C for 15 seconds, annealing at 60°C for 1 minute, 35 cycles; Extension at 72°C for 10 minutes;

[0039] (6) Separate the PCR products by 1% agarose gel electrophoresis, select recombinant clones, and send them to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing to obtain the 504 bp gene sequence of Laodelphax striatellus LsSGSP. The obtained sequence is shown as SEQ ID NO.5.

[0040] Example 2. Synthesis of dsRNA of Laodelphax striatellus LsSGSP gene

[0041] PCR amplification and purification with T7 primers

[0042] (1) Using the recombinant plasmid or bacterial solution containing the Laodelphax striatellus LsSGSP gene as a template, amplify the target gene using primers with the T7 promoter sequence (underlined sequence of the LsSGSP-F primer) (shown in Table 2). The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The amplification system is Max Buffer 100 μl, dNTP Mix (10 mM each) 4 μl, Max Super-Fidelity DNA Polymerase 4 μl, 4 μl each of upstream and downstream (SEQ ID NO.6 and SEQ ID NO.7), 4 μl of recombinant plasmid or bacterial solution, and finally make up to 200 μl with ddH 2 O. The PCR amplification conditions are: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 1 minute, 35 cycles; post-extension at 72°C for 10 minutes.

[0043] Table 2. Primers with T7 promoter sequence

[0044]

[0045]

[0046] (2) Separate the amplification products by agarose gel electrophoresis and recover them using a DNA agarose gel recovery kit to finally obtain a large amount of single LsSGSP gene fragments containing the T7 promoter.

[0047] Synthesis and purification of dsRNA

[0048] Use the T7 High Yield RNA Transcription Kit from Novizan Biotech Co., Ltd. to synthesize and purify the dsRNA of the LsSGSP gene. The specific method is as follows:

[0049] (1) Using the LsSGSP gene fragment containing the T7 promoter obtained by PCR amplification as the DNA template to synthesize dsRNA. The reaction system is as follows: 2 μl of 10×Reaction Buffer, 2 μl of ATP solution, 2 μl of UTP solution, 2 μl of CTP solution, 2 μl of GTP solution, 2 μl of enzyme mix, 1 μg of DNA template, and make up to 20 μl with RNase-free water. After the reaction system is prepared, mix well and react overnight at 37°C;

[0050] (2) Add 1 μl of TURBO DNase to the reaction system to eliminate the DNA in the reaction system, and react at 37°C for 15 min;

[0051] (3) Denature the reacted sample at 65°C for 5 min;

[0052] (4) Measure the concentration of dsRNA with Nanodrop and determine the quality of dsRNA by 1% agarose gel electrophoresis ( Figure 2 );

[0053] (5) Store the dsRNA of the LsSGSP gene at -80°C for later use.

[0054] Example 3. Introduction of dsRNA of the LsSGSP gene into Laodelphax striatellus

[0055] Use the microinjection method to introduce the dsRNA of the LsSGSP gene into Laodelphax striatellus. The specific method is as follows:

[0056] (1) Take the third instar (for mortality statistics) and fourth instar (for honeydew determination) nymphs of Laodelphax striatellus, and anesthetize them with CO 2 for 10 s;

[0057] (2) Use a capillary puller (P 97, Sutter Instrument) to pull the glass capillary (Wuhan Microprobe Scientific Instruments Co., Ltd.) to an appropriate size. The set program parameters are: heat = 800, pull = 150, vel = 150, time = 80;

[0058] (3) Use a micropipette tip (Eppendorf) to inject the dsRNA of the LsSGSP gene into the glass capillary respectively;

[0059] (4) Install the glass capillary with the sample added onto a microinjector (Eppendorf), and introduce the dsRNA into Laodelphax striatellus under a stereomicroscope. The parameters of the microinjector are set as follows: injection pressure 1300 pah, injection time 0.3 s, compensation pressure 10 pah;

[0060] (5) Using the same method, the dsRNA of the Aequorea victoria green fluorescent protein (GFP) gene was introduced into the body of Laodelphax striatellus as a negative control;

[0061] (6) After the Laodelphax striatellus into which the dsRNA had been introduced woke up, it was transferred onto rice plants.

[0062] Example 4. Statistics of the mortality rate of Laodelphax striatellus

[0063] The injected Laodelphax striatellus nymphs were reared on rice seedlings. Approximately 30 Laodelphax striatellus were placed on each group of rice plants, and three replicates were made. The mortality rate was counted daily. The rearing conditions for Laodelphax striatellus were as follows: temperature 26°C ± 0.5°C, relative humidity 50% ± 5%, and photoperiod 16h:8h (day:night);

[0064] The experimental results were as Figure 3 shown. There was a significant difference (p < 0.01) in the survival curve of Laodelphax striatellus into which the dsRNA of the LsSGSP gene had been introduced (dsLsSGSP) compared with the control (dsGFP), indicating that the dsRNA of the LsSGSP gene has potential application value for the control of Laodelphax striatellus.

[0065] Example 5. Determination of the honeydew secretion amount of Laodelphax striatellus

[0066] The feeding amount of planthoppers is positively correlated with the honeydew secretion amount. Therefore, the feeding situation of Laodelphax striatellus was confirmed by measuring the honeydew secretion amount of Laodelphax striatellus under different treatments.

[0067] Rice seedlings at the 4 - 5 leaf stage were taken, and parafilm was fixed on the rice stem by the double - layer clamping method. The 5th - instar Laodelphax striatellus nymphs treated with dsRNA were placed into the parafilm rearing chamber at a rate of 10 per group. The rearing chamber should ensure a certain space so that Laodelphax striatellus can not only contact the rice but also move freely. After the parafilm rearing chamber was sealed, it was placed in an artificial climate chamber for 24 h, with the temperature controlled at 26°C ± 0.5°C, the relative humidity at 50% ± 5%, and the photoperiod at 16h:8h (day:night).

[0068] After being placed in the artificial climate chamber for 24 h, the Laodelphax striatellus in the parafilm rearing chamber were carefully taken out, and the weight of the parafilm containing honeydew was weighed using an electronic balance (sensitivity 0.0001 g). Subsequently, the honeydew was wiped off with absorbent paper, and the weight of the parafilm without honeydew was weighed again. The mass difference between the two was the honeydew amount secreted by Laodelphax striatellus. Twenty independent biological replicates were made for each treatment.

[0069] The experimental results were as Figure 4As shown, the honeydew secretion of Laodelphax striatellus introduced with dsRNA of LsSGSP (dsLsSGSP) was significantly lower than that of the control (dsGFP) (p < 0.01), indicating that the LsSGSP gene may affect the feeding of Laodelphax striatellus, thereby leading to abnormal phenotypes of Laodelphax striatellus.

[0070] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Use of a reagent for silencing the salivary gland-specific gene LsSGSP of Laodelphax striatellus in the preparation of a Laodelphax striatellus control agent, Characterized in that: The amino acid sequence encoded by the salivary gland-specific gene LsSGSP of Laodelphax striatellus is as shown in SEQ ID NO.

2.

2. The use according to claim 1, Characterized in that: The reagent for silencing the salivary gland-specific gene LsSGSP of Laodelphax striatellus is dsRNA of the salivary gland-specific gene LsSGSP of Laodelphax striatellus, and the nucleotide sequence of the dsRNA is as shown in SEQ ID NO.

5.

3. Use of a reagent for silencing the salivary gland-specific gene LsSGSP of Laodelphax striatellus in reducing the honeydew secretion amount of Laodelphax striatellus, Characterized in that: The amino acid sequence encoded by the salivary gland-specific gene LsSGSP of Laodelphax striatellus is as shown in SEQ ID NO.

2.

4. Method for controlling Laodelphax striatellus, Characterized in that: Silencing the expression of the salivary gland-specific gene LsSGSP of Laodelphax striatellus in the body of Laodelphax striatellus, and the amino acid sequence encoded by the salivary gland-specific gene LsSGSP of Laodelphax striatellus is as shown in SEQ ID NO.2.

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