Application of a dsRNA in inhibiting the reproductive ability of Bemisia tabaci

Abstract

The invention provides an application of dsRNA in inhibiting the reproductive ability of whitefly, belonging to the technical field of pesticides; the dsRNA is ds BtInR1 、ds BtInR2 The present invention discovered two insulin receptor genes in Bemisia tabaci BtInR1 , BtInR2 , and it was clear that when the knockdown BtInR1 , BtInR2 After the expression level is reduced, it can affect the JH signaling pathway by interfering with the insulin signaling pathway, and finally inhibit the reproductive ability of the whitefly, providing a theoretical and application basis for the development and production of green biological pesticides to control whiteflies.

Description

Technical Field

[0001] The invention relates to the technical field of pesticides, in particular to the application of dsRNA in inhibiting the reproductive ability of whitefly. Background Art

[0002] Bemisia tabaci Bemisia tabaci ) is a serious global pest with a wide host range, large egg-laying capacity, rapid resistance development and the ability to spread a variety of plant viruses. When a whitefly outbreak occurs, it can cause damage to a variety of field and room temperature crops, including cash crops and ornamental plants, causing significant economic losses.

[0003] In whiteflies, insulin or insulin-like peptides bind to insulin receptors (InR) to activate the insulin signaling pathway, thereby regulating insect metabolism, tissue differentiation, lifespan and fertility. The insulin receptor plays a key role in the insulin signaling pathway, and its basic structure is formed by two α2 subunits and two β subunits connected by disulfide bonds.

[0004] Juvenile hormone (JH) is a sesquiterpenoid substance that plays an important role in maintaining larval morphology, regulating insect age, and reproduction. JH includes JH Ⅰ, JH Ⅱ, JH Ⅲ and juvenile hormone analogs Methoprene, JHB3, JHSB3, etc. that exist in nature. Among them, JH Ⅲ is the main form of juvenile hormone in most insects, including whiteflies. The JH signaling pathway can regulate the synthesis of yolk protein in the fat body. Yolk protein is a necessary substance for the development of oocytes and can promote reproductive activities of female insects.

[0005] The insulin signaling pathway can cooperate with the juvenile hormone signaling pathway to jointly regulate various important physiological processes in insects. In view of the above theoretical basis, the present invention intends to explore a method that can regulate the metabolism, lifespan or fertility of whiteflies, so as to control whiteflies through a green biological approach. Summary of the invention

[0006] The present invention uses bioinformatics analysis technology to identify two whitefly insulin receptor genes BtI , BtR , and it was clear that when the knockdown BtI , BtR After the expression level is reduced, it can affect the JH signaling pathway by interfering with the insulin signaling pathway, and finally inhibit the reproductive ability of the whitefly, providing a theoretical basis for the development and production of green biological pesticides to control whiteflies.

[0007] The technical solution of the present invention is as follows:

[0008] A use of dsRNA in inhibiting the reproductive ability of whitefly, wherein the dsRNA is ds BtI 、ds BtR One or more of; wherein, the ds BtI The nucleotide sequence of ds BtR The nucleotide sequence is shown in SEQ ID NO.8.

[0009] Preferably, the application method is to feed ds BtI or ds BtR , inhibiting the reproductive capacity of female whiteflies.

[0010] Further preferably, the inhibiting the reproductive capacity of female whiteflies includes: inhibiting the ovarian development of female whiteflies and reducing the egg laying amount of female whiteflies.

[0011] Preferably, the feeding ds BtI or ds BtR The method is: use a mixture containing ds BtI or ds BtR Whiteflies were fed with sucrose water.

[0012] Preferably, the concentration of sucrose in the sucrose water is 0.17-0.23 mg / L.

[0013] Preferably, the concentration of sucrose in the sucrose water is 0.2 mg / L.

[0014] Preferably, the ds BtI or ds BtR The concentration in sucrose water is 250ng / µL.

[0015] Beneficial effects:

[0016] The present invention discovered two insulin receptor genes of Bemisia tabaci BtI , BtR , and it was clear that when the knockdown BtI , BtR After the expression level is reduced, it can affect the JH signaling pathway by interfering with the insulin signaling pathway, and finally inhibit the reproductive ability of the whitefly, providing a theoretical and application basis for the development and production of green biological pesticides to control whiteflies. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the analysis of the conserved domain of amino acid composition of BtInR1 and BtInR2;

[0018] Figure 2is the phylogenetic tree of InR amino acid sequences of different species;

[0019] Figure 3 RNAi in Bemisia tabaci BtI , BtR The expression level of

[0020] Figure 4 is the JH titer in female whiteflies after RNAi;

[0021] Figure 5 RNAi in female whiteflies Bt , BtKr-h1 The expression level of

[0022] Figure 6 The ovarian development of female whiteflies after RNAi;

[0023] Figure 7 is the number of ovarioles of female whiteflies after RNAi;

[0024] Figure 8 RNAi in female whiteflies BqV , BqI The expression level of

[0025] Fig. 9 is the egg laying amount of female whiteflies after RNAi. DETAILED DESCRIPTION

[0026] The following is explained in conjunction with specific embodiments:

[0027] Source of experimental materials:

[0028] Trizol reagent: purchased from Thermo Fisher Scientific;

[0029] PrimeScript™ II 1st Strand cDNA Synthesis Kit: purchased from Bio-Rad Biotechnology Co., Ltd.

[0030] ApexHF HS DNA Polymerase: purchased from Apex Biotech;

[0031] TranscriptAid T7 High Yield Transcription Kit: purchased from Thermo Fisher Scientific;

[0032] PrimeScript™ RT reagent Kit with gDNA eraser: purchased from Bio-Rad Biotechnology Co., Ltd.

[0033] ELISA kit: Shanghai ELISA Biotechnology Co., Ltd.;

[0034] The primers involved in the examples were all commissioned to be synthesized by Qingke Biotechnology Co., Ltd.

[0035] The Bemisia tabaci QS population involved in the examples was collected from cotton fields in Jinan City, Shandong Province in 2012.

[0036] Embodiment 1: BtI , BtR Sequence identification and analysis

[0037] Based on the conserved sequence of insect InR (insulin receptor) sequences, two insulin receptor genes were screened from the genome of Bemisia tabaci by searching the National Center of Biotechnology Information (NCBI) database online. BtI , BtR ,in, BtI The amino acid sequence (BtInR1) is shown in SEQ ID NO.1, BtR The amino acid sequence (BtInR2) is shown in SEQ ID NO.2.

[0038] The amino acid composition conserved domain analysis of the two sequences of BtInR1 and BtInR2 was performed. Figure 1 As shown. Figure 1 It can be seen that the sequence similarity between BtInR1 and BtInR2 is only 27.63%, but they both contain five conserved structures: ligand L binding domain (Receptor Domain), furin-like cysteine ​​rich domain (Furin-like Cysteine ​​Rich Domain), fibronectin type 3 domain (Fibronectin type 3 domain), catalytic domain of insulin receptor-like protein tyrosine kinases (Catalytic domain of insulin receptor-like protein tyrosine kinases) and ATP-binding site (ATP-binding site).

[0039] Phylogenetic analysis of the two amino acid sequences of BtInR1 and BtInR2 was performed: 28 InR amino acid sequences of 23 insects in the NCBI database were selected and compared with the BtInR1 and BtInR2 sequences of whiteflies using the MUSCLE program in MEGA 5.0 software. The phylogenetic tree was constructed using the maximum likelihood method, with 1000 replications selected for Bootstrap and 50% cutoff value for the phylogenetic tree. The phylogenetic tree was edited using the online phylogenetic tree beautification software ChiPlot (https: / / www.chiplot.online / ). Among them, the 28 InR sequences of 23 insects are as follows: Drosophila barbatelli ( Drosophila busckii )DbInR, Drosophila melanogaster ( Drosophila melanogaster )DmInR, Glossina punctatus ( Glossina morsitans )GmmInR, Anopheles sinensis ( Anopheles sinensis )AsInR, Anopheles daphni Anopheles darlingi )AdInR, Aedes aegypti AaInR, Bicyclus anynana )BaInR, Peacock butterfly ( Nymphalidae Inachis io )NiInR, Bombyx mori ( Bombyx mori )BmInR, Spodoptera litura ( Spodoptera litura )SlInR, Plutella xylostella( Plutella xylostella )PxInR, Rhizosiphum graminearum ( Rhopalosiphum padi )RpInR1, RpInR2, brown citrus aphid ( Aphis citricidus )AcInR1, AcInR2, pea aphid ( Acyrthosiphon pisum )ApInR1, ApInR2, red stink bug ( Pyrrhocoris apterus )PaInR1, PaInR2, brown planthopper ( Nilaparvata lugens )NlInR1, NlInR2, broad-horned castaneum ( Gnathocerus cornutus )GcInR, Tribolium castaneum( Tribolium castaneum )TcInR, Monochamus alternatus ( Monochamus alternatus )MaInR, jumping sickle ant ( Harpegnathos Saltalor )HsInR, fungus ant ( Cyphomyrmex costatus )CcInR, Bi's egg-horned ant ( Ooceraea biroi )ObInR, European Bear Peak( Bombus terrestris )BotInR.

[0040] The constructed phylogenetic tree is as follows Figure 2 As shown. Figure 2It can be seen that the amino acid sequence of InR is highly conserved among insect species, that is, the InR sequences of different insects are clustered according to the order of the insects they belong to. In addition, two different InRs (InR1 and InR2) were identified in some insects of Hemiptera and Coleoptera. These two InRs are two independent branches in the phylogenetic tree, indicating that the two InRs of insects are relatively independent in evolution. Among them, BtInR1 and BtInR2 (blue dotted boxes) of Bemisia tabaci are clustered with the two InR genes of Hemiptera insects in the phylogenetic tree.

[0041] Embodiment 2: BtI , BtR Knockdown of expression level

[0042] 1. Synthetic dsRNA

[0043] (1) Total RNA from QS population of whiteflies was extracted using Trizol reagent, and the first-strand cDNA was synthesized using PrimeScript™ II 1stStrand cDNA Synthesis Kit.

[0044] (2) Using the first-strand cDNA as a template, amplify ds BtI Gene fragments and ds BtR Gene fragments; among them, the amplified ds BtI The primers used for the gene fragments are shown in SEQ ID NOs.3-4, respectively, to amplify ds BtR The primers used for the gene fragments are shown in SEQ ID NOs.5~6 respectively.

[0045] (3) ds BtI Gene fragments and ds BtR The gene fragments were used as templates and synthesized using the TranscriptAidT7 High Yield Transcription Kit. BtI , BtR Double-stranded RNA (dsRNA);

[0046] The dsRNA synthesis system is: 5×TranscriptAid Reaction Buffer 5μL, ribonucleotide (A / G / C / U, 100mM) 2μL each, template 1μg, enzyme-free water to 20μL, and mix thoroughly.

[0047] (4) Incubate the above dsRNA synthesis system at 37°C for 4 h; after the incubation, add 2 μL DNase I and incubate in a 37°C water bath for 15 min; add 2 μL 0.5M EDTA and incubate at 65°C for 10 min; add enzyme-free water to make up to 500 μL, then add 200 μL chloroform and mix gently, let stand for 10 min; centrifuge at 12000g and 4°C for 15 min, and take the supernatant; add 200 μL chloroform to the supernatant, centrifuge, and take the supernatant; add 1 / 10 volume of sodium acetate solution (3M, pH 5.2) Add 2.5 times the volume of ethanol to the supernatant and place at -80℃ for 2.5h; centrifuge at 12000g and 4℃ for 30min, discard the supernatant, add 1mL of 75% ethanol solution, and resuspend the precipitate; centrifuge at 10000g and 4℃ for 10min, discard the supernatant, wait for the remaining ethanol in the tube to dry, and add 200μL of enzyme-free water to dissolve, and obtain the target product ds BtI 、ds BtR The nucleotide sequences are shown in SEQ ID NOs.7~8 respectively.

[0048] At the same time, the dsRNA (ds GFP ) was used as a control group for subsequent RNA interference (RNAi) experiments.

[0049] 2. RNAi

[0050] ds BtI 、ds BtR and ds GFP Dissolved in 20% (0.2 mg / L) sucrose water respectively, so that the final concentration of each dsRNA is 250 ng / µL, to obtain feeding solution; added the feeding solution into a feeding device, which is made of a 50 mL centrifuge tube, the four sides and the bottom of the tube are closed by an insect-proof net, and the top is sealed by a centrifuge tube cap; separated the feeding solution from about 500 female whiteflies of the QS population by a polytetrafluoroethylene membrane, the mouthparts of the whitefly can pierce the polytetrafluoroethylene membrane and absorb the feeding solution on the inside of the membrane; after feeding for 72 hours, the surviving whiteflies were collected for subsequent experiments.

[0051] 3. qPCR

[0052] Detection of live whiteflies by qPCR BtI , BtR The specific steps are as follows:

[0053] Total RNA from surviving whiteflies after RNAi was extracted using Trizol reagent, and then reverse transcribed using PrimeScript™ RTreagent Kit with gDNA eraser. Reverse transcription was performed according to the kit instructions to synthesize first-strand cDNA. Succinate dehydrogenase complex A (SDHA) and heat shock protein 40 (HSP40) were used as two internal reference genes for mRNA expression analysis. Each treatment was repeated three times, using 2 -ΔΔCt Relative quantitative analysis of RNAi in Bemisia tabaci BtI , BtR The relative expression of

[0054] The qPCR reaction system (20 μL) was as follows: TB Green Advantage Premix 10 μL, Primer-F 0.5 μL, Primer-R 0.5 μL, cDNA 2 μL, ddH 2 O 7 μL.

[0055] The qPCR amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 60°C for 34 s, and 40 cycles.

[0056] BtI , BtR The qPCR detection results of genes are as follows Figure 3 As shown. Figure 3 Available, when taking ds BtI After 72 hours, the whitefly BtI The expression level of BtR There was no significant change in the expression level of BtR Afterwards, the whitefly BtR The expression level of BtI There was no significant change in the expression level.

[0057] The above results showed that the target gene dsRNA was successfully knocked down in whiteflies after feeding for 72 hours. BtI , BtR expression level.

[0058] Example 3: Verification BtI , BtR Effects of knockdown on JH synthesis, expression of key genes in the JH signaling pathway, and reproductive capacity in Bemisia tabaci

[0059] 1. JH titer

[0060] ELISA method was used to determine BtI , BtRThe juvenile hormone content (JH titer) in whiteflies after being knocked down, the specific steps are as follows:

[0061] 100 female whiteflies after RNAi were taken, 50 μL PBS buffer (0.01M, pH=7.4) was added, and the mixture was fully ground. The mixture was centrifuged at 3000 g and 4°C for 10 min, and the supernatant was taken as the test sample. According to the ELISA kit operating instructions, 50 μL of the test sample and 100 μL of horseradish peroxidase (HRP)-labeled antibody were added to the ELISA plate, incubated at 37°C for 60 min, and the liquid was discarded. The plate was washed 5 times with washing solution, 50 μL of substrate A and substrate B were added to the ELISA plate, incubated at 37°C for 15 min, and the reaction was terminated by adding the stop solution to obtain the reaction solution. The absorbance value (OD) of the reaction solution at a wavelength of 450 nm was measured using an Infinite 200 Pro ELISA reader. 450 ). The juvenile hormone content-absorbance value standard curve was drawn using the juvenile hormone standard, and the JH titer in the reaction solution was calculated. Three biological replicates were set for each treatment group.

[0062] The results of JH titer determination are as follows Figure 4 As shown. Figure 4 Available, when BtI , BtR After being knocked down, the JH titer of female whiteflies decreased significantly. Specifically, compared with the control group (0.73 pg / female), when feeding on ds BtI After 72 hours, the JH titer of female whiteflies decreased to 0.41 pg / female; BtR After 72 hours, the JH titer of female whiteflies decreased to 0.45 pg / female. BtI , BtR The reduction of expression level significantly inhibited the biosynthesis of JH in female whiteflies.

[0063] 2. Expression levels of key genes in the JH signaling pathway

[0064] In order to verify the role of the insulin signaling pathway in the JH signaling pathway, this experiment selected two key genes in the JH signaling pathway of Bemisia tabaci. Bt and BtKr-h1 , detected by qPCR BtI , BtR After being knocked down, Bt and BtKr-h1 The change of relative expression level in female whiteflies, the specific determination method is as shown in Example 2. Bt and BtKr-h1 The nucleotide sequences are shown in SEQ ID NOs.9~10 respectively.

[0065] qPCR test results Figure 5 As shown. Figure 5 Available, when BtI , BtR After being knocked down, the female whitefly Bt , BtKr-h1 The expression level was significantly decreased. Specifically, compared with the control group, the knockdown BtI back, Bt , BtKr-h1 The expression levels of BtR back, Bt , BtKr-h1 The expression levels of α and β were significantly reduced to 0.54 and 0.60 times, respectively. BtI , BtR The reduction in expression level can inhibit the expression of key genes in the JH signaling pathway by interfering with the synthesis of JH.

[0066] 3. Ovarian Development

[0067] The ovaries of insects are composed of ovarioles, which have the functions of storing and proliferating cells and absorbing necessary nutrients for reproductive cells to grow and develop to the mature stage. Studying the structure of the insect reproductive system can intuitively demonstrate its physiological adaptation mechanism. Selected female QS whiteflies from the first eclosion population at 2 days old were subjected to RNAi according to the method in Example 2. After feeding ds BtI 、ds BtR and ds GFP After the 5th, 8th and 10th days, the ovaries of B. tabaci at different developmental stages were dissected in PBS buffer (0.01M, pH=7.4), and the number of ovarian tubes was recorded using an SN-60R4K ultra-high-definition camera to compare the development of the ovaries of B. tabaci at different stages and under different treatments.

[0068] Ovarian development Figure 6 As shown. Figure 6 Compared with the control group, the BtI 、ds BtR After the 5th, 8th and 10th days, the ovarian growth of female whiteflies was significantly restricted, the volume of the entire ovary was reduced, and the yolk deposition in the mature ovarian tube was reduced.

[0069] The results of ovarian tube count are as follows Figure 7 As shown. Figure 7 Compared with the control group, the BtI 、ds BtR After the 5th and 8th days, the number of ovarioles of female whiteflies decreased significantly.

[0070] The above results show that BtI , BtRThe reduction of expression level significantly inhibited the ovarian development of female whiteflies.

[0071] 4. Bemisia tabaci vitellogenin ( BqV ) and Bemisia tabaci vitellin receptor ( BqI ) expression level

[0072] Insect yolk protein receptor (VgR) is a key protein in the uptake and transport of vitellogenin (Vg), and plays a key role in insect vitellogenesis and oogenesis. BtI , BtR The effect on the reproductive capacity of Bemisia tabaci was studied by selecting 2-day-old female QS population Bemisia tabaci and performing RNAi according to the method in Example 2. BtI 、ds BtR and ds GFP After 72 hours, the BqV and BqI The change of relative expression level in Bemisia tabaci, the specific determination method is referred to Example 2. BqV and BqI The nucleotide sequences are shown in SEQ ID NOs.11~12 respectively.

[0073] qPCR test results Figure 8 As shown. Figure 8 Available, when BtI , BtR After being knocked down, the female whitefly BqV , BqI The expression level was significantly decreased. Specifically, compared with the control group, the knockdown BtI back, BqV , BqI The expression levels of BtR back, BqV , BqI The expression levels of α and β were significantly reduced to 0.55 and 0.59 times, respectively. BtI , BtR The reduction in expression levels will inhibit the expression of key genes for yolk and oogenesis in female whiteflies.

[0074] 5. Egg laying

[0075] Two-day-old females of the QS population of Bemisia tabaci were selected for RNAi according to the method in Example 2. BtI 、ds BtR and ds GFPAfter 72 hours, unmated females were selected and paired with male whiteflies from the QS population. After pairing, the females were raised alone in a hydroponic cup with cotton seedlings. Their survival status was checked every day, and their egg production was recorded every five days until the females died.

[0076] The results are as follows Fig. 9 As shown. Fig. 9 Compared with the average egg laying amount of single female insects in the control group (86±5 eggs / female insect), the knockdown BtI After knockdown, the egg laying rate of single female insects was significantly reduced to 70±5 eggs per female insect. BtR After that, the number of eggs laid by a single female insect decreased significantly to 54±8 eggs / female insect. BtI , BtR The reduction of expression level significantly inhibited the egg laying of whitefly.

Claims

1. A use of dsRNA in inhibiting the reproductive ability of Bemisia tabaci, characterized in that: The dsRNA is ds BtI 、ds Bt One or more of; wherein, the ds BtI The nucleotide sequence of ds Bt The nucleotide sequence is shown in SEQ ID NO.8, and the whitefly is a female whitefly.

2. The use according to claim 1, characterized in that The application method is to feed ds BtI or ds Bt , inhibiting the reproductive capacity of female whiteflies.

3. The use according to claim 2, characterized in that The method of inhibiting the reproductive capacity of female whiteflies includes: inhibiting the ovarian development of female whiteflies and reducing the egg-laying amount of female whiteflies.

4. The use according to claim 2, characterized in that The feeding ds BtI or ds Bt The method is: use a mixture containing ds BtI or ds Bt Whiteflies were fed with sucrose water.

5. The use according to claim 4, characterized in that The concentration of sucrose in the sucrose water is 0.17-0.23 mg / L.

6. The use according to claim 4, characterized in that The concentration of sucrose in the sucrose water is 0.2 mg / L.

7. The use according to claim 4, characterized in that The ds BtI or ds Bt The concentration in sucrose water is 250ng / µL.

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