Method for preventing and controlling bemisia tabaci by inhibiting ilvE gene of symbiotic bacteria

Abstract

The invention provides a method for preventing and controlling bemisia tabaci by inhibiting an ilvE gene of symbiotic bacteria, and belongs to the technical field of genes. According to the invention, the ilvE gene translation initiation region antisense PNA sequence is designed in a targeted manner and is connected with the cell penetrating peptide, and the ilvE gene translation initiation region antisense PNA sequence is injected into the bemisia tabaci body, so that the level of branched chain amino acid of the bemisia tabaci can be obviously reduced, and an efficient and green prevention and control method is provided for inhibiting the growth and reproduction of the bemisia tabaci.

Description

Technical Field

[0001] The present invention relates to the field of gene technology, and in particular to a method for controlling whitefly by inhibiting the ilvE gene of symbiotic bacteria. Background Art

[0002] Bemisia tabaci is an important pest that harms global agriculture. It causes serious economic losses by spreading plant viruses and directly feeding on plants. Insects and their symbiotic bacteria are interdependent. Insects provide nutrients and energy sources for symbiotic bacteria, and symbiotic bacteria provide insects with essential nutrients, help insects digest food, participate in nitrogen cycle, and maintain a normal physiological environment. There is also complementarity in nutrient metabolism pathways between symbiotic bacteria and insects, which provides impetus for the co-evolution of the two. The physiological functions of whiteflies are also highly dependent on their symbiotic bacteria. The symbiotic bacteria in whiteflies include Portiera, Hamiltonella, Wolbachia, Cardinium, Rickettsia, Fritschea, and Arsenophonus. Studies have shown that the symbiotic bacteria Portiera in whiteflies has lost the gene encoding the synthesis of branched-chain amino acid terminal transaminase (ilvE). The loss of key biosynthetic steps by symbiotic bacteria may help the host control nutrient production and symbiotic bacteria growth, thereby facilitating the survival and reproduction of whiteflies.

[0003] At present, chemical pesticides are mainly used to control whiteflies, but chemical pesticides have no selectivity for symbiotic bacteria, which can easily lead to drug resistance and ecological and environmental risks. Gene silencing technology can accurately intervene in the symbiotic bacteria of whiteflies, directly inhibiting the growth and reproduction of whiteflies, which is of great significance for achieving green control.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an application of an ilvE gene in controlling Bemisia tabaci, by silencing the ilvE gene of the symbiotic bacteria of Bemisia tabaci, thereby inhibiting the synthesis of branched-chain amino acids of Bemisia tabaci, thereby inhibiting the growth and reproduction of Bemisia tabaci.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides an application of an ilvE gene in preventing and controlling Bemisia tabaci. The growth and reproduction of Bemisia tabaci can be inhibited by silencing the ilvE gene.

[0008] Preferably, the ilvE gene is the ilvE gene in Rickettsia, a symbiotic bacterium of whiteflies.

[0009] Preferably, the nucleotide sequence of the ilvE gene is as shown in SEQ ID NO:1.

[0010] Preferably, the silencing of the ilvE gene can reduce the level of branched-chain amino acids in Bemisia tabaci.

[0011] Preferably, the silencing of the ilvE gene can reduce the synthesis of the ilvE enzyme in the symbiotic bacterium Rickettsia.

[0012] Preferably, the PNA sequence for silencing the ilvE gene is shown as SEQ ID NO:2.

[0013] The present invention also provides a method for silencing the ilvE gene in the above application, comprising the following steps: (a) designing a PNA sequence, wherein the PNA sequence is complementary to the base region from -6 to +6 of the translation start codon of the ilvE gene of Rickettsia;

[0014] (b) connecting a cell-penetrating peptide to the N-terminus of the PNA to obtain PNA_ilvE;

[0015] (c) dissolving the PNA_ilvE in a solution containing CaCl 2 In an enzyme-free aqueous solution, a PNA_ilvE solution is prepared;

[0016] (d) injecting the PNA_ilvE solution into female cryptic species MED of Bemisia tabaci to silence the expression of the ilvE gene.

[0017] Preferably, the cell penetrating peptide in step (b) is an arginine-rich cell penetrating peptide RXRRXRRXRRXRXB, wherein R is arginine, X is 6-aminohexanoic acid, and B is β-alanine.

[0018] Preferably, the concentration of the PNA_ilvE solution in step (c) is 18-22 μM.

[0019] Preferably, the expression level of ilvE and the level of branched-chain amino acids in whitefly can be reduced by injecting PNA_ilvE solution.

[0020] The silencing efficiency of the present invention is high. After one day of injection, the expression of ilvE can be significantly reduced by 78.8%, and the levels of leucine, isoleucine and valine are significantly reduced. The antisense PNAs designed by the present invention have no other off-target genes, ensuring the specificity of gene silencing and avoiding non-target interference with the host or other symbiotic bacteria. The present invention targets the metabolic pathway of symbiotic bacteria and reduces the impact on non-target organisms and the ecological environment. In addition, the CPP design optimizes the delivery efficiency while maintaining low cytotoxicity, avoiding the induction of host immune response or imbalance of the symbiotic system, and realizing green control of whiteflies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The PNA_ilvE and PNA_mm sequences designed in Example 1 (wherein R is arginine, X is 6-aminohexanoic acid, B is β-alanine, and the bases marked in red are changed to be mismatched with the original complementary bases of the gene ilvE);

[0022] Figure 2 This is a graph showing the effect of silencing ilvE on the expression level of ilvE in Example 2;

[0023] Figure 3 This is a graph showing the effect of silencing ilvE on BCAAs levels in Example 3. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] The ilvE gene is shown in SEQ ID NO: 1:

[0026]

[0027] Example 1

[0028] Design an antisense PNA sequence SEQ ID NO: 2: TGTCATTGTTAT that is complementary to the -6 to +6 base region of the translation start codon (ATG) of the ilvE gene of Rickettsia. Attach an arginine-rich cell-penetrating peptide (CPP) (RXR) to the N-terminus of the PNA 4 XB, PNA_ilvE was obtained, two bases in the ilvE gene were changed to make them mismatched with the original complementary bases of the ilvE gene, and PNA_mm was obtained as a control group (SEQ ID NO: 3: TGTGATTGTTTT). The designed PNA_ilvE and PNA_mm sequences are shown in Figure 1 As shown, it was synthesized and purified by PANAGENE Inc. (Daejeon, Korea).

[0029] Dissolve PNA-ilvE and PNA-mm in 100 μL of enzyme-free water to prepare a 200 μM stock solution, and store it in a -20°C refrigerator for later use. Since calcium ions can enhance the penetration of PNA into cells, PNA_ilvE and PNA_mm were diluted with 12 mM CaCl 2 Dilute to 20 μM in enzyme-free aqueous solution.

[0030] In order to further clarify the function of Rickettsia gene ilvE, PNA technology was used to silence ilvE. After completing the sequence design of PNA for silencing ilvE. Whole genome searches of Portiera, Hamiltonella and Rickettsia showed that the 12bp PNA_ilvE was in the translation start codon or Shine-Dalgarno region (SD, ribosome binding site sequence in messenger RNA in bacteria and archaea), and no other off-target genes matching its sequence were found except the target gene ilvE.

[0031] Example 2 Effect of silencing ilvE on ilvE expression

[0032] The female cryptic species MED of Bemisia tabaci 1 day after emergence was injected with 12 mM CaCl prepared in Example 1. 2PNA_ilvE and PNA_mm were diluted to 20 μM in an enzyme-free aqueous solution. Samples were collected on the first day after the injection of 20 μM PNA_mm and PNA_ilvE. Ten female insects were used as a biological replicate to detect changes in ilvE expression. The reaction conditions were: first, 95°C for 30 s; second, 95°C for 5 s, 60°C for 30 s, and 40 cycles; finally, a melting curve was added, 95°C for 10 s, and 65°C for 5 s. The primers are shown in Table 1; the reaction system is shown in Table 2 below, and the results are shown in Figure 2 shown.

[0033] Table 1 qRT-PCR primers for detection

[0034]

[0035] Table 2 Reaction system

[0036] TBGreenPremixExTaqII(TliRNaseHPlus) 10μL Upstream primer (10 μM) 0.8μL Downstream primer (10 μM) 0.8μL cDNA 2μL <![CDATA[ddH 2 The]]> 6.4μL

[0037] Depend on Figure 2 It can be seen that the change in ilvE expression was detected on the first day after the injection of 20 μM PNA_mm and PNA_ilvE. The results showed that on the first day after the injection of PNAs, the expression of ilvE was significantly reduced by 78.8% ( Figure 2 , P=0.00096<0.001), it can be seen that the method of the present invention significantly and effectively silenced the ilvE gene and significantly reduced the expression level of the ilvE gene.

[0038] Example 3 Effect of silencing ilvE on BCAAs

[0039] The female cryptic species MED of Bemisia tabaci 1 day after emergence was injected with 12 mM CaCl prepared in Example 1. 2 PNA-ilvE and PNA-mm were diluted to 20 μM in an enzyme-free aqueous solution. Samples were collected on the first day after the injection of 20 μM PNA_mm and PNA_ilvE. Each biological replicate consisted of 50 female insects. Amino acids were extracted and BCAAs were analyzed. Figure 3 shown.

[0040] The method for extracting amino acids is as follows: take the samples to be tested, 50 heads in a group, transfer them to a centrifuge tube, add 3 to 5 grinding beads, add 50 μL 1×PBS buffer and grind for 1 minute; centrifuge at 18000×g at 4°C for 3 minutes; take 39 μL of supernatant and add 11 μL 40 mmol / L HCl solution, incubate on ice for 30 minutes; centrifuge at 18000×g at 4°C for 15 minutes, take 20 μL of supernatant and store it in a -80°C refrigerator for protein determination; filter the remaining supernatant through a 0.45 μm filter plate (Millipore), and store it in a -80°C refrigerator for amino acid determination.

[0041] Amino acid content (pmol / μg) = total amount of substance (pmol) / protein content (μg)

[0042] Depend on Figure 3 It can be seen that compared with the control injected with PNA_mm, the levels of leucine, isoleucine and valine decreased by 33.8%, 37.4% and 30.4% respectively after injection of PNA_ilvE (P=0.006, 0.031 and 0.011 all <0.05).

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of the ilvE gene in controlling Bemisia tabaci, characterized in that: By silencing the ilvE gene, the growth and reproduction of whiteflies can be inhibited.

2. The use according to claim 1, characterized in that The ilvE gene is the ilvE gene in the Rickettsia symbiotic bacteria of whitefly.

3. The use according to claim 2, characterized in that The nucleotide sequence of the ilvE gene is shown in SEQ ID NO:

1.

4. The use according to claim 2 or 3, characterized in that The silencing of the ilvE gene can reduce the level of branched-chain amino acids in Bemisia tabaci.

5. The use according to claim 2 or 3, characterized in that: The silencing of the ilvE gene can reduce the synthesis of the ilvE enzyme in the symbiotic bacterium Rickettsia.

6. The use according to claim 1, characterized in that The PNA sequence for silencing the ilvE gene is shown in SEQ ID NO:

2.

7. A method for silencing the ilvE gene for use according to any one of claims 1 to 6, characterized in that: The following steps are involved: (a) designing a PNA sequence that is complementary to the -6 to +6 base region of the translation start codon of the ilvE gene of Rickettsia; (b) connecting a cell-penetrating peptide to the N-terminus of the PNA to obtain PNA_ilvE; (c) dissolving the PNA_ilvE in an enzyme-free aqueous solution containing CaCl2 to prepare a PNA_ilvE solution; (d) injecting the PNA_ilvE solution into female cryptic species MED of Bemisia tabaci to silence the expression of the ilvE gene.

8. The method according to claim 7, characterized in that The cell penetrating peptide in step (b) is an arginine-rich cell penetrating peptide RXRRXRRXRRXRXB, wherein R is arginine, X is 6-aminohexanoic acid, and B is β-alanine.

9. The method according to claim 8, characterized in that The concentration of the PNA_ilvE solution in step (c) is 18-22 μM.

10. The method according to claim 9, characterized in that The expression level of ilvE and the level of branched-chain amino acids in whiteflies can be reduced by injecting PNA_ilvE solution.

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