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Application of nilaparvata lugens NLSP7 as target in prevention and control of nilaparvata lugens

A brown planthopper and target technology, applied in the biological field, can solve problems in a specific period or in a specific tissue, and achieve the effect of increasing the content

Active Publication Date: 2021-09-03
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But on the other hand, the use of RNA interference to study salivary proteins also has certain limitations. For example, certain genes of some insects only appear in specific ages, periods or tissues of insects; such as Manduca sextai and Bombyx mori mori) are difficult to be silenced in biological tissues other than hemolymph (Eleftherianos et al., 2007; Miller et al., 2008; Huang Xiaohui, 2016); on the other hand, some salivary protein genes are an indispensable part of the normal development of insects , if the RNA interference of these genes will cause the insects to die quickly, it is difficult to conduct subsequent experiments on the role of the genes in insect feeding

Method used

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  • Application of nilaparvata lugens NLSP7 as target in prevention and control of nilaparvata lugens
  • Application of nilaparvata lugens NLSP7 as target in prevention and control of nilaparvata lugens
  • Application of nilaparvata lugens NLSP7 as target in prevention and control of nilaparvata lugens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Cloning of embodiment 1 brown planthopper NLSP7 gene

[0055] Take 30 brown planthoppers fed with RH rice for multiple generations, and extract total RNA. The total RNA of the female adults of brown planthoppers was extracted using the Trizol method. The specific operations are as follows:

[0056] 1) Put the N. lugens samples into grinding tubes, freeze them rapidly in liquid nitrogen, add 100 μL Trizol reagent and grind them thoroughly on ice immediately.

[0057] 2) Add 900 μL of Trizol reagent, pipette and wash the remaining N. lugens tissue on the grinding rod, mix well, and let stand at room temperature for 5 minutes.

[0058] 3) Add 200 μL Trichloromethane (trichloromethane), shake rapidly for 15 seconds, and let stand at room temperature for 20 minutes.

[0059] 4) Centrifuge at 12,222 g for 15 min at 4°C.

[0060] 5) Aspirate the supernatant, add 500 μL Dimethylcarbinol (isopropanol), shake rapidly for 15 seconds, and let stand at room temperature for 20 minu...

Embodiment 2

[0077] Example 2 NLSP7 gene in different tissues and different ages in brown planthopper

[0078] Six different tissues (including fat body, head, foot, midgut, ovary, and salivary gland) of brachypterous female adults of N. lugens subcultured on rice variety RH were dissected under a body microscope using tweezers and insect needles in a clean environment , each treatment was repeated 3 times. Extract the total RNA of each tissue respectively, reverse transcribe after being the cDNA of the first strand (the specific method is the same as embodiment 1), use the quantitative reagent of Roche Company to carry out the fluorescent quantitative PCR technique to detect, the components of the fluorescent quantitative PCR reaction solution are as follows: Table 3 shows.

[0079] Table 3 Components of Fluorescent Quantitative PCR Reaction Solution

[0080]

[0081] The above reaction solution was dispensed into 3 wells of a 384-well plate, about 10 μL in each well.

[0082] PCR r...

Embodiment 3

[0091] The preparation of embodiment 3dsRNA

[0092] 1) Use the complete cDNA sequence obtained by inversion in Example 1 as a template, and NLSP7-F2 and NLSP7-R2 as primers for PCR amplification. The PCR amplification system is shown in Table 4. PCR reaction procedure: ① Pre-denaturation at 95°C for 5 minutes ; ② 35 cycles of 95°C for 30s, 57°C for 30s, and 72°C for 40s; ③ 72°C extension for 10 minutes; the final PCR amplification product 1 was stored in a 4°C refrigerator.

[0093] Table 4 PCR reaction system

[0094]

[0095] NLSP7-F2: ATGAGGGCTGCCCTGATT (SEQ ID NO. 4);

[0096] NLSP7-R2: TAGACAACCTGTGGTCCA (SEQ ID NO. 5);

[0097] Wherein, the underlined region is the T7 RNA polymerase promoter sequence.

[0098] 2) Use the plasmid containing green fluorescent protein GFP (GenBank: ACY56286) as a template, GFP-F, GFP-R as primers to perform PCR amplification to obtain PCR amplification product 2. The PCR reaction system is as shown in Table 5. The PCR reaction Proce...

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Abstract

The invention relates to the biotechnology field, and discloses an application of nilaparvata lugens NLSP7 as a target in prevention of control of nilaparvata lugens. The invention discloses the application of the nilaparvata lugens NLSP7 as the target in prevention and control of the nilaparvata lugens for the first time. By silencing the NLSP7 gene, trophic behavior and food intake of the nilaparvata lugens can be influenced, thereby allowing the nilaparvata lugens to generate a food refusal effect, and thus achieving the purpose of prevention and control of nilaparvata lugens. The invention discloses the application of the nilaparvata lugens NLSP7 as the target in increasing tricin content in rice for the first time. The tricin content in the rice (especially rice with high tricin content) can be increased by silencing the NLSP7 gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of brown planthopper NLSP7 as a target in the control of brown planthopper. Background technique [0002] Brown planthopper (Nilaparvata lugens (Stal)) belongs to the family Hemiptera (Hemiptera: Delphacidae). It is a rice pest that can migrate long distances and has strong adaptability to the environment. It is currently endangering my country and many Southeast Asian countries. The primary pest of rice in countries such as Sogawa (Sogawa, 1982; Velusamy et al., 1986). The brown planthopper has a single choice of host plants, and only feeds and lays eggs on rice or wild rice (Dyck et al., 1979; Hong Xiaoyue et al., 2007; Wang et al., 2008). The brown planthopper seriously affects the growth and development of rice mainly by sucking the phloem juice of the host rice. The salivary sheath formed when the brown planthopper feeds will block the vascular bundle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01N63/60A01P7/04A01H5/00A01H6/46
CPCC12N15/113C12N15/8243C12N15/8286A01N63/60C12N2310/14
Inventor 张振飞袁龙宇巩固
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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