CN108707603B

CN108707603B - A kind of ESCRT-III core subunit Snf7 double-stranded RNA and preparation method and application thereof

Info

Publication number: CN108707603B
Application number: CN201810332093.6A
Authority: CN
Inventors: 张文庆; 黎丹
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2018-04-13
Filing date: 2018-04-13
Publication date: 2021-07-20
Anticipated expiration: 2038-04-13
Also published as: CN108707603A

Abstract

The present invention provides an ESCRT-III core subunit Snf7 double-stranded RNA, the nucleotide sequence of the sense strand of the transcription template is shown in SEQ ID NO: 1.

Description

ESCRT-III core subunit Snf7 double-stranded RNA and preparation method and application thereof

Technical Field

The invention belongs to the field of insect control, and particularly relates to double-stranded RNA (dsRNA) of a transport essential element sorting compound III core subunit Snf7, a preparation method and application of the dsRNA.

Background

ESCR-III, known as transport essential complex III (the endosomal absorbed complex for transport-III), is composed primarily of the four "core" subunits Vps20, Snf7/Vps32, Vps24 and Vps2, and is a key component in eukaryotic cells to accomplish endosomal membrane invagination to form multivesicular bodies (MVBs). The primary function of ESCR-III is to recruit de-ubiquitinated (de-ubiquitin) enzymes, facilitating degradation of membrane proteins labeled with ubiquitin (ubiquitin). In addition to being involved in MVB formation, ESCT-III is also involved in cellular vital activities such as internalization, transport, sorting, autophagy and cell division of cell transmembrane proteins, which are closely related to MVB formation (Babst et al, 2002; Res et al, 2007; Capalbo et al, 2012; Tang et al, 2015). Therefore, ESCT-III plays an important role in the growth and development of all eukaryotes.

Snf7 belongs to a family of evolutionarily conserved proteins, consisting of 240 amino acids in yeast, Shrb (Shrub) as the homolog in Drosophila, and CHMP4 as the homolog in human, which is present in any species other than archaea (Babst et al, 2002; McMillan et al, 2016). Snf7 is a soluble coiled-coil protein comprising a highly structured "tetranuclear"A helical domain. The functions of Snf7 include: 1) autophagy; 2) interaction with Deubiquitinase (DUB) recruits DUB to the ESCRT pathway; 3) multivesicular endosomal vesicles (ILV) form MVBs; 4) cell division (Lata et al, 2008; obita et al, 2007; ramaseshadri et al, 2013;

etc., 2014).

Although Snf7 is an essential protein in most biological life activities, it has been studied less in insects, among which drosophila and Western Corn Rootworm (WCR) are the most studied. Sweeney et al discovered that Shrb plays an important role in the neural morphogenesis of insects through studies on Drosophila Shrub (i.e., Drosophila Snf7) mutants (Sweeney et al, 2006). Baum et al found that very low doses of dsDvSnf7 resulted in WCR lethality through RNAi experiments with Western corn rootworm (Baum et al, 2007). Subsequently, bolgnesi et al interfered with the expression of Snf7 gene in WCR larvae by RNAi technique and found that the experimental group larvae were significantly hindered from growing compared to the control group and that feeding the larvae at doses of 50ng/ml and 1000ng/ml for 24h resulted in 90.3% and 94.6% lethality, respectively (bolgnesi et al, 2012). After feeding dsDvSnf7 to corn rootworm, ubiquitinated protein was found to accumulate in larval tissues, preventing cell autophagy of midgut and fat bodies leading to larval death (Ramaseshadri et al, 2013).

Currently, there are many studies on insect insecticides, but the major focus is on insect-specific genes such as Ecdysone receptor (EcR) and chitin synthase (CHS) (Wu et al, 2012; Wang et al, 2012). The main advantage of these insecticides is that they are a broad spectrum insecticide against most insects, but they are relatively safe and have limited killing efficiency against insects, which greatly limits the use of insect-specific genetic insecticides. Since Snf7 plays a crucial role in the growth and development of insects, researchers have designed specific fragments to obtain efficient and safe narrow-spectrum pesticide only aiming at western corn rootworm. Through specific fragment design of the housekeeping gene of the brown planthopper Snf7, the screening method is helpful for screening new high-efficiency safe narrow-spectrum insecticides against the brown planthopper.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the high-efficiency safe insecticidal dsRNA with remarkable effect on controlling brown planthopper.

The invention also aims to provide a preparation method of the high-efficiency and safe insecticidal dsRNA.

Still another object of the invention is to provide an application of the high-efficiency and safe insecticidal dsRNA in preparation of drugs for controlling brown planthopper.

Still another object of the present invention is to provide a method for controlling brown planthopper using the highly efficient and safe insecticidal dsRNA.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides ESCRT-III core subunit Snf7 double-stranded RNA, wherein the nucleotide sequence of the sense strand of the transcription template is shown as SEQ ID NO: 1 is shown.

The invention also provides a preparation method of the ESCRT-III core subunit Snf7 double-stranded RNA, which comprises the following steps:

1) extracting total RNA of brown planthopper: grinding imagoes in liquid nitrogen, and extracting total RNA by a TRIzol method;

2) first strand cDNA Synthesis: reverse transcribing 1. mu.g of total RNA to cDNA;

3) preparing an intermediate fragment of a brown planthopper Snf7 homologous gene Nl020727 gene, and preparing a vector containing the intermediate fragment;

4) and 3) taking the vector obtained in the step 3) as a template, and amplifying to obtain a target fragment, namely the ESCRT-III core subunit Snf7 double-stranded RNA.

Specifically, the specific operation of step 3) is as follows:

step 1, polymerase chain reaction: the reagent comprises pEASY-020727 plasmid DNA1.5 mu L, 10 mu M of the Nl020727 gene specific upstream primer Nl 020727-F2 mu L, 10 mu M of the Nl020727 gene specific downstream primer Nl 020727-R2 mu L, 2 x Phanta Max Buffer 25 mu L, dNTP mix 1 mu L, Phanta Max Super-Fidelity DNA Ploymerase 1 mu L and double distilled water 17.5 mu L; mixing the reagents to obtain a mixed solution, wherein the total volume is 50 mu L; the polymerase chain reaction comprises the following specific steps: the mixture was pre-denatured at 95 ℃ for 3min, then entered the following cycle: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 50s for 30 cycles, and final extension at 72 ℃ for 7 min; wherein the sequence of an upstream primer Nl020727-F for amplifying the Nl020727 gene specific fragment is as follows: 5'-CCCTTTCACTACTACTCATCTTCA-3', respectively; the sequence of the downstream primer Nl020727-R is as follows: 5'-GTTCTTCTTCTTCTTTTTCTTCCT-3', respectively;

step 2, amplification product purification: purifying the PCR amplified fragment by using a magenta gel recovery kit to obtain a purified product;

and 3, obtaining an intermediate carrier: cloning the purified product to a pEASY-Blunt Zero vector according to a ligation reaction system, transforming to Trans1-T1 competent cells, and culturing overnight on an LB plate containing 100ug/mL ampicillin to obtain a colony; the connection reaction system is as follows: the purified product was 4. mu.L, pEASY-Blunt Zero 1. mu.L, in a total volume of 5. mu.L;

and 4, plasmid purification: and (3) inoculating the bacterial colony to an LB liquid culture medium, carrying out shaking table overnight culture at 37 ℃, collecting bacterial liquid, and extracting plasmids to obtain the plasmids containing the Nl020727 gene.

Specifically, the specific operation of step 4) is as follows:

step a, designing the following specific primers:

dsNl020727-F：5’-AATGAAGGGAAGAGAGATCGTGCCAAA-3’

dsNl020727-R：5’-TACAGCCTCATCATCCTCAGCAGTCA-3’

dsNl020727-T7F：

5’-GGATCCTAATACGACTCACTATAGGAATGAAGGGAAGAGAGATCGTGCCAAA-3’

dsNl020727-T7R：

5’-GGATCCTAATACGACTCACTATAGGTACAGCCTCATCATCCTCAGCAGTCA-3’

and b, using a vector containing the intermediate fragment of the Nl020727 gene as a template, and respectively amplifying by using the specific primers in the step a according to the following reaction conditions to obtain a target fragment:

a reaction system is as follows:

b, the reaction system is as follows:

wherein, the PCR instrument amplification program comprises: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 35s for 30 cycles; further extension for 10min at 72 ℃;

wherein, double-stranded RNA corresponding to the Nl020727 gene is synthesized by a dsRNA synthesis kit.

The invention also provides a pesticide which comprises the ESCRT-III core subunit Snf7 double-stranded RNA as an active ingredient.

The invention also provides application of the ESCRT-III core subunit Snf7 double-stranded RNA in preparation of a medicament for preventing and treating brown planthopper.

The invention also provides a method for preventing and treating brown planthopper by using the ESCRT-III core subunit Snf7 double-stranded RNA, which comprises the following steps: the ESCRT-III core subunit Snf7 type double-stranded RNA is used for injecting brown planthopper.

Preferably, the final concentration of the ESCRT-III core subunit Snf7 double-stranded RNA injected is 0.025 μ g/μ L to 0.2 μ g/μ L.

The existing main methods for controlling the brown planthopper comprise agricultural control, chemical control and biological control, and the control work of the brown planthopper is difficult to develop continuously due to the resistance effect brought by chemical drugs, environmental pollution and other problems. The ESCRT-III core subunit Snf7 double-stranded RNA (dsRNA) is adopted, and a transcription template of the double-stranded RNA is a brown planthopper Snf7 homologous gene (Nl 020727). According to the invention, double-stranded RNA is obtained by screening the conserved gene of the brown planthopper Snf7, and the double-stranded RNA is used for controlling the brown planthopper, so that the normal development and reproduction of the brown planthopper can be effectively inhibited, the control effect is obvious, and the method does not generate drug resistance and environmental pollution, and is safe and environment-friendly.

Drawings

FIG. 1 is a graph showing the relative expression level changes of mRNA of brown planthopper after dsNl020727 injection.

FIG. 2 is a graph of the corrected mortality change for brown planthopper after dsNl020727 injection.

FIG. 3 is a graph of protein changes of brown planthopper after injection of dsNl 020727.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

dsRNA was prepared according to the following steps:

1. selecting brown planthopper adults for 1 day

2. Extraction of total RNA: taking 1 adult, grinding in liquid nitrogen, and extracting total RNA by a TRIzol method.

Synthesis of first strand cDNA: mu.g of total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit from Takara with the gDNA Eraser Kit instructions.

4. The procedure for preparing the intermediate fragment of Nl020727 is described below.

(1) Polymerase Chain Reaction (PCR) reagents (Vazyme)

Max Super-Fidelity DNA Polymerase) and reaction conditions:

the polymerase chain reaction comprises the following specific steps: the mixture was pre-denatured at 95 ℃ for 3min and then subjected to the following cycle: denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, and elongation at 72 ℃ for 50s for 30 cycles, and finally elongation at 72 ℃ for 7 min.

Wherein the sequence of an upstream primer Nl020727-F for amplifying the Nl020727 gene specific fragment is as follows: 5'-CCCTTTCACTACTACTCATCTTCA-3' (SEQ ID NO: 2); the sequence of the downstream primer Nl020727-R is as follows: 5'-GTTCTTCTTCTTCTTTTTCTTCCT-3' (SEQ ID NO: 3);

(2) and (3) purifying an amplification product: the PCR amplified fragment was purified using the magenta gel recovery kit.

(3) Obtaining an intermediate vector: the purified product was obtained according to the following ligation reaction system, intermediate support: cloning the purified product to pEASY-Blunt Zero vector (Beijing all-purpose gold Biotechnology Co., Ltd.) according to the following ligation reaction system, transforming to Trans1-T1 competent cells (Beijing all-purpose gold Biotechnology Co., Ltd.), and culturing on LB plate containing 100ug/mL ampicillin overnight;

the ligation reaction system is as follows:

purified product 4. mu.L

pEASY-Blunt Zero 1μL

Total volume 5. mu.L

(4) And (3) plasmid purification: selecting 5 bacterial plaques, carrying out colony PCR verification, selecting correct bacterial colonies, inoculating the bacterial colonies to an LB liquid culture medium, carrying out shaking table overnight culture at 37 ℃, collecting bacterial liquid, and extracting plasmids according to a magenta plasmid DNA extraction kit.

(5) Sequencing and homology detection: the nucleotide sequence of the Nl020727 gene is obtained by analyzing the sequencing result, and the length is 710bp (shown as SEQ ID NO: 12). The nucleotide sequence of ESCRT-III core subunit Snf7 double-stranded RNA is shown as SEQ ID NO: 1, 312bp in length.

The plasmid containing the Nl020727 gene was designated as pEASY-020727.

Example 2

(1) Specific primers with the T7 promoter were designed for in vitro synthesis of dsRNA for Nl020727, while dsGFP was synthesized as a control using primers for the green fluorescent protein gene.

dsNl020727-F：5’-AATGAAGGGAAGAGAGATCGTGCCAAA-3’(SEQ ID NO：4)

dsNl020727-R：5’-TACAGCCTCATCATCCTCAGCAGTCA-3’(SEQ ID NO：5)

dsNl020727-T7F：

5’-GGATCCTAATACGACTCACTATAGGAATGAAGGGAAGAGAGATCGTGCCAAA-3’

(SEQ ID NO：6)

dsNl020727-T7R：

5’-GGATCCTAATACGACTCACTATAGGTACAGCCTCATCATCCTCAGCAGTCA-3’(SEQ ID NO：7)

dsGFP-F：5’-AAGGGCGAGGAGCTGTTCACCG-3’(SEQ ID NO：8)

dsGFP-R：5’-CAGCAGGACCATGTGATCGCGC-3’(SEQ ID NO：9)

dsGFP-T7F：

5’-GGATCCTAATACGACTCACTATAGGAAGGGCGAGGAGCTGTTCACCG-3’(SEQ ID NO：10)

dsGFP-T7R：

5’-GGATCCTAATACGACTCACTATAGGCAGCAGGACCATGTGATCGCGC-3’(SEQ ID NO：11)

(2) Using the plasmid named pEASY-020727 obtained in example 1 as a template, the target fragment was amplified under the following reaction conditions using the primers designed in step 1.

A reaction system is as follows:

b, the reaction system is as follows:

PCR instrument amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 35s for 30 cycles; further extension for 10min at 72 ℃;

(1) synthesis of the kit by dsRNA (T7 RiboMAX)^TMExpress RNAi System, Promega, USA) synthesized dsRNA corresponding to Nl020727, named dsNl 020727.

(2) Brown planthopper was injected with dsNl020727 at final concentrations of 0.025 μ g/. mu.L, 0.05 μ g/. mu.L, 0.1 μ g/. mu.L and 0.2 μ g/. mu.L in four gradients with green fluorescent protein dsGFP as control.

(3) Collecting brown planthoppers of the female short-

wing adults

24, 48 and 72 hours after the injection in the step (2), and detecting the mRNA relative expression change of the Nl020727 gene by a fluorescent quantitative PCR method. As shown in FIG. 1, the mRNA expression levels were found to be significantly reduced after injection of dsNl020727 at concentrations of 0.025. mu.g/. mu.L to 0.2. mu.g/. mu.L. In addition, the mortality rate of the brown planthopper after injecting different doses of dsNl02072 is counted, and the results are shown in figure 2, the mortality rate of the brown planthopper is continuously increased within 13 days after injecting four doses of dsRNA, and the mortality rate at 13 days is as high as 70%, which indicates that the dsNl02072 prevents the normal development of the brown planthopper.

(4) Brown planthoppers 1 day and 5 days after 0.05 μ g/μ L dsNl020727 injection are collected, and the variation of the Nl020727 protein expression is detected by a Western blot method, and as a result, as shown in fig. 3, the expression level of the Nl020727 protein in the brown planthopper is found to be remarkably reduced along with the increase of time after the brown planthopper is injected with the dsNl 020727. The result shows that the dsNl020727 fragment is injected into brown planthopper to effectively interfere the expression of target gene and block the protein synthesis of Nl 020727.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention are intended to be covered by the protection scope of the present invention.

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Claims

1. An ESCRT-III core subunit Snf7 double-stranded RNA that interferes with Snf7 gene expression, wherein the nucleotide sequence of the sense strand of its transcription template is as shown in SEQ ID NO: 1.

2. the preparation method of ESCRT-III core subunit Snf7 class double-stranded RNA according to claim 1, is characterized in that comprising the following steps:

1) Extract the total RNA of N. lugens: take the adults, grind them in liquid nitrogen, and extract the total RNA by the TRIzol method;

2) Synthesize the first strand of cDNA: reverse transcribe 1 μg of total RNA into cDNA;

3) prepare the intermediate fragment of the N1020727 gene of the N. lugens Snf7 homologous gene, and prepare the vector containing the intermediate fragment;

4) using the vector obtained in step 3) as a template, amplify to obtain the target fragment, that is, the ESCRT-III core subunit Snf7 double-stranded RNA;

The concrete operation of described step 3) is as follows:

Step 1. Polymerase chain reaction: the reagents include 1.5 μL of cDNA, 2 μL of the N1020727 gene-specific upstream primer N1020727-F at 10 μM, 2 μL of the N1020727 gene-specific downstream primer N1020727-R at 10 μM, and 2×Phanta Max Buffer 25 μL, 1 μL of dNTP mix, 1 μL of Phanta Max Super-Fidelity DNA Ploymerase, and 17.5 μL of double-distilled water; the reagents were mixed to obtain a mixed solution, and the total volume was 50 μL; the specific steps of the polymerase chain reaction were as follows: The solution was pre-denatured at 95 °C for 3 min, and then entered the following cycle: denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 50 s, a total of 30 cycles, and finally extended at 72 °C for 7 min; wherein, the N1020727 gene-specific fragment was amplified The sequence of the upstream primer N1020727-F is: 5'-CCCTTTCACTACTACTCATCTTCA-3'; the sequence of the downstream primer N1020727-R is: 5'-GTTCTTCTTCTTCTTTTTCTTCCT-3';

Step 2. Purification of amplified products: use Magen gel recovery kit to purify PCR amplified fragments to obtain purified products;

Step 3. Obtaining an intermediate vector: The purified product was cloned into the pEASY-Blunt Zero vector according to the ligation reaction system, transformed into Trans1-T1 competent cells, and cultured on an LB plate containing 100 μg/mL ampicillin overnight to obtain colonies ; The ligation reaction system is: 4 μL of purified product, 1 μL of pEASY-Blunt Zero, and a total volume of 5 μL;

Step 4. Plasmid purification: inoculate the colony in LB liquid medium, culture it on a shaker at 37°C overnight, collect the bacterial liquid, and extract the plasmid to obtain the plasmid pEASY-020727 containing the N1020727 gene;

The concrete operation of described step 4) is as follows:

Step a. Design the following specific primers:

dsNl020727-F: 5'-AATGAAGGGAAGAGAGATCGTGCCAAA-3'

dsNl020727-R: 5'-TACAGCCTCATCATCCTCAGCAGTCA-3'

dsNl020727-T7F:

5’-GGATCCTAATACGACTCACTATAGGAATGAAGGGAAGAGAGATCGTGCCAAA-3’

dsNl020727-T7R:

5’-GGATCCTAATACGACTCACTATAGGTACAGCCTCATCCTCAGCAGTCA-3’

Step b, using the plasmid containing the N1020727 gene as a template, and using the specific primers in the step a to amplify according to the following reaction conditions, respectively, to obtain the target fragment:

A reaction system is as follows:

The reaction system of B is as follows:

PCR amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 35s, 30 cycles; extension at 72°C for 10 min;

Wherein, the double-stranded RNA corresponding to the N1020727 gene was synthesized by a dsRNA synthesis kit.

3. The application of the ESCRT-III core subunit Snf7-like double-stranded RNA of claim 1 in the preparation of a medicine for preventing and treating N. lugens.

4. A method for preventing and treating N. lugens, characterized in that: using the ESCRT-III core subunit Snf7 double-stranded RNA of claim 1 to inject N. lugens.

5 . The method according to claim 4 , wherein the final concentration of the ESCRT-III core subunit Snf7-like double-stranded RNA injected is 0.025 μg/μL to 0.2 μg/μL. 6 .

6. A brown planthopper insecticide, characterized in that it comprises the ESCRT-III core subunit Snf7 double-stranded RNA of claim 1 as an active ingredient.