CN113122540B - RNAi target gene capable of killing grubs efficiently and application of RNAi target gene - Google Patents

RNAi target gene capable of killing grubs efficiently and application of RNAi target gene Download PDF

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CN113122540B
CN113122540B CN202110528880.XA CN202110528880A CN113122540B CN 113122540 B CN113122540 B CN 113122540B CN 202110528880 A CN202110528880 A CN 202110528880A CN 113122540 B CN113122540 B CN 113122540B
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赵丹
郭巍
康占海
陆秀君
李瑞军
刘兆瑞
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Heibei Agricultural University
Graduate School of CAAS
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Abstract

The invention discloses an RNAi target gene capable of killing grubs efficiently and application thereof, and belongs to the field of biotechnology and agricultural application. The nucleotide sequence of RNAi is shown in SEQ ID NO.13, and the obtaining steps are as follows: (1) Extracting total RNA of the grubs, and performing reverse transcription to obtain a first cDNA chain; (2) Carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and primers shown in SEQ ID NO.1 and SEQ ID NO. 2; (3) Taking the PCR product in the step (2) as a template, and carrying out PCR by using primers shown in SEQ ID NO. 3-6 to obtain a positive template and a reverse template for synthesizing dsRNA; (4) And recovering the electrophoresis product after the reaction is finished, and transcribing and synthesizing the dsRNA by using the recovered electrophoresis product as a template. After the dsRNA is injected or fed for 72 hours, the silencing efficiency of HpCDA1 of grub larvae is higher than 95 percent, and the mortality rate is higher than 90 percent.

Description

RNAi target gene capable of killing grubs efficiently and application of RNAi target gene
Technical Field
The invention belongs to the field of biotechnology and agricultural application, and particularly relates to an RNAi target gene capable of efficiently killing grubs and application thereof.
Background
Grubs are soil-inhabiting pests which are recognized to be difficult to control at home and abroad, and cause serious harm to crops, fruit trees, forest trees and the like. Chemical pesticide control destroys ecological environment and crop quality, so that the grub harm control by using a biological control method becomes inevitable, and development of a new pest control strategy of a biological control target becomes a research hotspot. The insect body wall is an important defense system for maintaining the growth and development of insects, wherein the epidermal layer in the body wall mainly comprises chitin and protein and can be used as a novel target of an insecticidal drug. Chitin Deacetylase (CDA) is an important enzyme in the chitin metabolic process, can convert chitin into chitosan by catalyzing deacetylation of N-acetamido-D-glucosamine (N-acetyl-b-D-glucosamine), and is an important biocontrol target by changing the chitin and destroying the growth mechanism of insects.
RNA interference (RNAi) is an endogenous specific post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA). After the dsRNA is introduced into an organism, the dsRNA is decomposed into 21-23 bp small interfering RNA (siRNA) by RNase III called Dicer in cells, the siRNA is combined with target mRNA under the action of a silencing complex (RISC), the target mRNA is degraded in sequence specificity, the synthesis of corresponding protein products is prevented, and the function of a target gene is lost. Since the discovery of such a mechanism for rapid and direct silencing of a specific gene, RNAi technology has rapidly become widely used in the field of gene function studies in non-model animals. RNAi is widely used as a tool for gene function research, especially in animals and plants with imperfect genetic manipulation tools.
Disclosure of Invention
One of the purposes of the invention is to provide an RNAi target gene which can kill grubs efficiently, and the nucleotide sequence of the RNAi target gene is shown in SEQ ID NO. 13.
The preparation method of the RNAi target gene comprises the following steps:
(1) Extracting total RNA of the grubs, and performing reverse transcription to obtain a first cDNA chain;
(2) Carrying out PCR by using the cDNA obtained in the step (1) as a template and primers shown in SEQ ID NO.1 and SEQ ID NO.2 to obtain a chitin deacetylase1 (HpCDA 1) full-length gene;
(3) Taking the PCR product in the step (2) as a template, and performing PCR amplification by using primers shown in SEQ ID NO. 3-6 to obtain a forward template and a reverse template which are used for amplifying dsRNA sequences;
(4) And after the reaction is finished, carrying out agarose gel electrophoresis, then recovering an electrophoresis product, and transcribing and synthesizing dsRNA by taking the recovered electrophoresis product as a template to obtain the RNAi target gene, wherein the RNAi target gene consists of a nucleotide sequence shown in SEQ ID NO. 13.
Further, the PCR reaction system in the step (2) is as follows: 25 muL: 2 XPrimeSTAR Max Premix 12.5 muL, 10 mumol/L upstream and downstream primers are 1 muL respectively, a template is 1 muL, and sterile distilled water is 10.5 muL.
Further, the PCR reaction conditions in step (2) are: 30s at 95 ℃;95 ℃ for 5s, 55 ℃ for 15 s,72 ℃ for 10 s,35 cycles.
Further, the PCR reaction system in step (3) is: 25 muL: 2 XPrimeSTAR Max Premix 12.5 muL, 10 mumol/L upstream and downstream primers are 1 muL respectively, a template is 1 muL, and sterile distilled water is 10.5 muL.
Further, the PCR reaction conditions in the step (3) are as follows: 5min at 95 ℃;95 ℃,30 s;60 ℃,30s;72 ℃,45s,5 cycles; 30s at 95 ℃; 30s at 50 ℃; 45s at 72 ℃,35 cycles; 72 ℃ for 10min.
The invention also aims to provide application of the RNAi target gene in grub prevention and control.
It is a further object of the present invention to provide a biological product containing the above-mentioned RNAi target gene.
Further, the biological product is an injection pesticide or a bait.
Compared with the prior art, the invention has the following beneficial effects:
through injecting and feeding dsRNA (RNAi target gene) of white grub chitin deacetylase1 (HpCDA 1), the mortality of the insect population and the gene silencing efficiency after 72h of taking in the dsRNA are counted, and Green Fluorescent Protein (GFP) is used as a control for carrying out the same treatment. The result shows that the silencing efficiency of HpCDA1 genes of grubs injected and fed with dsRNA is higher than 95%, and the mortality rate of worm openings is higher than 90%. The invention provides a high-efficiency target gene for biological control of grubs.
Drawings
FIG. 1 shows grubs after injection of dsGFP and dsHpCDA for 1h in example 2HpCDA1Relative expression level of gene.
FIG. 2 shows the morphology of dsHpCDA1 72h injected grub in example 2.
FIG. 3 is a morphological diagram of the control dsGFP grub in example 2.
Detailed Description
Example 1
HpCDA1 specific sequences
The chitinase deacetylase gene of grubsHpCDA1The gene sequence is submitted to GenBank, the accession number is MG189704, the gene length is 1593bp, 530 amino acids are coded, the predicted protein molecular weight is 60.1kDa, and the structural domain analysis result shows that HpCDA1 comprises a chitin binding region (ChBD), a low-density lipoprotein receptor region (LDLa) and a chitin deacetylation catalytic region (CDA).
2. HpCDA1Gene specific sequence amplification
According to sequence information, specific primers of the HpCDA1 full-length gene are designed, the sequences of the primers are shown in Table 1, the primers comprise SEQ ID NO.1 and SEQ ID NO.2, and a target fragment with the length of 1593bp can be amplified.
TABLE 1 Gene clone specific primers
Figure 600826DEST_PATH_IMAGE002
3. Feeding test insects
The grub is bred by using artificial feed in a plant protection institute insect biochemistry and molecular biology laboratory of Hebei agriculture university, wherein the breeding temperature is 26 +/-1 ℃, the relative humidity is 60-70%, and the illumination is 16 percent.
4. Extraction of RNA
(1) Extracting total RNA of white grub whole worm, weighing larva tissue 14 mg, and extracting the total RNA according to the instruction of the TIANGERNNNAprep pure animal tissue total RNA extraction kit. The specific RNA extraction operation steps are as follows:
(1) and (3) homogenization treatment: adding 300 μ l of lysis solution RL into every 10-20 mg tissue (before use, whether beta-mercaptoethanol is added or not is checked), and grinding the tissue thoroughly with a grinding pestle (if the tissue is hard to grind thoroughly, an electric or glass homogenizer can be selected); subsequently, 590. Mu.l RNase-Free ddH was added to the homogenate 2 O and 10 mul of protease K, evenly mixing, and treating for 10-20 min at 56 ℃. Note that: the amount of tissue must not exceed 20 mg otherwise it will lead to reduced RNA yield and quality.
(2) Centrifuging at 12,000 rpm (13,400 Xg) for 2-5 min, and taking the supernatant to perform the following operations.
(3) Slowly adding 0.5 times of the volume of the supernatant of absolute ethyl alcohol, uniformly mixing (at this time, precipitation possibly occurs), transferring the obtained solution and the precipitation into an adsorption column CR3 (the adsorption column is placed in a collecting pipe), centrifuging at 12,000 rpm (13,400 Xg) for 30-60 sec, discarding waste liquid in the collecting pipe, and placing the adsorption column back into the collecting pipe.
(4) 350 μ l deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000 rpm (-13,400 Xg) for 30-60 sec, the waste liquid was discarded, and the adsorption column was returned to the collection tube.
(5) Preparing DNase I working solution: add 10. Mu.l DNase I stock into a new RNase-Free centrifuge tube, add 70. Mu.l RDD buffer, mix gently.
(6) Add 80. Mu.l of DNase I working solution to the center of the adsorption column CR3, and leave it at room temperature for 15min.
(7) 350 μ l deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000 rpm (-13,400 Xg) for 30-60 sec, the waste liquid was discarded, and the adsorption column was returned to the collection tube.
(8) Add 500. Mu.l of rinsing solution RW (check whether ethanol was added before use) to the adsorption column CR3, leave it at room temperature for 2 min, centrifuge at 12,000 rpm (-13,400 Xg) for 30-60 sec, discard the waste, and return the adsorption column CR3 to the collection tube.
(9) And repeating the step 8.
Centrifuge at 12,000 rpm (-13,400 Xg) for 2 min, and discard the waste liquid. The adsorption column CR3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.
⑪ the adsorption column CR3 is transferred into a new RNase-Free centrifuge tube, 30. Mu.l of RNase-Free ddH2O is suspended and dripped into the middle part of the adsorption membrane, and the adsorption membrane is placed at room temperature for 2 min and centrifuged at 12,000 rpm (13,400 Xg) for 2 min to obtain an RNA solution. Then, 1ul of the obtained RNA solution was diluted 10 times, and the purity and concentration of RNA were measured using a spectrophotometer. And 1ul of the same diluted 10-fold was run through agarose gel for integrity testing.
5. Synthesis of cDNA
According to the Promega cDNA Synthesis Kit (Promega) Kit instructioncDNA was synthesized. Specifically, a reverse transcription system was prepared as follows: 1ul of the above-mentioned total RNA,1ul of oligo dT,3ul of nucleic acid free-water, in a PCR apparatus at 70 ℃ for 5min; then 5 × Realtion Buffer,2.5ul mM MgCl was added 2， 1ul Mix，0.5ul Inhibitor，1ul RT，6ul ddH 2 And (O). In a PCR instrument at 25 ℃ for 5min,42 ℃ for 1h and 70 ℃ for 15min. After synthesis, the cDNA was stored in a freezer at-20 ℃.
6. Synthesis of dsRNA
Design of amplification with reference to T7 RiboMAX Express RNAi System SpecificationHpCDA1Gene and controlGFPPrimers for the forward and reverse Linear DNA of the gene are shown in Table 2 and include SEQ ID NO.3 to SEQ ID NO.10. Using SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 as primers and 1593bp obtained in step 2HpCDA1PCR is carried out by taking the gene as a template to obtain a synthetic target geneHpCDA1 The dsRNA of (a) requires a forward and reverse template. PCR was performed using SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 as a set of primers and pGR plasmid as a template to obtain forward and reverse templates required for synthesis of control GFP dsRNA. PCR reaction system (25. Mu.L): 2 XPrimeSTAR Max Premix 12.5 muL, 10 mumol/L up-and-down primer 1 muL, template 1 muL and sterile distilled water 10.5 muL. PCR reaction procedure: 5min at 95 ℃;95 ℃,30 s;60 ℃,30s;72 ℃,45s,5 cycles; 30s at 95 ℃; 30s at 50 ℃;72 ℃,45s,35 cycles; 72 ℃ for 10min. After the reaction is finished, agarose gel electrophoresis is respectively carried out, then the kit is used for recovering electrophoresis products, and the transcription kit is used for synthesizing dsRNA by taking the recovered positive and reverse products as templates. dsRNA synthesis transcription system (20 µ L): riboMAX TM Express T7X Buffer 10 muL, positive and negative templates are respectively 5 muL (2 mug), T7 Express Enzyme Mix 2 muL, and sterile distilled water is 3 muL. In-vitro transcription was performed at 37 ℃ in a PCR instrument and 4 h to synthesize single-stranded ssRNA in the forward and reverse directions. Mixing the positive and reverse ssRNA products with the same volume, gently mixing, keeping at 70 deg.C for 10min, and naturally cooling to room temperature. Adding RNaseA Solution and RNaseFree DnaseSoltion, 37 deg.C, 30 min, and removing DNA template and ssRNA. Adding 2.5 times volume of 95% ethanol and 1/10 volume of 3 mol/LNaAc (pH 5.2), gently mixing, and ice-coolingThe dsRNA was purified by centrifugation at 12000 rpm for 10min at 5min. Removing supernatant, adding 500mL, precooling 70% ethanol, washing precipitate, centrifuging at 12000 rpm for 3 min, removing supernatant, drying in a super clean bench, adding 100 mu L of Nucleasefree ddH 2 O dissolves the dsRNA.
The mass and fragment size of the dsRNA (dsHpCDA 1 and dsGFP) obtained by the above amplification were electrophoretically examined. The concentration of dsHpCDA1 detected by a spectrophotometer is 6950.8 mug/ml, the OD value 260/280 is 2.08, the concentration of dsGFP is 5750.5 mug/ml, the OD value 260/280 is 2.03, a PCR strip is single and has no other miscellaneous bands, the size and the concentration meet the requirements, and the PCR strip can be used for RNAi tests.
TABLE 2 dsRNA specific primers
Figure 111442DEST_PATH_IMAGE003
Example 2
2-year-old 1-day grubs were picked, and dsHpCDA1 synthesized as described above was injected into the grubs with dsGFP as a control, at the junction between the 2 nd and 3 rd contra-podal flank of the abdomen. The amount of dsRNA injected per test insect was 10ug (2 ul,5000 ng/ul). The experiment was set up with 3 biological replicates, 20 nymphs per biological replicate, for a total of 60 test insects. And (4) feeding the test insects in a 24-pore plate, and counting the mortality rate and the gene silencing efficiency of the test insects after injection for 72 hours.
Extracting total RNA of the white grubs injected with dsGFP and dsHpCDA for 72h respectively, carrying out reverse transcription to synthesize a cDNA first strand, measuring the expression quantity of the HpCDA1 injected with dsGFP and dsHpCDA1 for 72h by using a qRT-PCR method by using HpCDA1F SEQ ID NO.11 and HpCDA1R SEQ ID NO.12 as primers, carrying out statistical analysis on silencing efficiency, and finding that the gene expression quantity of the white grubs HpCDA1 injected with dsHpCDA1 is inhibited, wherein the calculated silencing rate is about 99% as shown in figure 1, and the white grubs HpCDA1 gene injected with dsGFP is not silenced.
The relative expression of the genes was obtained as follows: and after the real-time fluorescent quantitative PCR obtains a result, analyzing a dissolution curve and then analyzing data by a method for comparing Ct values. Three repeats were set for each cDNA, with β -actin as the reference gene. SPSS software was used to analyze the significance of the differences between samples, and the relative expression was calculated using the following equation:
2 △△Ct =2 - [ (Ct destination Gene-Ct internal reference Gene)]Treatment group- [ (Ct target gene-Ct internal reference gene)]Control group
The Holotrichia parallela (larva) injected with dsHpCDA1 has dark or even black body, slow growth, slow action, hypomotility, and shrivelled epidermis, and has molting difficulty and death, and the state is shown in FIG. 2. The holotrichia parallela (larvae) status of the control group is shown in fig. 3. The mortality rate of the larvae injected with dsHpCDA1 for 72h was 100%, which was significantly higher than that of the control group by 3.33%.
TABLE 3
Figure DEST_PATH_IMAGE005
Example 3
In the embodiment, the injection mode in the embodiment 2 is replaced by a grub feeding mode, 20 mug (2 mug, 5000 ng/mug) of dsRNA is mixed into 2g of artificial feed (corn straw powder, 500ml of water is added into sugarcane powder to be boiled to be eroded, residues are smashed, 300ml of water is taken to be added into sugar and agar, water is added to be boiled, the mixture is poured into the components, and the artificial feed is obtained after the mixture is subjected to constant volume to 1L to be fed for 72 hours, and then the death rate of the test insects and the gene silencing efficiency are counted.
After 72 hours of feeding dsGFP and dsHpCDA, extracting the total RNA of the grub respectively, and statistically analyzing the silencing efficiency in the same way as in example 2 and 72 hours, the result shows that the gene expression quantity of the grub HpCDA1 is inhibited, the calculated silencing rate is about 95.51%, the larva mortality rate is 91.67%, while the grub HpCDA1 gene fed with dsGFP is not silenced, and the mortality rate is 0.
From the examples 2 and 3, it is clear that the silencing of the HpCDA1 gene can be realized by both the implementation methods, the death rate of the grubs is more than 90%, and the application prospect is very high.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
SEQUENCE LISTING
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<120> RNAi target gene capable of killing grubs efficiently and application thereof
<130> 2021.04.09
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Claims (9)

1. The dsRNA is highly lethal to grubs, and the preparation method of the dsRNA comprises the following steps: taking the nucleotide shown in SEQ ID NO.13 as a template, taking SEQ ID NO.3-4 and SEQ ID NO.5-6 as primers, respectively carrying out PCR amplification to obtain a forward product and a reverse product, and then transcribing to synthesize dsRNA.
2. The dsRNA of claim 1, which is prepared by a process comprising the steps of:
(1) Extracting total RNA of the grubs, and performing reverse transcription to obtain a first cDNA chain;
(2) Performing PCR amplification by using the DNA obtained in the step (1) as a template and primers shown in SEQ ID NO.1 and SEQ ID NO. 2;
(3) Respectively carrying out PCR amplification by taking the PCR product in the step (2) as a template and SEQ ID NO.3-4 and SEQ ID NO.5-6 as primers to obtain a positive template and a reverse template which are used for amplifying a dsRNA sequence;
(4) And recovering the electrophoresis product after the reaction is finished, and transcribing and synthesizing dsRNA by using the recovered electrophoresis product as a template to obtain the RNAi target gene.
3. The dsRNA of claim 2, wherein the PCR reaction system in step (2) is: 25. mu L:2 XPrimeSTAR Max Premix 12.5 muL, 10 mumol/L upstream and downstream primers are 1 muL respectively, a template is 1 muL, and sterile distilled water is 10.5 muL.
4. The dsRNA of claim 3, wherein the PCR reaction conditions in step (2) are: 95. 30s; 95.5 s at 56 ℃ and 45s at 72 ℃ and 10 s at 72 ℃ for 35 cycles.
5. The dsRNA of claim 2, wherein in step (3) the PCR reaction system is: 25 muL: 2 XPrimeSTAR Max Premix 12.5 muL, 10 mumol/L upstream and downstream primers are 1 muL respectively, a template is 1 muL, and sterile distilled water is 10.5 muL.
6. The dsRNA of claim 5, wherein the PCR reaction conditions in step (3) are: 5min at 95 ℃;95 ℃,30 s;60 ℃,30s;72 ℃,45s,5 cycles; 30s at 95 ℃; 30s at 50 ℃;72 ℃,45s,35 cycles; 72 ℃ for 10min.
7. The dsRNA of any one of claims 1 to 6 for grub control.
8. A biological product comprising the dsRNA of any one of claims 1-6.
9. The biologic product of claim 8, wherein said biologic product is an injectable pesticide or bait.
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