DsRNA for efficiently preventing and treating phyllotreta striolata and application of dsRNA

Abstract

The invention relates to the technical field of biology, and discloses dsRNA for efficiently preventing and treating phyllotreta striolata and application of the dsRNA. According to the dsRNA for efficiently preventing and treating phyllotreta striolata, the dsRNA is obtained by transcription of an aryl alkylamine n-acyltransferase gene, and the dsRNA has an RNA sequence as shown in SEQ ID NO. 1. The gene sequence of the aryl alkylamine n-acyltransferase is as shown in SEQ ID NO. 2. According to the dsRNA for efficiently preventing and treating phyllotreta striolata and the application of the dsRNA, the provided target gene aryl alkylamine n-acyltransferase gene has specificity and is highly expressed in digestive tissues of adult phyllotreta striolata, so that the difficulty that the dsRNA reaches the target tissues can be reduced, the interference effect of the dsRNA on the phyllotreta striolata can be improved, and the aim of preventing and treating the adult phyllotreta striolata can be fulfilled. Further, the control effect of the dsRNA on the phyllotreta striolata is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a dsRNA for efficiently preventing and controlling yellow striped flea beetles and an application thereof. Background Art

[0002] The widespread use of chemical pesticides has led to the rapid development of resistance in the yellow striped flea beetle, so other pest control methods need to be developed. RNA interference (RNAi) technology has broad application prospects in plant breeding. RNA interference technology has become a common molecular tool in various research fields, including gene function research. Due to its specificity, simplicity and high efficiency, RNAi has received great attention in medical treatment and pest control in the medical and agricultural fields.

[0003] One of the key challenges in using RNAi to control insect pests is to effectively deliver the target double-stranded RNA (dsRNA) molecules into the pest organism. Common methods for introducing dsRNA in insect RNAi research include direct injection and oral administration. Oral ingestion, especially through feeding, is the choice for most biological control applications. However, the penetration of exogenous dsRNA is different in different tissues, such as the cells of the ovary and nervous system, which are reported to have low permeability to dsRNA, which poses a challenge to RNAi target design.

[0004] First, dsRNA needs to be ingested through the mouth and absorbed through the intestine to reach the target tissue; the degradation of dsRNA in the intestinal environment and the distance between the target tissue and the intestine need to be considered. Second, although the general target of spraying is plant leaves, the developmental stages of the yellow flea beetle other than adults do not parasitize on plant leaves. Therefore, dsRNA spraying is only lethal to adults, because the target gene needs to be continuously expressed in the adult stage and has important physiological functions. Summary of the invention

[0005] The purpose of the present invention is to provide a dsRNA and its application for effectively controlling yellow striped flea beetles. The provided target gene arylalkylamine n-acyltransferase gene has specificity and is highly expressed in the digestive tissues of yellow striped flea beetle adults, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on yellow striped flea beetles, and thus improve the control effect of dsRNA on yellow striped flea beetles.

[0006] To achieve the above object, the present invention provides a dsRNA for effectively controlling yellow striped flea beetle, wherein the dsRNA is transcribed from an arylalkylamine N-acyltransferase gene, and the dsRNA has an RNA sequence as shown in SEQ ID NO.1.

[0007] Furthermore, the gene sequence of arylalkylamine N-acyltransferase is shown in SEQ ID NO.2.

[0008] The present invention also provides a primer set for preparing the above dsRNA, the primer set comprising an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4.

[0009] The present invention also provides the use of the above-mentioned dsRNA for effectively controlling yellow striped flea beetles in controlling yellow striped flea beetles.

[0010] The present invention also provides the use of the above-mentioned dsRNA for effectively preventing and controlling the yellow striped flea beetle in the preparation of a drug for preventing and controlling the yellow striped flea beetle.

[0011] The present invention also provides a drug for preventing and controlling the yellow striped flea beetle, comprising a dsRNA targeting an arylalkylamine N-acyltransferase gene, wherein the dsRNA is the above-mentioned dsRNA.

[0012] The present invention also provides a method for preventing and controlling yellow striped flea beetles, which comprises spraying a drug containing dsRNA on the surface of plant stems or leaves, wherein the drug containing dsRNA is the above-mentioned drug.

[0013] The advantages and positive effects of the dsRNA for efficiently controlling the yellow striped flea beetle and its application described in the present invention are: 1. The target gene arylalkylamine n-acyltransferase gene provided by the present invention is specific and highly expressed in the digestive tissue of adult yellow striped flea beetles, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on yellow striped flea beetles, and thus improve the control effect of dsRNA on yellow striped flea beetles.

[0014] 2. The present invention provides dsRNA that can specifically target genes, and verifies the control effect of a certain concentration of dsRNA on the yellow striped flea beetle, indicating that dsRNA can be used as an effective substance for biological control of the yellow striped flea beetle.

[0015] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of correlation analysis of different repetitions of different samples in the dsRNA for effectively controlling yellow striped flea beetles and its application examples of the present invention; Figure 2 A Venn diagram in an embodiment of a dsRNA for effectively controlling yellow striped flea beetles of the present invention and its application shows the number of commonly expressed and specifically expressed genes within and between groups; Figure 3It is a volcano map of the specific highly expressed genes in the intestine and other tissues of the yellow striped flea beetle in the dsRNA for effectively preventing and controlling the yellow striped flea beetle of the present invention and its application examples; Figure 4 It is a bubble diagram of KEGG enrichment analysis clustering in a dsRNA for effectively controlling yellow striped flea beetle and its application examples of the present invention; Figure 5 The present invention provides a dsRNA for effectively controlling yellow striped flea beetles and the use thereof, and the expression levels of two candidate genes in the intestines and other body tissues of yellow striped flea beetles and the detection results of gene expression after feeding dsRNA for 48 hours, wherein A is the expression level of psaaNAT-like in the intestines and other body tissues of yellow striped flea beetles, and B is the down-regulation of psaaNAT-like expression after feeding dsRNA (1000 ng / μL) for 48 hours; Figure 6 The present invention is a dsRNA for effectively controlling yellow striped flea beetles and a dsRNA-sucrose solution dosing device in an application example thereof, wherein A is a conceptual diagram of the dosing device, B is a physical diagram of the dosing device, and C is an enlarged diagram of point ① in B; Figure 7 The present invention is a dsRNA for effectively controlling yellow striped flea beetles and the survival rate of different dsRNA concentration sucrose solutions after continuous feeding for 7 days, wherein the dsRNA concentration of A is 5000 ng / μL, the dsRNA concentration of B is 1000 ng / μL, and the dsRNA concentration of C is 500 ng / μL; Figure 8 The experimental settings and survival rates of foliar application of psaaNAT-like dsRNA in a dsRNA for effectively controlling yellow striped flea beetles and its application examples of the present invention, wherein A is the experimental settings for foliar application of psaaNAT-like dsRNA, and B is the survival rate of leaves treated with insect feeding for 5 days (limited duration of detached leaves); Fig. 9 The present invention is a dsRNA for effectively controlling yellow striped flea beetles and its application examples, and the damage of insects to leaves after 5 days of treatment with psaaNAT-like RNAi and mus -lta RNAi, wherein A is the damage of insects to leaves after 5 days of treatment with psaaNAT-like RNAi, and B is the damage of insects to leaves after 5 days of treatment with mus -lta RNAi; Fig.10 for Fig. 9After magnification, the morphological differences of the excrement of the yellow flea beetle, where Ⅰ in A is an enlarged view of the leaves of the psaaNAT-likeRNAi group, ① and ② are partial enlargements of the leaves in Ⅰ, Ⅱ in B is an enlarged view of the leaves of the mus -lta RNAi group, ③ and ④ are partial enlargements of the leaves in Ⅱ; Fig.11 The dsRNA for effectively controlling yellow striped flea beetles and its application examples of the present invention show the dynamic changes in the number of yellow striped flea beetles on leaves after applying the dsRNA, wherein the red and blue shadows represent the standard error at each time point; Fig.12 The present invention discloses a dsRNA for efficiently controlling yellow striped flea beetles and its application examples, and shows morphological changes in the intestinal tissue of insects after feeding control dsRNA (mus-lta) and psaaNAT-like dsRNA, wherein A is the control mus-ltaRNAi group and B is the psaaNAT-like RNAi group. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0018] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0019] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0020] Example 1. Insect breeding and plant materials: The yellow flea beetle (Phyllotreta striolat) used in the experiment was from a sensitive population raised in the laboratory of Qingdao Agricultural University, which had been raised indoors for more than 5 years. Cabbage was selected as the host plant for the experiment. The rearing conditions were 16L:8D photoperiod and 25℃. Before treatment, the adults were collected using an aspirator and treated in a refrigerator at 4℃ for 5 minutes before being treated individually.

[0021] 2. Transcriptome analysis of intestinal-specific expressed genes: More than 600 adult yellow-banded flea beetles 1 month after eclosion were collected, immediately frozen in liquid nitrogen, and dissected to separate the digestive tract. The intestinal tissue and the remaining tissue were collected separately and distributed in 4 centrifuge tubes, with more than 100 flea beetles in each tube. Transcriptome sequencing was performed on these samples. Sequencing was performed on the Illumina platform at Novogene Biotechnology Co., Ltd., Beijing, China. RNA integrity was assessed using the RNA Nano 6000Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Raw reads were processed using Fastp software.

[0022] The completeness of the de novo assembled transcriptomes was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool. Mapped reads for each sample were assembled using a reference-based approach using StringTie (v1.3.3b), and differential expression analysis was performed using the DESeq2 R package (1.20.0).

[0023] Transcriptome results and intestinal-specific expressed genes: The reliability and reproducibility of transcriptome sequencing samples were high. The intra-group correlation based on gene FPKM values ​​was good. The intra-group correlation between the four groups of digestive tract samples and the four groups of systemic residual tissue samples was high, while the correlation between the digestive tract and other tissue groups was low, which confirmed the reliability of the samples and data ( Figure 1 ). The Venn diagram of co-expressed genes shows the number of genes that are exclusively expressed or co-expressed in the two treatment groups. After integrating the data from different replicates, it was found that 277 genes were expressed only in the intestine and 9572 genes were expressed in the intestine and other tissues. The intra-group Venn diagram of the 4 replicates showed that the gene expression of the 4 replicate samples in each group had high reproducibility, indicating that the sequencing and analysis results have high accuracy and reference value ( Figure 2 The volcano plot illustrates the distribution of differentially expressed genes in the intestine compared to other tissues. The results show that 2,370 genes are significantly upregulated in the intestine (indicated by red dots), which is a key source for subsequent target selection ( Figure 3 As shown). After annotation and KEGG enrichment analysis of intestinal up-regulated genes, it was found that the intestinal highly expressed genes were mostly related to nutritional metabolic pathways such as fatty acid degradation, galactose metabolism, folate biosynthesis, riboflavin metabolism, pyruvate metabolism, porphyrin metabolism, and cellular energy metabolic pathways such as oxidative phosphorylation and citric acid cycle (TCA cycle) ( Figure 4 These pathways correspond to the basic physiological functions of the intestine.

[0024] The expression levels of the target psaaNAT-like in the adult intestine and the whole body were further detected. The expression of psaaNAT-like in the intestine was high, and the expression of psaaNAT-like in the intestine was about 2.5 times that in the body, with a significant difference (t = 4.643, df = 6, p = 0.0035; Figure 5 (A). After 48 h of sucrose feeding with 1000 ng / μL dsRNA, all target genes were significantly down-regulated. The average down-regulation rate of psaaNAT-like was about 83% (t = 2.795, df = 6, p = 0.03145; Figure 5 (middle B).

[0025] 3. In vitro synthesis and feeding of dsRNA: Based on transcriptome data, genes with high basal expression levels in intestinal tissue (FPKM value of about 10,000, gut vs Body log 2 foldchange>3) were selected. qRT-PCR and dsRNA synthesis primers were designed (Table 1).

[0026] Table 1 Primer sequences ;

[0027] Among them, T7 sequence: 5'-taatacgactcactataggg-3'.

[0028] The synthesis of dsRNA was performed using a T7 promoter primer synthesis kit (TranscriptAid T7 high-yield transcription kit, 91257083, Thermo Fisher Scientific, Waltham, MA; USA). The synthesized dsRNA was quantitatively adjusted to 6000 ng / μL and stored at -20°C. The mus-lta dsRNA from mice was selected as a control.

[0029] dsRNA was administered by sucrose solution feeding. dsRNA (ds-psaaNAT and ds-mus-lta) was dissolved in sucrose solution and the final concentrations were adjusted to 500 ng / μL, 1000 ng / μL and 5000 ng / μL, with a final concentration of 0.5 g / mL. There were 3 groups for each concentration, for a total of 6 dsRNA groups. The dsRNA-sucrose solution (12 μL) was pipetted into a 200 μL sponge PCR tube. The prepared device was placed in a 90 mm culture dish and maintained in humidity with wet filter paper ( Figure 6 Subsequently, 50 starved yellow flea beetles were placed in 5 culture dishes and fed for 7 consecutive days, and the dsRNA-sucrose solution was replaced every 2 days.

[0030] Effects and mortality of psaaNAT-like fed with sucrose solution: After 7 days of sucrose feeding, the lethal effects of different concentrations of dsRNA were observed. At a concentration of 5000 ng / μL, the mortality of psaaNAT-like dsRNA treatment was significantly higher than that of control dsRNA treatment (p<0.0001, log-rank); 50% of the death time was observed at 144 h and 156 h, respectively, among which the psaaNAT-like interference group reached the highest, with mortality rates of 60% and 58% ( Figure 7 At a concentration of 1000 ng / μL, the mortality rate of psaaNAT-like dsRNA treatment was significantly higher than that of control treatment (p<0.0001, log-rank); 50% of the death time was observed at 96h and 132h, respectively, among which the psaaNAT-like interference group had the highest mortality rate of 82%, with the best lethal effect ( Figure 7 At 500 ng / μL, the mortality rate of psaaNAT-like dsRNA treatment was also significantly higher than that of the control treatment (p = 0.0057, log-rank); only the mortality rate of the psaaNAT-like interference group reached 50%, which occurred at 168 h, indicating that the lethal effect was relatively poor ( Figure 7 (C). The killing effect of dsRNA at a concentration of 1000 ng / μL was the best.

[0031] 4. Application on leaf surface simulates the effect of field application: 4.1 Blade surface application: The psaaNAT-like dsRNA with the best lethal effect after sucrose feeding was selected for the experiment. New leaves of Chinese cabbage were selected, with a length of 1 to 1.5 cm. 50 μL of dsRNA solution with a concentration of 2000 ng / μL was applied to both sides of the leaves. After drying, the petiole was immersed in 1.8 mL ddH 2 O (1.5 mL centrifuge tube). Leaves and 50 hungry flea beetles were placed in 5 small cages (5.5 cm in diameter; 10 cm in height) with the bottom directly touching the leaves. After 5 days of feeding flea beetles, the leaves did not change ( Figure 8 Middle A).

[0032] Leaf surface application of psaaNAT-like dsRNA significantly increased beetle mortality (p < 0.0001, log-rank). However, the 5-day treatment did not reach 50% mortality, with a final mortality of 42%, which was 10 times higher than the control group treated with mus-lta dsRNA (4%) ( Figure 8Middle B). Observation of damaged leaves showed that the psaaNAT-like dsRNA-treated group had fewer and smaller pinholes, more and larger pinholes, and empty holes and defective connections compared with the control group ( Fig. 9 A and B in the figure). Microscopic observation revealed that the morphology and distribution of the feces excreted by beetles on the leaves were significantly different: the feces of the control group were normal, dry, and elongated, evenly distributed on the leaves, and there were few feces aggregates, while the feces of the psaaNAT-like dsRNA-treated group were irregular, moist, and shapeless, and a large number of feces aggregated together ( Fig.10 A and B).

[0033] 4.2 Avoidance effect of psaaNAT-like dsRNA: This test used the leaf surface application method. After allowing flea beetles to feed freely for 48 hours, the number of flea beetles was counted every 30 minutes for 12 hours (25 groups) with 5 replicates.

[0034] Observation and recording of the number of beetles that stayed or fed on the leaf surface for 12 h in the five leaf treatment dsRNA treatment groups showed that there was no significant difference in the number of beetles between the treatment group (psaaNAT-like) and the control group (mus-lta) (p = 0.782; univariate repeated measures, general linear model; Fig.11 ).

[0035] 4.3 Cross-HE staining of midgut: Tissue sections and cross-sections of flea beetles were stained on day 4 after RNAi. Samples were fixed overnight in 10% (w / v) buffered formalin, dehydrated, embedded in paraffin, and sectioned. Slides were soaked in xylene twice for 20 minutes, in 100% alcohol twice for 5 minutes, and finally in 75% (v / v) alcohol for 5 minutes. They were then rinsed with water, soaked in hematoxylin solution for 3-5 minutes, and rinsed with water again. Sections were differentiated with acid alcohol, rinsed with ammonia, and rinsed with slow tap water. Sections were stained with 85% (v / v) alcohol for 5 minutes, 95% (v / v) alcohol for 5 minutes, and eosin for 5 minutes. Sections were dehydrated, mounted in 100% alcohol 3 times for 5 minutes, in xylene 2 times for 5 minutes, and finally mounted with colorless transparent resin. Digital images of the sections were acquired using a Nikon DS-Ri1 (Nikon, Tokyo, apan) with a Nikon 80i microscope system (Nikon, Tokyo, Japan) and Nis-Elements software v3.22.14 (Build 736, Nikon, Tokyo, Japan).

[0036] Fig.12As shown in A and B, the specific microstructural changes of intestinal tissue under the action of psaaNAT-like RNAi are shown. Compared with the control group, after psaaNAT-like RNAi treatment, intestinal cells showed more fragmented structures; it was difficult to determine whether they belonged to tissue or food residues. In addition, the intestinal cavity space of individuals after psaaNAT-like RNAi was relatively larger than that of mus-lta RNAi, and the amount of food consumed was less, which may be related to feeding inhibition. However, this difference was not significant compared with the intestinal state caused by starvation, because the cross-sectional area of ​​the intestinal sections of the starvation group was significantly reduced.

[0037] 5. Data Analysis: All experimental data were analyzed using SPSS (version 22; SPSS Inc., Chicago, IL, USA). KM survival curves were drawn using the ggsurvplot R package on the bioinformatics.com.cn website (last accessed on November 10, 2023), and the p value was calculated using the “log-rank” method.

[0038] The above comprehensive analysis shows that the target gene arylalkylamine n-acyltransferase gene provided by the present invention has specificity and is highly expressed in the digestive tissue of the adult yellow striped flea beetle, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on the yellow striped flea beetle, and thus improve the control effect of dsRNA on the yellow striped flea beetle. The dsRNA that can specifically target genes provided by the present invention and the control effect of dsRNA at a certain concentration on the yellow striped flea beetle are verified, indicating that dsRNA can be used as an effective substance for biological control of the yellow striped flea beetle.

[0039] Therefore, the present invention adopts the above-mentioned dsRNA and its application for highly controlling yellow striped flea beetle, and the provided target gene arylalkylamine n-acyltransferase gene has specificity and is highly expressed in the digestive tissue of yellow striped flea beetle adults, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on yellow striped flea beetle, and thus improve the control effect of dsRNA on yellow striped flea beetle.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A dsRNA for effectively controlling the yellow striped flea beetle, characterized in that: The dsRNA is transcribed from an arylalkylamine N-acyltransferase gene, and has an RNA sequence as shown in SEQ ID NO.

1.

2. A dsRNA for effectively controlling the yellow striped flea beetle according to claim 1, characterized in that: The gene sequence of arylalkylamine n-acyltransferase is shown in SEQ ID NO.

2.

3. A primer set for preparing the dsRNA according to claim 1, characterized in that: The primer set includes an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.

4.

4. Use of the dsRNA for efficiently controlling the yellow striped flea beetle according to claim 1 or 2 in controlling the yellow striped flea beetle.

5. Use of the dsRNA for effectively controlling the yellow striped flea beetle according to claim 1 or 2 in the preparation of a drug for controlling the yellow striped flea beetle.

6. A drug for preventing and controlling the yellow striped flea beetle, characterized in that: The invention comprises a dsRNA targeting an arylalkylamine N-acyltransferase gene, wherein the dsRNA is the dsRNA according to claim 1 or 2.

7. A method for controlling yellow striped flea beetle, characterized in that: The drug containing dsRNA is sprayed on the surface of plant stems or leaves, wherein the drug containing dsRNA is the drug described in claim 6.

Images