An dsRNA for effectively controlling Phyllotreta striolata and its application

Abstract

The present invention relates to the field of biotechnology, and discloses a dsRNA for efficiently controlling Phyllotreta striolata and its application. A dsRNA for efficiently controlling Phyllotreta striolata, the dsRNA is transcribed from an arylalkylamine N-acyltransferase gene, and the dsRNA has an RNA sequence as shown in SEQ ID NO.1. The arylalkylamine N-acyltransferase gene sequence is as shown in SEQ ID NO.2. By using the dsRNA for efficiently controlling Phyllotreta striolata and its application as described above, the provided target gene, the arylalkylamine N-acyltransferase gene, has specificity and is highly expressed in the digestive tissues of adult Phyllotreta striolata, which can reduce the difficulty of the dsRNA reaching the target tissue, improve the interference effect of the dsRNA on Phyllotreta striolata, and further improve the control effect of the dsRNA on Phyllotreta striolata.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a dsRNA for efficiently controlling Phyllotreta striolata and its application. Background Art

[0002] The widespread use of chemical pesticides has led to the rapid development of resistance in Phyllotreta striolata. Therefore, 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 controlling pests using RNAi is to effectively deliver the target double-stranded RNA (dsRNA) molecule 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 biocontrol applications. However, the permeability of exogenous dsRNA is different in different tissues, such as cells in the ovary and nervous system. These tissues are reported to have low permeability to dsRNA, which poses a challenge to RNAi target design.

[0004] Firstly, dsRNA needs to be ingested orally 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. Secondly, although the general target of spraying is the plant leaves, the developmental stages of Phyllotreta striolata other than adults do not parasitize on the plant leaves. Therefore, dsRNA spraying only kills 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 object of the present invention is to provide a dsRNA for efficiently controlling Phyllotreta striolata and its application. The provided target gene, arylalkylamine N-acyltransferase gene, is specific and highly expressed in the digestive tissues of Phyllotreta striolata adults, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on Phyllotreta striolata, and further improve the control effect of dsRNA on Phyllotreta striolata.

[0006] To achieve the above object, the present invention provides a dsRNA for efficiently controlling Phyllotreta striolata, and the dsRNA is transcribed from the arylalkylamine N-acyltransferase gene and 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, which includes 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 application of the above dsRNA for highly effectively controlling Phyllotreta striolata in the control of Phyllotreta striolata.

[0010] The present invention also provides the application of the above dsRNA for highly effectively controlling Phyllotreta striolata in the preparation of drugs for controlling Phyllotreta striolata.

[0011] The present invention also provides a drug for controlling Phyllotreta striolata, which contains dsRNA targeting the arylalkylamine N - acyltransferase gene, and the dsRNA is the above dsRNA.

[0012] The present invention also provides a method for controlling Phyllotreta striolata, which sprays the drug containing dsRNA on the surface of the plant stalk or leaf, and the drug containing dsRNA is the above drug.

[0013] The advantages and positive effects of the dsRNA for highly effectively controlling Phyllotreta striolata and its application according to the present invention are as follows:

[0014] 1. The target gene arylalkylamine N - acyltransferase gene provided by the present invention is specific and highly expressed in the digestive tissues of adult Phyllotreta striolata, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on Phyllotreta striolata, and further improve the control effect of dsRNA on Phyllotreta striolata.

[0015] 2. The present invention provides dsRNA that can specifically target genes, and verifies the control effect of dsRNA at a certain concentration on Phyllotreta striolata, indicating that dsRNA can be used as an effective substance for biological control of Phyllotreta striolata.

[0016] The technical solutions of the present invention will be further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the result of correlation analysis of different repetitions of different samples in the embodiment of dsRNA for highly effectively controlling Phyllotreta striolata and its application according to the present invention;

[0018] Figure 2 It is the Venn diagram showing the number of co - expressed and specifically expressed genes within and between groups in the embodiment of dsRNA for highly effectively controlling Phyllotreta striolata and its application according to the present invention;

[0019] Figure 3 Volcano plot of specifically highly expressed genes in the intestine and other tissues of Phyllotreta striolata in the example of dsRNA for highly efficient control of Phyllotreta striolata and its application of the present invention;

[0020] Figure 4 Bubble plot of KEGG enrichment analysis clustering in the example of dsRNA for highly efficient control of Phyllotreta striolata and its application of the present invention;

[0021] Figure 5 Detection results of the expression levels of two candidate genes in the intestine and other body tissues of Phyllotreta striolata and the gene expression levels 48 hours after feeding dsRNA in the example of dsRNA for highly efficient control of Phyllotreta striolata and its application of the present invention, where A is the expression level of psaaNAT-like in the intestine and other body tissues of Phyllotreta striolata, and B is the down-regulation of psaaNAT-like expression 48 hours after feeding dsRNA (1000 ng / μL);

[0022] Figure 6 DsRNA-sucrose solution administration device in the example of dsRNA for highly efficient control of Phyllotreta striolata and its application of the present invention, where A is the conceptual diagram of the administration device, B is the physical diagram of the administration device, and C is the enlarged view of ① in B;

[0023] Figure 7 Survival rates after continuous feeding with sucrose solutions of different dsRNA concentrations for 7 days in the example of dsRNA for highly efficient control of Phyllotreta striolata and its application of the present invention, where 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;

[0024] Figure 8 Experimental setup and survival rate of foliar application of psaaNAT-like dsRNA in the example of dsRNA for highly efficient control of Phyllotreta striolata and its application of the present invention, where A is the experimental setup of foliar application of psaaNAT-like dsRNA, and B is the survival rate 5 days after the insects feed on the treated leaves (limited duration of detached leaves);

[0025] Figure 9 Damage to leaves by insects 5 days after psaaNAT-like RNAi and mus-lta RNAi treatments in the example of dsRNA for highly efficient control of Phyllotreta striolata and its application of the present invention, where A is the damage to leaves 5 days after psaaNAT-like RNAi treatment, and B is the damage to leaves 5 days after mus-lta RNAi treatment;

[0026] Figure 10 For Figure 9 After magnification, the morphological differences of the excrement of Phyllotreta striolata. Among them, in A, Ⅰ is the enlarged view of the leaf of the psaaNAT-like RNAi group, and ① and ② are the partial enlargements of the leaf in Ⅰ; in B, Ⅱ is the enlarged view of the leaf of the mus-lta RNAi group, and ③ and ④ are the partial enlargements of the leaf in Ⅱ.

[0027] Figure 11 This is the dynamic change of the number of Phyllotreta striolata on the leaves after applying dsRNA in the example of the dsRNA for highly effective control of Phyllotreta striolata and its application of the present invention, where the red and blue shadows represent the standard error at each time point.

[0028] Figure 12 This is the morphological change of the intestinal tissue of insects after feeding the control dsRNA (mus-lta) and psaaNAT-like dsRNA in the example of the dsRNA for highly effective control of Phyllotreta striolata and its application of the present invention. Among them, A is the control mus-lta RNAi group, and B is the psaaNAT-like RNAi group. Detailed implementation mode

[0029] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0030] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0031] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are all commercially available.

[0032] Example

[0033] 1. Insect rearing and plant materials:

[0034] The Phyllotreta striolata used in the experiment was sourced from a sensitive population reared in the laboratory of Qingdao Agricultural University, which had been continuously reared indoors for more than 5 years. Chinese cabbage was selected as the host plant for the experiment. The rearing conditions were a photoperiod of 16L:8D and a temperature of 25°C. Before treatment, adults were collected using a suction device and individually treated after being placed in a refrigerator at 4°C for 5 minutes.

[0035] 2. Transcriptome analysis of intestine-specifically expressed genes:

[0036] More than 600 adult Phyllotreta striolata that emerged 1 month ago were collected, immediately frozen in liquid nitrogen, and dissected to isolate the digestive tract. Intestinal tissues and remaining tissues 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. The sequencing was carried out on the Illumina platform of Novogene Biotechnology Co., Ltd. in Beijing, China. The RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The raw reads were processed using the Fastp software.

[0037] The Benchmarking Universal Single-Copy Orthologs (BUSCO) tool was used to evaluate the integrity of the de novo assembled transcriptome. A reference-based method was adopted, and the mapped reads of each sample were assembled using StringTie (v1.3.3b), and differential expression analysis was performed using the DESeq2 R package (1.20.0).

[0038] Transcriptome results and intestine-specific expressed genes: The reliability and repeatability of the transcriptome sequencing samples were high. The within-group correlation based on the gene FPKM values was good. The within-group correlation of the four groups of digestive tract samples and the four groups of whole-body residual tissue samples was relatively high, while the between-group correlation between the digestive tract and other tissues was relatively low, confirming the reliability of the samples and data ( Figure 1 as shown). The Venn diagram of co-expressed genes shows the number of genes expressed only or co-expressed in the two treatment groups. After integrating different repeated data, it was found that 277 genes were expressed only in the intestine, and 9,572 genes were expressed in both the intestine and other tissues. The within-group Venn diagram of 4 repeats shows that the gene expression of 4 repeated samples within each group has high repeatability, indicating that the sequencing and analysis results have high accuracy and reference value ( Figure 2 as shown). The volcano plot illustrates the distribution of differentially expressed genes in the intestine compared with other tissues. The results show that 2,370 genes were significantly upregulated in the intestine (represented by red dots), which is a key source for subsequent target selection ( Figure 3 as shown). After annotating and performing KEGG enrichment analysis on the upregulated genes in the intestine, it was found that the highly expressed genes in the intestine were mostly related to nutritional metabolic pathways such as fatty acid degradation, galactose metabolism, folate biosynthesis, riboflavin metabolism, pyruvate metabolism, and porphyrin metabolism, as well as cellular energy metabolic pathways such as oxidative phosphorylation and the citric acid cycle (TCA cycle) ( Figure 4 as shown). These pathways correspond to the basic physiological functions of the intestine.

[0039] Further detect the expression levels of the target psaaNAT-like in the adult intestine and the whole body. It was highly expressed in the intestine. The expression level of psaaNAT-like in the intestine was about 2.5 times that in the body, showing a significant difference (t = 4.643, df = 6, p = 0.0035; Figure 5 as shown in A). After feeding with 1000 ng / μL dsRNA by the sucrose feeding method for 48 h, the target genes were all significantly downregulated. The average downregulation rate of psaaNAT-like was about 83% (t = 2.795, df = 6, p = 0.03145; Figure 5 as shown in B).

[0040] 3. In vitro synthesis and feeding of dsRNA:

[0041] Based on the transcriptome data, genes with high basal expression levels in intestinal tissues (FPKM value about 10,000, gut vs Body log2foldchange > 3) were selected. qRT-PCR and dsRNA synthesis primers were designed (Table 1).

[0042] Table 1 Primer sequences

[0043] ;

[0044] Among them, the T7 sequence: 5 ‘ -taatacgactcactataggg -3’.

[0045] The synthesis of dsRNA used 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℃. Mus-lta dsRNA from mice was selected as a control.

[0046] dsRNA was administered by the sucrose solution feeding method. dsRNA (ds-psaaNAT and ds-mus-lta) was dissolved in the 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, 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 petri dish and the humidity was maintained with a wet filter paper ( Figure 6as shown in A, B, and C). Subsequently, 50 starved Phyllotreta striolata were placed in 5 petri dishes and continuously reared for 7 d, and the dsRNA-sucrose solution was changed every 2 days.

[0047] Effect of sucrose solution feeding on psaaNAT-like and mortality: After 7 d of sucrose feeding, the lethal effects of different concentrations of dsRNA were observed. At a concentration of 5000 ng / μL, the mortality rates of the psaaNAT-like dsRNA treatment were all higher than those of the control dsRNA treatment, with a significant difference (p < 0.0001, log-rank); the 50% death times were observed at 144 h and 156 h respectively, and the highest values were reached in the psaaNAT-like interference group, with mortality rates of 60% and 58% ( Figure 7 as shown in A). At a concentration of 1000 ng / μL, the mortality rates of the psaaNAT-like dsRNA treatment were all significantly higher than those of the control treatment (p < 0.0001, log-rank); the 50% death times were observed at 96 h and 132 h respectively, and the highest values were reached in the psaaNAT-like interference group, with a mortality rate of 82%, showing the best lethal effect ( Figure 7 as shown in B). At a concentration of 500 ng / μL, the mortality rates of the psaaNAT-like dsRNA treatment were also significantly higher than those of the control treatment (p = 0.0057, log-rank); only the mortality rate in the psaaNAT-like interference group reached 50%, which occurred at 168 h, indicating a relatively poor lethal effect ( Figure 7 as shown in C). The dsRNA with a concentration of 1000 ng / μL had the best killing effect.

[0048] 4. Application on the leaf surface to simulate the field application effect:

[0049] 4.1 Application on the leaf surface:

[0050] The psaaNAT-like dsRNA with the best lethal effect after sucrose feeding was selected for the experiment. New leaves of Chinese cabbage were selected, and the length of the new leaves was 1 - 1.5 cm. 50 μL of the dsRNA solution with a concentration of 2000 ng / μL was applied to both sides of the leaves respectively. After drying, the petioles were immersed in 1.8 mL of ddH2O (1.5 mL centrifuge tube). The leaves and 50 starved flea beetles were placed in 5 small cages (diameter 5.5 cm; height 10 cm), and the bottom was in direct contact with the leaves. The flea beetles were fed for 5 days, and the leaves did not change ( Figure 8 as shown in A).

[0051] Foliar application of psaaNAT-like dsRNA significantly increased beetle mortality (p < 0.0001, log-rank). However, the 5-day treatment did not reach 50% mortality, and the final mortality was 42%, which was 10 times higher than the mortality of the control group treated with mus-lta dsRNA (4%) ( Figure 8 in B). Observation of damaged leaves showed that the psaaNAT-like dsRNA treatment group had fewer and smaller pinholes, more and larger pinholes, and connected cavities and defects compared to the control group ( Figure 9 in A and B). Microscopic observation revealed significant differences in the morphology and distribution of feces excreted by beetles on the leaves: the feces of the control group were normal, dry, and slender, evenly distributed on the leaves with less fecal aggregation, while the feces of the psaaNAT-like dsRNA treatment group were irregular, moist, shapeless, and had a large amount of feces aggregated together ( Figure 10 in A and B).

[0052] 4.2 Avoidance effect of psaaNAT-like dsRNA:

[0053] In this experiment, the foliar application method was used. After allowing the flea beetles to freely feed for 48 hours, the number of beetles was counted every 30 minutes for 12 consecutive hours (25 groups), with 5 replicates.

[0054] Observation and recording of the number of beetles staying or feeding on the leaf surface for 12 hours in the 5 groups of leaves treated with dsRNA 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; Figure 11 ).

[0055] 4.3 Cross HE staining of the midgut:

[0056] On the 4th day after RNAi, tissue sections and cross-sections of the flea beetles were stained. The samples were fixed overnight in 10% (w / v) buffered formalin, dehydrated, embedded in paraffin, and sectioned. The 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. Then they were rinsed with water, soaked in hematoxylin solution for 3 - 5 minutes, and rinsed with water again. The sections were differentiated with acidic alcohol, rinsed with ammonia water, and rinsed with slow running tap water. The sections were stained with 85% (v / v) alcohol for 5 minutes, 95% (v / v) alcohol for 5 minutes, and eosin for 5 minutes. The sections were dehydrated, soaked in 100% alcohol three times for 5 minutes, in xylene twice for 5 minutes, and finally mounted with colorless transparent resin. Digital images of the sections were obtained using a Nikon DS-Ri1 (Nikon, Tokyo, Japan) with a Nikon 80i microscope system (Nikon, Tokyo, Japan) and Nis-Elements software v3.22.14 (Build 736, Nikon, Tokyo, Japan).

[0057] Figure 12 As shown in A and B, it shows the specific microscopic structural changes in the intestinal tissue under the action of psaaNAT-like RNAi. Compared with the control group, after psaaNAT-like RNAi treatment, more fragmented structures appeared in the intestinal cells; it was difficult to determine whether they belonged to the tissue or food residues. In addition, the intestinal lumen space of individuals after psaaNAT-like RNAi was relatively larger than that of mus-lta RNAi, and the food intake was less, which might be related to feeding inhibition. However, compared with the intestinal state caused by starvation, this difference was not significant because the cross-sectional area of the intestinal sections in the starvation group decreased significantly.

[0058] 5. Data analysis:

[0059] All experimental data were analyzed using SPSS (version 22; SPSS Inc., Chicago, IL, USA). The KM survival curve was plotted using the ggsurvplot R software package on the website bioinformatics.com.cn (last accessed on November 10, 2023), and the p-value was calculated using the "log-rank" method.

[0060] From the above comprehensive analysis, it can be seen that the target gene arylalkylamine N-acyltransferase gene provided by the present invention is specific and highly expressed in the digestive tissues of adult Phyllotreta striolata, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on Phyllotreta striolata, and further improve the control effect of dsRNA on Phyllotreta striolata. The dsRNA provided by the present invention that can specifically target genes, and the control effect of dsRNA at a certain concentration on Phyllotreta striolata has been verified, indicating that dsRNA can be used as an effective substance for biological control of Phyllotreta striolata.

[0061] Therefore, the present invention adopts the above-mentioned dsRNA for highly efficient control of Phyllotreta striolata and its application. The provided target gene arylalkylamine N-acyltransferase gene is specific and highly expressed in the digestive tissues of adult Phyllotreta striolata, which can reduce the difficulty of dsRNA reaching the target tissue, improve the interference effect of dsRNA on Phyllotreta striolata, and further improve the control effect of dsRNA on Phyllotreta striolata.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

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

1.

2. A dsRNA for 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 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 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 the plant stems or leaves, wherein the drug containing dsRNA is the drug described in claim 6.

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