Method for preventing and controlling epilepis angustifolia population based on RNAi (Ribonucleic Acid Interfere) and intestinal microorganism horizontal propagation characteristics

Abstract

The invention relates to the technical field of biological prevention and control, in particular to a salix babylonia population prevention and control method based on RNAi and intestinal microorganism horizontal propagation characteristics. The vector is constructed, double-stranded RNA is successfully expressed in the pseudomonas putida, the RNAi effect is triggered when the latifolious blue beetle larvae eat leaves containing the pseudomonas putida, the fatality rate is remarkably increased, the antifeedant behavior is initiated, meanwhile, the individual development of pests and the expression of key genes are inhibited, and the survival and reproductive capacity of the pests is weakened in multiple layers. According to the invention, rapid horizontal transmission and prevention and control among pest populations are realized by virtue of bacterium-mediated RNAi, a new path is opened up for the field of prevention and control of plant diseases and insect pests, and the biological insecticide is expected to be developed into an efficient biological insecticide, effectively controls the pest populations and conforms to the green and environment-friendly agricultural development concept.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and particularly relates to a method for controlling the population of Plagiodera versicolora based on RNAi and the horizontal transmission characteristics of gut microbiota. Background Art

[0002] In the field of agricultural pest control, Plagiodera versicolora, as one of the main pests of Salicaceae plants, its effective control has always been the focus of research and practice. Currently, the control measures for Plagiodera versicolora mainly include three categories: chemical control, biological control, and physical control. However, these measures all have certain limitations and challenges in practical applications.

[0003] Chemical control, as a traditional pest control method, relies on the use of various chemical pesticides. Although it can rapidly reduce the pest population in the short term, long-term use not only easily leads to the development of pest resistance, reducing the control effect, but also the pesticide residues may cause irreversible damage to the ecological environment and non-target organisms. In addition, the high cost of chemical pesticides and the potential threat to human health are also issues that cannot be ignored. Biological control focuses on using natural factors such as natural enemy insects and microbial agents to control pests. Although this method is more environmentally friendly in theory, in practical applications, the survival and reproduction of natural enemy insects are greatly affected by the environment, making it difficult to stably control the pest population; while the effect of microbial agents is often affected by various factors such as temperature and humidity, resulting in unstable control effects. Physical control, such as manual killing and setting up insect-proof nets, can reduce the pest population to a certain extent, but its operation is cumbersome, the efficiency is low, and the control effect on small larvae or pests with strong concealment is limited. In addition, the high cost of physical barriers such as insect-proof nets also limits their wide application in large-scale agricultural production. In the actual environment, due to factors such as the behavior habits, distribution status of pests, and the implementation conditions of control measures, there will always be some pests escaping control, becoming the source of pest population regeneration. This limitation not only reduces the control effect but also increases the risk of the development of pest resistance.

[0004] At present, there are still many deficiencies in the control of Plagiodera versicolora, such as unstable control effects, negative impacts on the ecological environment and non-target organisms, and it is difficult for control measures to comprehensively cover the pest population. These defects not only affect the safety and efficiency of agricultural production but also pose a potential threat to ecological environment protection. Therefore, it is particularly important to explore new, more efficient and environmentally friendly methods for controlling Plagiodera versicolora. Summary of the Invention

[0005] In view of this, the present invention proposes a method for controlling the population of Plagiodera versicolora based on RNAi and the horizontal transmission characteristics of intestinal microorganisms. The present invention proposes an innovative strategy for controlling the population of Plagiodera versicolora, which combines RNA interference (RNAi) technology with the horizontal transmission characteristics of the intestinal microorganism Pseudomonas putida (abbreviated as P. putida). By constructing a vector, double-stranded RNA (dsRNA) was successfully expressed in Pseudomonas putida. When Plagiodera versicolora feeds on host plants containing this bacterium, the RNAi effect will be triggered. In addition, P. putida can colonize in the intestine of Plagiodera versicolora and, by virtue of its horizontal transmission ability in the pest population, combined with RNAi technology, effectively control the population of Plagiodera versicolora.

[0006] The technical solution of the present invention is realized as follows:

[0007] In the first aspect, the present invention provides a method for controlling the population of Plagiodera versicolora based on RNAi and the horizontal transmission characteristics of intestinal microorganisms, including the following steps:

[0008] S(1-1) Using specific primer pairs SEQ ID NO: 1-2, amplify scaffold 1 from the pBBR403gfp plasmid;

[0009] S(1-2) Using specific primer pairs SEQ ID NO: 3-4, with the pYQ11 plasmid as a template, amplify target fragment 1; SEQ ID NO: 16 is included in the target fragment 1; SEQ ID NO: 16 encodes double-stranded RNA targeting and silencing the expression of the Actin gene of Plagiodera versicolora;

[0010] S(1-3) Using specific primer pairs SEQ ID NO: 5-6, with the pUC18T-mini-Tn7T-Gm plasmid as a template, amplify target fragment 2; the target fragment 2 contains the T7 polymerase gene sequence as shown in SEQ ID NO: 18;

[0011] S(1-4) Simultaneously transform the scaffold 1, target fragment 1, and target fragment 2 into Escherichia coli competent DH5α; culture without selection pressure for 2 h, screen positive clones and perform plasmid extraction and sequence identification to obtain the target plasmid pYS10;

[0012] S(2) Transform the plasmid pYS10 into Pseudomonas putida competent cells, culture without selection pressure for 2 h, screen positive clones, verify by PCR and identify by sequencing to obtain the engineered strain P. putida-ACT;

[0013] The larvae of Plagiodera versicolora were fed with leaves treated with the P. putida-ACT bacterial solution, and the viable bacteria concentration on the leaves was ≥ 10 9 CFU / cm 2 。

[0014] In a second aspect, the present invention provides the application of the described method in the prevention and control of plant diseases and pests.

[0015] In a third aspect, the present invention provides the application of the described method in the preparation of biopesticide products.

[0016] The beneficial effects of the present invention at least include the following:

[0017] (1) By utilizing the horizontal transmission characteristics of the specific gut microbe P. putida, the present invention significantly increases the lethality rate against the larvae of Plagiodera versicolora, induces antifeedant behavior, effectively weakens the survival ability of the pests and their harm to plants, thereby enhancing the overall efficacy of pest control. The present invention not only directly leads to an increase in pest mortality, but also inhibits the individual development of pests, and further affects the physiological functions and normal physiological functions of pests by downregulating the expression of key genes, weakening the survival and reproductive abilities of pests from multiple levels.

[0018] (2) By using the bacteria-mediated RNAi technology, the present invention realizes the rapid horizontal transmission and population control among pest populations, providing a new perspective for the large-scale application of RNAi technology in the field of plant disease and pest control. In addition, the present invention is expected to be used for the development of new biological insecticides, and by utilizing its multiple effects on Plagiodera versicolora (such as direct insecticidal, growth inhibition, feeding influence, etc.), it can effectively control the population quantity of pests. At the same time, by deeply studying its action mechanism, optimizing the construction of engineering strains, and improving the control effect, it provides more effective means and theoretical support for population control technology, meeting the requirements of green and environmental-friendly agricultural development.

[0019] Table 1 Sequence information

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Appendix 7 Explanation

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 Schematic diagram of the structure of pYS10 and pYS18 plasmids provided in the embodiments of the present invention;

[0028] Figure 2 A Northern bolt test result diagram of dsRNA expression of the engineering bacteria provided in the embodiment of the present invention; it shows the Northern blot test result of the P. putida-YS10 strain sample;

[0029] Figure 3 A survival curve of blue leaf beetles fed with different engineered strains provided in an embodiment of the present invention; wherein the P. putida group represents the survival of blue leaf beetles fed with ordinary P. putida bacterial solution; P. putida-YS10 represents the survival of blue leaf beetles fed with P. putida-ACT bacterial solution; P. putida-YS18 represents the survival of blue leaf beetles fed with P. putida-GFP bacterial solution;

[0030] Figure 4 A diagram showing feeding of willow leaf beetles fed with different engineered strains on isolated willow leaves three days later according to an embodiment of the present invention;

[0031] Figure 5 A statistical graph of the weight of the blue leaf beetles fed with different engineered strains provided in an embodiment of the present invention; the ordinate represents the average weight of the blue leaf beetle larvae; different letters (a, b) are marked above the bar graph in the figure, indicating that there are significant differences between the groups;

[0032] Figure 6 The relative expression level of the Actin gene in the blue leaf beetle fed with different engineered strains three days after the embodiment of the present invention;

[0033] Figure 7 The experimental model and result diagram of horizontal transmission of P. putida-403 between willow blue leaf beetle populations provided in the embodiment of the present invention;

[0034] Figure 8Survival curve of P. putida during horizontal transmission among Plagiodera versicolora populations provided by the embodiments of the present invention; among them, the experimental group is the larvae transmitted by P. putida strain (T-P.putida group); the control groups are respectively the larvae transmitted by P. putida-YS10 strain (T-P.putida-YS10 group), and the larvae transmitted by P. putida-YS18 strain (T-P.putida-YS18 group);

[0035] Figure 9 Graph of the feeding on detached willow leaves three days after the horizontal transmission of P. putida among Plagiodera versicolora populations provided by the embodiments of the present invention;

[0036] Figure 10 Statistical graph of the body weight of Plagiodera versicolora based on the horizontal transmission of P. putida among Plagiodera versicolora populations provided by the embodiments of the present invention;

[0037] Figure 11 Relative expression level of Actin gene in vivo three days after the horizontal transmission of P. putida among Plagiodera versicolora populations provided by the embodiments of the present invention. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase. The methods used in the following embodiments are all conventional methods in the art unless otherwise specified. The acquisition routes of various biological materials described in the embodiments are only provided to achieve the specific public purpose of the experiment and should not be a limitation on the source of biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments. The genes, proteins or their fragments involved in the present invention can be natural purified products, chemically synthesized products, or products produced using recombinant techniques from prokaryotic or eukaryotic hosts (such as bacteria, yeast, plants, etc.).

[0039] In some specific embodiments of the present application, the plasmid extraction kit, gel extraction kit, and PCR product purification and recovery kit used were all purchased from Omega. The PCR Mix was provided by Beijing Tsingke Biotechnology Co., Ltd. All reagents required for qT-PCR analysis were sourced from Yeasen Biotechnology Co., Ltd. All antibiotic products were purchased from Solarbio Science & Technology Co., Ltd. The molecular marker and various rapid restriction endonucleases were purchased from Takara Bio Inc. In addition, all other chemical reagents met the national analytical purity standard. All primer sequences were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The plasmid pBBR403gfp is described in the reference (Xiao Yujie. The role of c-di-GMP and its metabolic enzymes BifA and GcbA in regulating biofilm formation and motility in Pseudomonas putida KT2440 [D], 2017); the plasmid pYQ11 is described in the reference (Zhang Y., Xu L., Li S., et al. Bacteria-Mediated RNA interference for management of Plagiodera versicolora (Coleoptera: Chrysomelidae) [J]. Insects, 2019, 10(12):415.); the plasmid pUC18T-mini-Tn7T-Gm is described in the reference (XIE M, WANG Q, ZHOU N, et al. Engineering a novel entomopathogenic strain Pseudomonas chlororaphis for efficient production of double-stranded RNAs and pest control [J]. Pest Management Science, 2025:ps.8699.); the strain Pseudomonas putida is described in the reference (XU L, XU S, SUN L, et al. Synergistic action of the gut microbiota in environmental RNA interference in a leaf beetle [J]. Microbiome, 2021, 9(1):98.). The plasmids and strains used in the present application can be obtained by the public from the Plastid Genetic Engineering Laboratory of the School of Life Sciences, Hubei University. The applicant undertakes to make the biological materials available to the public within twenty years from the filing date.

[0040] Data analysis and chart drawing were both completed using GraphPad Prism 6 and SPSS 19.0 software. During the bioassay, the survival of Plagiodera versicolora was statistically analyzed using the Kaplan-Meier survival curve, and the log-rank test was used to evaluate whether the differences between treatment groups were significant. The larval weight data were the average values of each group, and ANOVA (combined with the LSD test) was used to determine the significance of the weight differences between different groups. For the qRT-PCR data, the relative expression levels of the target genes were calculated according to the 2 -ΔΔCT method, and ANOVA (combined with the LSD test) was also used to analyze the significance of the expression differences between different groups. All of the above statistical analyses showed significant differences at P < 0.05, which was statistically significant.

[0041] This invention is the result of the "National Key R&D Program of China" project, and the project number is (2023YFC2607000).

[0042] Example 1

[0043] This application discloses two engineering bacteria of Pseudomonas putida (P. putida) and their preparation methods. The two engineering bacteria can respectively express interfering double-stranded RNA (dsRNA) targeting the Actin gene and the GFP gene. The preparation method includes preparing the pYS10 and pYS18 plasmids, transforming the pYS10 plasmid and the pYS18 plasmid into P. putida respectively, and screening to obtain P. putida-ACT and P. putida-GFP.

[0044] 1. Construction of the pYS10 plasmid

[0045] The pYS10 plasmid is a plasmid with the pBBR403gfp plasmid backbone, into which the dsRNA expression sequence targeting the Actin gene of Plagiodera versicolora is inserted by homologous recombination, and at the same time, the T7 polymerase gene is inserted. The specific steps are as follows:

[0046] (1) Amplify the backbone 1 from the pBBR403gfp plasmid using the specific primer pair Y10-GJ-F and Y10-GJ-R (as shown in SEQ ID NO: 1-2);

[0047] (2) Using the pYQ11 plasmid as a template, amplify the target fragment 1 using the specific primer pair ACT-F and ACT-R (as shown in SEQ ID NO: 3-4); the target fragment 1 contains the dsRNA expression sequence targeting the Actin gene of Plagiodera versicolora (as shown in SEQ ID NO: 16);

[0048] (3) Using the pUC18T-mini-Tn7T-Gm plasmid as a template, the target fragment 2 was amplified using the specific primer pair T7-F and T7-R (as shown in SEQ ID NO: 5-6); the target fragment 2 contains the T7 polymerase gene sequence (as shown in SEQ ID NO: 18).

[0049] (4) Then, the backbone 1, target fragment 1 and 2 were simultaneously transformed into Escherichia coli competent DH5α by heat shock method. Subsequently, they were cultured without selection pressure for 2 h, and then spread on an LB plate containing Gent (Gentamicin, final concentration 50 mg / L) to screen for positive clones. Subsequently, the plasmid gene sequence and correct assembly were determined by PCR detection and gene sequencing, and thus the target plasmid pYS10 was obtained.

[0050] The formula of the liquid LB medium is: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and the pH is adjusted to 7.4; the formula of the LB plate includes the components of the liquid LB plus 15 g / L of agar powder.

[0051] 2. Construction of the pYS18 plasmid

[0052] pYS18 is a plasmid that replaces the Actin sequence in the pYS10 plasmid with a sequence interfering with the expression of the GFP gene dsRNA through homologous recombination. The specific steps are as follows:

[0053] (1) The backbone 2 was amplified from pYS10 using the specific primer pair Y18-F and Y18-R (as shown in SEQ ID NO: 7-8).

[0054] (2) The target fragment 3 was amplified from the pYQ11 plasmid using the specific primer pair GFP-F and GFP-R (shown in SEQ ID NO: 9-10). The target fragment 3 contains the GFP gene dsGFP expression sequence (as shown in SEQ ID NO: 17).

[0055] (3) Then, the backbone 2 and the target fragment 3 were simultaneously transformed into Escherichia coli competent DH5α by heat shock method. Subsequently, they were cultured without selection pressure for 1 h, and then spread on an LB plate containing Gent (50 mg / L) to screen for positive clones. Subsequently, the plasmid gene sequence and correct assembly were determined by PCR detection and gene sequencing, and thus the target plasmid pYS18 was obtained.

[0056] 3. Preparation of the P.putida-ACT engineered bacteria

[0057] The engineered strain P.putida-ACT is obtained by introducing plasmid pYS10 into P.putida, which can express dsRNA interfering with the Actin gene of Plagiodera versicolora. After feeding Plagiodera versicolora, the Actin gene is down-regulated, leading to the death of Plagiodera versicolora. The specific steps are as follows:

[0058] pYS10 was introduced into P.putida competent cells by electroporation. Then, the cells were cultured without selection pressure for 2 h, and spread on LB plates containing Gent (50 mg / L) to screen for positive clones. Subsequently, the clones were verified by PCR and sequenced to obtain the engineered strain P.putida-ACT. The method for preparing the P.putida competent cells is as follows: The Pseudomonas putida strain was cultured in liquid LB for 8 h, and then co-treated with a sterile 15% (m / m) glycerol solution on ice for 30 min for standby. After collecting the cells by freezing centrifugation (5000 rpm; 10 min), pre-cooled 15% glycerol solution was added. The mixture was vortexed for 30 s and then left standing on ice for 30 min. After centrifugation again (5000 rpm; 10 min), the cells were collected, 1 mL of sterile 15% glycerol solution was added, and the mixture was pipetted evenly and then aliquoted into 100 μL and stored at -80 °C for standby. 10 μL of the target plasmid was added to the above-mentioned competent cells, mixed evenly and then transferred to an electroporation cuvette. The electroporation method is as follows: The electroporation cuvette was inserted into the electroporation chamber for electroporation (voltage: 2300 V). Subsequently, the electroporated competent cells were transferred into liquid LB.

[0059] 4. Preparation of the engineered strain P.putida-GFP

[0060] The engineered strain P.putida-GFP is obtained by introducing plasmid pYS18 into P.putida, which can express dsRNA targeting the GFP gene as a control. The specific steps are as follows:

[0061] pYS18 was introduced into P.putida competent cells by the above-mentioned electroporation method. Then, the cells were cultured without selection pressure for 2 h, and spread on LB plates containing Gent (50 mg / L) to screen for positive clones. Subsequently, the clones were verified by PCR and sequenced to obtain the engineered strain P.putida-GFP.

[0062] 5. Expression test of dsRNA

[0063] (1) Method

[0064] The Northern hybridization probe sequence (as shown in SEQ ID NO: 15) was transcribed from plasmid pYS10, and it was labeled respectively using the DIG RNA Labeling Kit (Roche) to obtain the DIG-labeled RNA probe.

[0065] Cultivate the above-obtained engineered strain until the OD 600 reaches 0.4, and induce the cultivation with 1 mM IPTG for 5 h. Take the bacterial liquid and extract the total RNA using an RNA extraction kit (TransZol UP, TransGen Biotech Co., Ltd., Beijing). Separate it by formaldehyde-denatured agarose gel electrophoresis, and then transfer it overnight by capillary blotting with filter paper to transfer the total RNA onto a nylon membrane.

[0066] Denature the RNA probe at 100 °C for 5 min, ice-bath for 5 min, and then add it to the hybridization solution of the hybridization membrane immersed with the transferred RNA for hybridization for more than 6 h. Crosslink with ultraviolet light to fix the RNA, and then wash the membrane to remove the unbound probe to reduce the background. Finally, develop to obtain the hybridization signal. Analyze the hybridization signal to identify the content and size of the specific RNA molecule.

[0067] (2) Results

[0068] The test results are as Figure 2 shown. The P. putida strain can produce a certain amount of dsRNA.

[0069] Example 2

[0070] 1. Test strains

[0071] 1×10 11 CFU (generally referring to CFU / mL, indicating the number of bacteria per unit volume) of P. putida-ACT bacterial liquid (obtained according to the preparation method of the P. putida-ACT engineered bacterium in Example 1).

[0072] Control group: 1×10 11 CFU of P. putida-GFP bacterial liquid (obtained according to the preparation method of the P. putida-GFP engineered bacterium in Example 1), which can express dsRNA interfering with the GFP gene.

[0073] 2. Test methods

[0074] (1) Cut the willow leaves into pieces of 1.5 cm × 4 cm, and smear 60 μL of the test strain (1×10 11 CFU / mL) bacterial liquid on each leaf (at this time, the final concentration of P. putida actually smeared on the leaf is 10 9 CFU / cm 2 ), and air-dry the leaves smeared with the bacterial liquid at room temperature.

[0075] (2) Place a piece of filter paper in a petri dish and add a little deionized water to keep the filter paper moist. Place the air-dried leaves in the petri dish and put 10 second-instar larvae of Plagiodera versicolora (purchased from Qushou Natural Interesting Insect Plant Museum, Xichuan County) in each petri dish. Each petri dish is a replicate, and 3 replicates are set for each treatment group (total number of larvae n = 30).

[0076] (3) To confirm whether the expression of dsRNA triggers RNAi in Plagiodera versicolora, larvae fed for three days were selected for qRT-PCR to detect the transcriptional level of the Actin gene. Total RNA of Plagiodera versicolora was extracted using Trans Zol reagent respectively. Using the mRNA among them as a template, with Random primers / Oligo(dT)18 primer, it was reverse transcribed into cDNA under the action of RNA-dependent DNA polymerase, that is, reverse transcriptase.

[0077] (4) Using cDNA as a template, the target gene was amplified by real-time fluorescence quantitative PCR (qRT-PCR) with specific primer pairs q-PV-ACT-F and q-PV-ACT-R (as shown in SEQ ID NO: 11-12).

[0078] The qRT-PCR amplification reaction system is as follows:

[0079] Component Volume (μL) cDNA 2 q-PV-ACT-F Primer 0.5 q-PV-ACT-R Primer 0.5 Hieff qPCR SYBR Green Master Mix 10 Water 7

[0080] The relative expression level of the target gene on the 3rd day was calculated by the 2 -ΔΔCT method. Among them, the internal reference gene 18S was amplified by qRT-PCR, and its primer pairs were q-PV-18s-F and q-PV-18s-R (as shown in SEQ ID NO: 13-14).

[0081] 2. Test results

[0082] As Figure 3As shown, in the experiment exploring the effect of P.putida-ACT bacterial solution on the survival rate of Plagiodera versicolora, we found that the survival rate of Plagiodera versicolora in the P.putida-YS10 group (i.e., the experimental group fed with P.putida-ACT bacterial solution) showed a downward trend since the first day of the experiment. By the fifth day of the experiment, the mortality rate of Plagiodera versicolora in this group had exceeded 60%, and there was a significant difference between the two groups at the statistical level compared with the P.putida group fed with ordinary P.putida bacterial solution. This result strongly indicates that the P.putida-ACT bacterial solution has a significant toxic effect on Plagiodera versicolora and can significantly reduce its survival rate. On the other hand, in the experiment evaluating the effect of P.putida-GFP bacterial solution, we found that the survival curves of the P.putida-YS18 group (i.e., the experimental group fed with P.putida-GFP bacterial solution) and the P.putida group almost completely overlapped, and there was no significant difference between the two at the statistical level. This finding shows that the non-target dsRNA expressed in the P.putida-GFP bacterial solution did not enhance the insecticidal ability of P.putida against Plagiodera versicolora, that is, this non-target dsRNA had no obvious effect on the survival status of Plagiodera versicolora.

[0083] Figure 4 The feeding situation of Plagiodera versicolora on willow leaves three days after feeding is shown. Compared with the control group, Plagiodera versicolora fed with the engineered strain P.putida-ACT showed obvious feeding refusal, while there was little difference between those fed with P.putida and P.putida-GFP. The results indicate that feeding P.putida expressing the target sequence dsRNA seriously affects the feeding of Plagiodera versicolora.

[0084] Figure 5The weight of Plagiodera versicolora after three days of feeding was shown. The weight of Plagiodera versicolora fed with the engineered strain P.putida-ACT was the lowest, showing a significant difference compared with that fed with P.putida and P.putida-GFP. The weight of Plagiodera versicolora fed with the P.putida-ACT bacterial solution (P.putida-YS10 group) decreased by about 44.4% compared with that fed with P.putida. This indicates that the bacterial solution may have an inhibitory effect on the growth and development of Plagiodera versicolora, restricting its weight gain. The weight of Plagiodera versicolora fed with the P.putida-ACT bacterial solution (P.putida-YS10 group) decreased by about 37.5% compared with that fed with P.putida-GFP. That is, compared with the other two groups, the weight of the P.putida-YS10 group showed a significant difference, indicating that this inhibitory effect is statistically significant. The average weights of Plagiodera versicolora in the P.putida group and the P.putida-YS18 group were relatively close, and there was no significant difference in weight between the two groups. This shows that feeding with the ordinary P.putida bacterial solution and the P.putida-GFP bacterial solution containing non-target dsRNA has a similar effect on the weight of Plagiodera versicolora, without obvious promoting or inhibitory effects.

[0085] It can be seen from this that the engineered strain P.putida-ACT can not only increase the mortality rate of Plagiodera versicolora (combined with Figure 3 the results), but also significantly inhibit its growth and development, resulting in slow weight gain. The expression of non-target dsRNA in the engineered strain P.putida-GFP has no significant effect on the weight gain of Plagiodera versicolora, and the effect is equivalent to that of the ordinary P.putida bacterial solution. Generally speaking, the engineered strain P.putida-ACT has multiple effects in controlling Plagiodera versicolora, providing more powerful evidence for its potential as a population prevention and control means.

[0086] As Figure 6 shown, the relative transcriptional level of the Actin gene in Plagiodera versicolora fed with P.putida-ACT was significantly down-regulated compared with the control group, and there was a significant difference. There was no significant difference in feeding with P.putida and P.putida-GFP, indicating that RNAi did occur in feeding with P.putida-ACT, and it also shows that the death of Plagiodera versicolora caused by feeding with P.putida-ACT is due to the silencing of the Actin gene.

[0087] Example 3

[0088] 1. Preparation of the engineered bacteria P.putida-403gfp

[0089] The engineered strain P.putida-403gfp is a P.putida strain expressing green fluorescent protein GFP, and its preparation method is as follows: Use an electroporator to transfer the pBBR403gfp plasmid into P.putida, then culture it without selection pressure for 2 h, and spread it on an LB plate containing Gent (50 mg / L) to screen for positive clones, thus obtaining the engineered strain P.putida-403gfp.

[0090] 2. Test the engineered strain P.putida-403gfp

[0091] (1) Method

[0092] Spread the P.putida-403gfp strain on willow leaves at a cell concentration of 1×10 9 CFU / cm 2 Let the leaves coated with the bacterial solution dry at room temperature. Place two willow leaves coated with the bacterial solution in each petri dish. Place a wet filter paper in the petri dish to maintain the humidity of the larval feeding environment. Put the early second-instar larvae of Plagiodera versicolora into the petri dish, and place 10 second-instar larvae in each petri dish.

[0093] Feed the early second-instar larvae of Plagiodera versicolora with the leaves coated with the P.putida-403gfp bacterial solution for 3 days. Then, mix the larvae that have been fed with the P.putida-403gfp bacterial solution and the early second-instar larvae that have not been fed with the strain at a ratio of 1:2 in a petri dish containing fresh willow leaves that have not been treated. A total of 15 larvae are placed in each petri dish. Mark the larvae that have been fed with the P.putida-403gfp bacterial solution for easy distinction. Sample and dissect the guts of the insects at 12 h and 24 h of transmission and observe the GFP fluorescence signal.

[0094] (2) Results

[0095] As Figure 7 shown, the guts of the larvae fed with the P.putida-403gfp bacterial solution have strong green fluorescence signals compared to the guts of the control larvae without treatment. It can be determined that there is a phenomenon of horizontal transmission of the P.putida strain among the Plagiodera versicolora population.

[0096] Example 4

[0097] 1. Test the strains

[0098] Experimental group: 1×10 11 CFU of P.putida-ACT bacterial solution.

[0099] Control group: 1×10 11 CFU of P.putida bacterial solution and 1×10 11CFU P.putida-GFP bacterial solution.

[0100] 2. Testing method

[0101] Cut the willow leaves into pieces of 1.5 cm × 4 cm. Apply 60 μL of the test strain bacterial solution with a concentration of 1×1011 CFU / mL to each piece of leaf, so that the final concentration of P.putida on the leaf reaches 10 9 CFU / cm 2 . Air-dry the leaves applied with the bacterial solution at room temperature. Place a filter paper in a petri dish and add a little deionized water to keep the filter paper moist.

[0102] Feed the second-instar larvae of Plagiodera versicolora with the air-dried excised willow leaves for 3 days. Then, mix the larvae that have been fed with the bacterial solution and the early second-instar larvae that have not been fed with the bacterial solution at a ratio of 1:2 in a new petri dish, and feed them with fresh willow leaves without applying any bacterial solution. After 3 days, take away the larvae that were previously fed with the bacterial solution, and the remaining Plagiodera versicolora larvae that have been transmitted. Then, continuously feed the remaining larvae with fresh willow leaves without applying any bacterial solution until the pupation stage.

[0103] 3. Experimental results

[0104] In terms of mortality rate, as Figure 8 shown, the larval mortality rate in the T-P.putida-YS10 group was significantly higher than that in the T-P.putida group and the T-P.putida-YS18 group. This data clearly indicates that the transmission of the P.putida-YS10 strain has a more significant adverse effect on the survival of the larvae, suggesting that this strain may contain certain substances or mechanisms that are toxic or harmful to the larvae.

[0105] In terms of feeding situation, Figure 9 shows that the larvae in the T-P.putida-YS10 group showed obvious feeding refusal behavior. This observation indicates that the transmission of the P.putida-YS10 strain may change the feeding preference of the larvae, or cause some adverse changes to the leaves eaten by the larvae, resulting in the larvae refusing to feed.

[0106] The analysis of the body weight situation is as Figure 10 shown, the body weight of the larvae in the T-P.putida-YS10 group was significantly lower than that in other groups. This significant difference indicates that the individual development of the larvae in this group was significantly inhibited. This inhibition may be due to insufficient nutrient intake caused by the larvae's feeding refusal, or the P.putida-YS10 strain itself has a direct negative impact on the growth and development of the larvae.

[0107] In terms of gene expression, Figure 11It was revealed that the expression level of the Actin gene in the larvae of the T-P.putida-YS10 group was significantly downregulated. As a key gene closely related to cell structure and function, the downregulation of the Actin gene expression may further affect the normal physiological functions of the larvae. Such changes in gene expression may be directly associated with physiological phenomena such as increased larval mortality and inhibited development.

[0108] It can be seen from this that the transmission of the P.putida-YS10 strain has a significant impact on the larvae of Plagiodera versicolora, including increasing mortality, inducing feeding refusal, inhibiting individual development, and downregulating the expression of key genes (such as the Actin gene). In contrast, the transmission of the P.putida strain and the P.putida-YS18 strain has relatively less impact on the larvae. These findings suggest that the P.putida-YS10 strain may exert a toxic effect on the larvae of Plagiodera versicolora through a certain specific mechanism, and thus has potential application value in the field of population control of Plagiodera versicolora. To further understand the mechanism of action of this strain and provide a scientific basis for its future development and utilization, it is necessary to carry out further research work.

[0109] The above results indicate that RNAi based on horizontal transmission of gut microbiota is effective for pest population control.

[0110] In summary, this application provides a brand-new perspective and method for controlling pests by bacteria-mediated RNAi. RNAi mediated by gut microbiota can be rapidly horizontally transmitted among populations and achieve population control, and is expected to be used for large-scale application of RNAi in controlling plant diseases and pests. This application has broad application prospects in the field of population control of Plagiodera versicolora. On the one hand, it can be developed into a new type of biological pesticide, and using its multiple effects on Plagiodera versicolora, such as direct insecticidal effect, growth inhibition, and feeding influence, etc., to effectively control the population quantity of Plagiodera versicolora and reduce its damage to plants such as willows. On the other hand, by further in-depth study of its mechanism of action, especially clarifying the specific roles of substances such as dsRNA in the processes of insecticidal effect, growth and development influence, and gene expression regulation, etc., it is expected to optimize the construction of engineering strains, improve the control effect, provide more effective means and theoretical support for population control technology, and at the same time reduce the use of chemical pesticides, meeting the requirements of green and environmental-friendly agricultural development.

[0111] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for controlling the population of blue beetles based on RNAi and the horizontal transmission characteristics of intestinal microorganisms, characterized in that: The steps include: S(1-1) backbone 1 was amplified from pBBR403gfp plasmid using specific primer pair SEQ ID NO: 1-2; S(1-2) using a specific primer pair SEQ ID NO: 3-4 and pYQ11 plasmid as a template to amplify and obtain a target fragment 1; the target fragment 1 contains SEQ ID NO: 16; the SEQ ID NO: 16 encodes a double-stranded RNA that targets and silences the expression of the Actin gene of the blue leaf beetle; S(1-3) using a specific primer pair SEQ ID NO: 5-6 and pUC18T-mini-Tn7T-Gm plasmid as a template to amplify the target fragment 2; the target fragment 2 contains a T7 polymerase gene sequence as shown in SEQ ID NO: 18; S(1-4) Simultaneously transforming the backbone 1, target fragment 1 and target fragment 2 into competent E. coli DH5α; culturing for 2 hours without selection pressure, screening positive clones and performing plasmid extraction and sequence identification to obtain the target plasmid pYS10; S(2) transforming plasmid pYS10 into Pseudomonas putida competent cells, culturing for 2 h without selection pressure, screening positive clones, verifying by PCR and sequencing, and obtaining the engineered strain P. putida-ACT; S(3) Feed the larvae of the blue leaf beetle with leaves treated with P. putida-ACT solution, and the concentration of live bacteria on the leaves is ≥10 9 CFU / cm 2 .

2. The method according to claim 1, characterized in that In step S(2), the preparation of Pseudomonas putida competent cells comprises the following steps: culturing Pseudomonas putida in liquid LB medium for 8 hours, then placing it on ice together with a sterile glycerol solution for 30 minutes for standby use; performing refrigerated centrifugation at 5000 rpm for 10 minutes, collecting the bacterial cells, adding the pre-cooled glycerol solution, shaking for 30 seconds, and standing on ice for 30 minutes; performing refrigerated centrifugation at 5000 rpm for 10 minutes again, collecting the bacterial cells, adding 1 mL of sterile glycerol solution, mixing, and then packaging, and placing it at -80°C for standby use; the mass ratio of the glycerol solution is 15%.

3. The method according to claim 2, characterized in that In step S(2), the transformation comprises the following steps: adding 10 μL of the target plasmid pYS10 to the Pseudomonas putida competent cells, mixing well and transferring to an electroporation cup, electrotransforming at 2300 V, and then transferring the cells to liquid LB culture medium.

4. The method according to claim 1, characterized in that: In steps S(1-4) and (2), the positive clones are screened by coating on LB solid culture medium containing 50 mg / L gentamicin.

5. The method according to claim 1, characterized in that Step S(3) further includes: adding the engineered strain P. putida-ACT to a bacterial solution with a concentration of 1×10 9 CFU / cm 2 Apply it on willow leaves, let the leaves dry and feed it to the larvae of willow blue leaf beetles.

6. The method according to claim 1, characterized in that Step S(3) also includes: selecting more than one third of the larvae in the population as treatment objects, and feeding them with leaves containing P. putida-ACT bacterial solution.

7. The method according to claim 6, characterized in that The larvae are in the early second instar.

8. Use of the method according to any one of claims 1 to 7 in plant disease and insect pest control.

9. The use according to claim 8, characterized in that: Used to prepare engineered bacteria for preventing and controlling plant diseases and insect pests.

10. Use of the method according to any one of claims 1 to 7 in the preparation of biological pesticide products.

Images