DsRNA of pinus massoniana gene11 gene and application of dsRNA

Abstract

The invention relates to the technical field of agricultural biology, in particular to dsRNA of a pinus massoniana gene11 gene and application of the dsRNA, and the dsRNA is composed of a sense RNA chain shown in SEQ ID NO: 1 and an antisense RNA chain reversely complementary with the sense RNA chain. According to the dsRNA of the pine caterpillars gene11 gene and the preparation method and application thereof, the dsRNA is injected into pine caterpillars, target genes of the pine caterpillars are efficiently silenced, the death rate of the pine caterpillars can be increased, feeding behavior taxis is reduced, food digestion is affected, and finally the prevention and treatment effect is achieved; and a new effective way is provided for preventing and treating the pine caterpillars.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a dsRNA of the gene11 gene of the pine caterpillar and its application. Background Technology

[0002] Pine caterpillars (Dendrolimus) are a collective term for insects belonging to the order Lepidoptera, family Phyllotyridae, and genus Dendrolimus. They are major forest pests that cause large-scale outbreaks and severe damage. A typical example is the pine caterpillar (Dendrolimus punctatus), whose larvae feed on pine needles. During outbreaks, entire pine forests can be completely devastated within days, appearing withered, charred, and burned, often referred to as "smokeless forest fires," causing significant economic losses to forestry. With the development of molecular biology techniques, modern pine caterpillar control urgently requires a green, efficient, and precise molecular regulation technology.

[0003] RNA interference (RNAi) via double-stranded RNA (dsRNA) refers to the phenomenon where an organism induces the degradation of target RNA through endogenous or exogenous small RNAs. RNA interference technology is highly specific, harmless to non-target species, and has great application potential. This technology has become an important option for developing new methods of forest pest management, and genes related to increased mortality and impacts on feeding and digestion can serve as promising candidate target genes for RNA interference in the larval stage of the pine caterpillar. Summary of the Invention

[0004] The purpose of this invention is to provide a dsRNA of the gene11 gene of the pine caterpillar and its application in the control of the pine caterpillar, so as to solve the problems of environmental pollution, increased insect resistance and poor safety to non-target organisms caused by existing chemical control methods, and to provide a new and effective way for green and precise control of the pine caterpillar.

[0005] To achieve the above objectives, the present invention provides the following technical solution: On one hand, the present invention provides a dsRNA of the gene11 gene of the pine caterpillar, wherein the dsRNA consists of the sense RNA strand shown in SEQ ID NO:1 and its antisense RNA strand, which is inversely complementary to the sense RNA strand. Further, the nucleotide sequence of the gene11 gene of the pine caterpillar targeted by the dsRNA is shown in SEQ ID NO:2.

[0006] Accordingly, the present invention provides a method for preparing the above-mentioned dsRNA, comprising the following steps: (1) Using the gene11 gene of the pine caterpillar shown in SEQ ID No.2 as a template, a DNA template for in vitro transcription was obtained by PCR amplification. (2) The DNA template obtained in step (1) is used for in vitro transcription to synthesize the dsRNA.

[0007] As a preferred embodiment of the present invention, in step (1), the primer pair used for PCR amplification is DS11-F (SEQ ID NO.3) and DS11-R (SEQ ID NO.4), and the 5′ end of DS11-F (SEQ ID NO.3) and DS11-R (SEQ ID NO.4) is respectively added with T7 promoter (SEQ ID NO.5) to ensure efficient in vitro transcription.

[0008] In a preferred embodiment of the present invention, in step (2), the in vitro transcription uses MEGAscript. ® The RNAi Kit is now complete. It is easy to use, has a high yield, and produces dsRNA with good integrity.

[0009] Furthermore, this invention also provides the application of the dsRNA in the preparation of biological agents for controlling pine caterpillars. Experiments have shown that after the dsRNA is administered to pine caterpillar larvae via microinjection, it can significantly downregulate the expression level of the gene11 gene, causing larval growth inhibition, increased mortality, and a sublethal effect, resulting in decreased food intake and reduced digestion and absorption capacity of the pine caterpillars.

[0010] As a preferred embodiment of the present invention, the dosage of the dsRNA is 10 μg per pine caterpillar larva, at which a significant gene silencing effect and control effect can be achieved.

[0011] As a preferred embodiment of the present invention, the pine caterpillar is in the middle of the 3rd instar, at which time the pine caterpillar larva has moderate epidermal permeability and is sensitive to RNAi response, resulting in high control efficiency.

[0012] Compared with the prior art, the present invention has the following beneficial effects: by injecting dsRNA into the body of the pine caterpillar, the present invention effectively silences the target gene of the pine caterpillar, which can increase the mortality rate of the pine caterpillar, reduce feeding behavior tropism and affect food digestion, and ultimately achieve the effect of prevention and control, providing a new and effective way to control the pine caterpillar. Attached Figure Description

[0013] Figure 1 This is an electrophoretic detection diagram of dsRNA in this invention; Figure 2 The expression level of the gene11 gene in the pine caterpillar after injection of dsRNA. Figure 3 Survival rate of pine caterpillars after injection of dsRNA; Figure 4 The relative growth rate of the pine caterpillar after injection of dsRNA; Figure 5 Food utilization rate of *Pinus massoniana* after dsRNA injection; Figure 6 The approximate consumption rate of pine caterpillars after dsRNA injection is given. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed under conventional conditions, such as the specific methods listed in Molecular Cloning: A Laboratory Manual (3rd Edition, by J. Sambrook et al.), or according to the kit and product instructions; the actual and biological materials mentioned are commercially available unless otherwise specified.

[0016] Example 1: Preparation of dsRNA to inhibit feeding and digestion by pine caterpillars A search of the genome data of the pine caterpillar (GenBank: GCA_012273795.1) revealed the gene11 (SEQ ID NO.2) on chromosome 15. Primer pairs DS11-F (SEQ ID NO.3) and DS11-R (SEQ ID NO.4) were designed targeting the specific sequence of the pine caterpillar gene11 (SEQ ID NO.2).

[0017] T7 promoter (SEQ ID NO.5) was added to the 5′ end of DS11-F (SEQ ID NO.3) and DS11-R (SEQ ID NO.4), respectively. Using the gene11 gene (SEQ ID NO.2) as a template, PCR amplification was performed using the modified primer pair to obtain a DNA template for in vitro transcription.

[0018] The PCR amplification program was set as follows: pre-denaturation at 94℃ for 3 min; followed by 35 cycles, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 50℃ for 30 s, and extension at 72℃ for 1 min; after the cycle, final extension at 72℃ for 10 min.

[0019] Using the green fluorescent protein (GFP) gene as a control, the corresponding dsRNA (dsGFP) was synthesized using the same method as a negative control for subsequent functional verification.

[0020] Refer to MEGAscript ® The RNAi Kit instructions were followed to synthesize dsRNA. The concentration of dsRNA was detected using a micro spectrophotometer. 1 μL of dsRNA was subjected to 1% agarose gel electrophoresis to confirm its integrity. It was then stored at -80°C for later use.

[0021] Please see Figure 1 , Figure 1 The title above lane 2, ds11, is short for dsgene11, corresponding to dsRNA. Figure 1 Lanes 1-3 were designated as Marker, dsRNA, and dsGFP, respectively. Electrophoresis results showed that both dsRNA and dsGFP exhibited non-diffuse degradation bands, indicating that double-stranded RNA of the gene11 gene from the pine caterpillar was obtained in this embodiment. Sequence verification and electrophoretic analysis confirmed that the synthesized dsRNA was 387 bp in length and the synthesized dsGFP was 431 bp in length.

[0022] Example 2: Detection of RNA interference effect on the gene11 gene of the pine caterpillar 10 μg of dsRNA was microinjected into healthy, uniformly growing 3rd instar pine caterpillars. The control group was injected with an equal amount of dsGFP. Samples were collected 24 h later, flash-frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted from the pine caterpillars using the RNAprep Pure Polysaccharide-Polyphenol Plant Total RNA Extraction Kit (provided by Tiangen Biotech Co., Ltd.). Then, 1 ng of RNA was extracted using GoScript. TM The Reverse Transcription System kit (provided by Promega, USA) was used to synthesize the first strand of cDNA. The cDNA product was diluted to 200 ng / μL, and used as a template for quantitative real-time RT-PCR to detect the expression level of the gene11 gene in the pine caterpillar after injection. The results are as follows: Figure 2 As shown.

[0023] Please see Figure 2 Compared with microinjection of dsGFP, the expression level of gene11 in pine caterpillars injected with microinjection of dsRNA decreased significantly within 24 h, indicating that dsRNA had a significant effect.

[0024] Example 3: Survival rate of *Pinus massoniana* after gene11 gene silencing. 10 μg of dsRNA was microinjected into the abdomen of mid-3rd instar larvae of the pine caterpillar (*Pinus massoniana*). An equal amount of dsGFP was injected into the control group, and a blank control (CK) was also included. A second injection was administered on the third day after the initial injection to ensure continued reduction of the *gene11* gene in the pine caterpillar. After injection, the larvae were fed fresh two-year-old pine needles, and the survival rate was observed and recorded daily (n=40). Around 10 days later, most larvae entered the 4th instar. Most larvae in the ds11 group failed to survive to the 4th instar, while those that did survive to the 4th instar were generally viable. Therefore, data was recorded up to day 12. Results are as follows: Figure 3 As shown.

[0025] Please see Figure 3 After interfering with the gene11 gene of the pine caterpillar, the survival rate of the third instar larvae was significantly lower than that of the dsGFP group and the CK group, indicating that low expression of the gene11 gene in the pine caterpillar affects the survival rate of the third instar larvae.

[0026] Example 4: Feeding status of pine caterpillars after gene11 gene silencing. Before the experiment, mid-third instar pine caterpillars were prepared and weighed using an analytical balance to ensure that the size and shape of the test insects remained within a certain range. After starvation for 6 hours and emptying their feces, their initial fresh weight was measured. 10 μg of dsRNA was microinjected into the abdomen of mid-third instar pine caterpillar larvae. The control group was injected with an equal amount of dsGFP, and a blank control (CK) was set up. Fresh two-year-old pine needles were used for insect rearing. After 48 hours, the remaining feed was removed and its fresh weight was measured. After starvation for 6 hours, the fresh weight of the larvae and the fresh weight of their feces were measured. A control experiment without insects was also set up to measure the weight change of the pine needles due to moisture loss during the experiment. Mortality occurred within 2 days after dsRNA injection; therefore, 15 replicates were set up for each group, and each insect was reared individually.

[0027] Calculate the relative growth rate, food utilization rate, and approximate consumption rate of the pine caterpillars in the experiment using the following formulas: In the formula, I: corrected feed intake; W: weight of the control feed without insects at the beginning of the experiment; L: weight of the control feed without insects at the end of the experiment; B: weight gain of the test insects. : Average weight of test insects during the experimental period; T: Number of experimental days; F: Amount of remaining feces.

[0028] like Figures 4 to 6As shown, compared with the GFP group and the CK group, the relative growth rate, food utilization rate and near consumption rate of the pine caterpillar were significantly reduced after injection of dsRNA. This indicates that interfering with the gene11 gene not only increases the mortality rate of the pine caterpillar, but also produces a sublethal effect, reducing the amount of food consumed by the pine caterpillar and decreasing its ability to digest and absorb food.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A dsRNA of the gene11 gene of the pine caterpillar, characterized in that: The dsRNA consists of the sense RNA strand shown in SEQ ID NO:1 and its antisense RNA strand, which is its anticomplement.

2. The dsRNA according to claim 1, wherein the nucleotide sequence of the gene11 gene of the pine caterpillar targeted by the dsRNA is shown in SEQ ID NO:

2.

3. A method for preparing dsRNA as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Using the gene11 gene of the pine caterpillar shown in SEQ ID No.2 as a template, a DNA template for in vitro transcription was obtained by PCR amplification. (2) The DNA template obtained in step (1) is used for in vitro transcription to synthesize the dsRNA.

4. The preparation method according to claim 3, characterized in that: In step (1), the primer pair used for PCR amplification is DS11-F and DS11-R, whose nucleotide sequences are shown in SEQ ID No.3 and SEQ ID No.4, respectively.

5. The preparation method according to claim 4, characterized in that: The 5′ ends of the DS11-F and DS11-R are respectively supplemented with T7 promoters, and the nucleotide sequences of the T7 promoters are shown in SEQ ID No.

5.

6. The preparation method according to any one of claims 3 to 5, characterized in that: In step (2), the in vitro transcription uses MEGAscript. ® The RNAi Kit is now complete.

7. The use of the dsRNA as described in claim 1 or 2 in the preparation of a biological agent for controlling the pine caterpillar.

8. The use according to claim 7, characterized in that: The dsRNA was administered to the larvae of the pine caterpillar via microinjection.

9. The use according to claim 8, characterized in that: The dosage of the dsRNA was 10 μg per pine caterpillar larva.

10. The use according to claim 8 or 9, characterized in that: The larvae of the pine caterpillar are in the middle of the third instar.

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