CN112410345B - Eggplant dioctadecylate ladybug FTZ-F1 gene and application thereof in preventing and controlling eggplant twenty-eight-star ladybug - Google Patents

Abstract

The invention discloses the FTZ-F1 gene of the solanum 28-star ladybug and its application in preventing and treating the solanum 28-star ladybug. The FTZ-F1 gene of the solanum 28-star ladybug has a nucleotide sequence such as SEQ Its dsRNA is shown in ID NO.1, and the nucleotide sequence of one strand of the dsRNA is shown in SEQ ID NO.2. The present invention obtains a FTZ‑F1 gene of ladybug Solanum 28, which plays an important role in molt and pupation of ladybug Solanum 28, directly feeding dsFTZ‑F1 finally leads to 28 Solanum The death of a ladybug. The method has many advantages such as high specificity, convenient operation, high insecticidal efficiency, and environmental friendliness, and has a good application prospect.

Description

FTZ-F1 Gene of Ladybug Solanum and Its Function in Controlling Ladybug Solanum application

technical field

The invention belongs to the technical field of insect pest prevention and control, and more specifically relates to the FTZ-F1 gene of the ladybird solanum and its application in preventing and controlling the ladybird solanum.

Background technique

Henosepilachna vigintioctopunctata belongs to the order Coleoptera and the ladybug family. It is one of the most serious economic pests in Asia. It hosts a wide range of plants. It mainly damages eggplant, potato and tomato and other solanaceous vegetables and cucurbit vegetables such as cucumbers. , Wax gourd and loofah etc. Its larvae and adults damage the leaves, calyxes and fruits of plants. They like to cluster on the back of the leaves and feed on the mesophyll. The damaged leaves usually form irregular transparent spots, and only reticular veins remain in severe cases. The ladybug has strong adaptability and fecundity, and has a wide distribution range in my country, especially in the south of the Yangtze River. In recent years, with the continuous expansion of planting area of vegetables such as Solanaceae, the occurrence and damage of ladybug Solanaceae have become more and more serious. Therefore, the prevention and control of this pest is urgent.

At present, the control of ladybug Solanum solani mainly relies on chemical insecticides. However, the long-term use of chemical insecticides will cause many problems such as pesticide residues, drug resistance and pest re-infestation. Therefore, it is urgent to find a new method for precise control and environmental friendliness to prevent and control the solanum beetle and reduce the impact on Dependence on chemical pesticides.

RNA interference (RNA interference, RNAi) is a phenomenon that is highly conserved during evolution and relies on the production of short fragments of RNAs (siRNAs), thereby promoting the efficient and specific degradation of homologous mRNAs. The inventor's team found in the previous research that the toxicity to ladybugs can be achieved by directly feeding suitable exogenous dsRNA. Therefore, according to the principle of RNAi technology, the development of exogenous dsRNA products that can be directly fed can be highly efficient and safe. It is easy to use and low in cost to prevent and control insect pests, and can realize precise prevention and control of ladybugs.

Contents of the invention

The object of the present invention is to overcome the deficiencies in the prior art, and provide a FTZ-F1 gene of ladybird solanum and its application in preventing and controlling ladybird solanum. The present invention utilizes the method of directly feeding FTZ-F1 gene dsRNA (dsFTZ-F1), so that the larvae of the 28-star eggplant beetle fail to molt or pupate and die, thereby achieving the purpose of efficient and precise control of the 28-star eggplant ladybird . The method has many advantages such as quick effect, convenient operation, high insecticidal efficiency, and environmental friendliness, and has a good application prospect.

The first object of the present invention is to provide a kind of FTZ-F1 gene of ladybug solani.

The second object of the present invention is to provide a dsRNA of the FTZ-F1 gene of ladybug solani.

The third object of the present invention is to provide the application of any one of the FTZ-F1 gene and/or its expression inhibitor in the prevention and control of ladybug solani, or in the preparation of products for preventing and controlling ladybug solani.

The fourth object of the present invention is to provide any of the FTZ-F1 gene and/or its expression inhibitor in inhibiting the growth of ladybug solanum and/or preparing products for inhibiting the growth of ladybird solanum Applications.

The fifth object of the present invention is to provide any of the FTZ-F1 gene and/or its expression inhibitor in promoting the death of ladybug Solanum 28 and/or preparing products that promote the death of ladybug 28 Applications.

The 6th object of the present invention is to provide a kind of method of preventing and treating ladybird solanum.

The seventh object of the present invention is to provide a kind of product of preventing and treating ladybird solanum.

In order to achieve the above object, the present invention is achieved through the following technical solutions:

The present invention discovers the FTZ-F1 gene of the lady beetle solani, and develops the technology of feeding the dsRNA (dsFTZ-F1) of the FTZ-F1 gene to prevent and control the lady beetle. In the present invention, eggplant leaves are respectively soaked in the solutions of dsFTZ-F1 and dsGFP, taken out and dried, and then fed to the newly hatched 1st instar larvae of ladybug Eggplant for 2 days, and then fed with eggplant leaves not treated with dsRNA. Feed, observe and record the mortality and developmental status of ladybug Solanum solanum; In addition, in order to measure the influence of dsFTZ-F1 on the pupation of ladybug Solanum 28, feed the new molting with a solution containing 100ng dsFTZ-F1 The pupation status of the 4th instar larvae was observed, and then the insecticidal activity of exogenous dsFTZ-F1 on Ladybug solani was comprehensively evaluated. Finally, the method of fluorescent quantitative PCR (RT-qPCR) was used to detect and analyze the expression level changes of FTZ-F1 gene in ladybugs that fed on dsFTZ-F1 and dsGFP Solanum. The results showed that most of the 1st instar larvae fed dsFTZ-F1 failed to molt and died, and all 4th instar larvae failed to pupate and died, and the gene expression of FTZ-F1 in larvae treated with dsFTZ-F1 decreased significantly. This shows that direct feeding of exogenous dsFTZ-F1 has a very significant and highly lethal effect on Ladybug solani.

Therefore, the following invention claims the following:

A FTZ-F1 gene of ladybug Solanum solani, the nucleotide sequence of which is shown in SEQ ID NO.1.

A dsRNA of the FTZ-F1 gene of ladybug Solanum solani, the nucleotide sequence of one strand of the dsRNA is shown in SEQ ID NO.2.

The application of any one of the FTZ-F1 gene and/or its expression inhibitor in the prevention and treatment of ladybug infestation and/or in the preparation of products for preventing and controlling the infestation of ladybird solanum.

Use of any of the FTZ-F1 gene and/or its expression inhibitors in inhibiting the growth of the ladybug solani and/or preparing a product for inhibiting the growth of the ladybug.

The use of any of the FTZ-F1 gene and/or its expression inhibitor in promoting the death of the ladybug solani and/or preparing a product for promoting the death of the ladybug.

Preferably, the expression inhibitor is dsRNA.

More preferably, the dsRNA is the dsRNA shown in SEQ ID NO.2.

The invention discloses a method for preventing and controlling the ladybird solanum, feeding exogenous dsRNA, and the dsRNA can silence/repress the expression of the FTZ-F1 gene.

Preferably, the dsRNA is the dsRNA shown in SEQ ID NO.2.

A product for preventing and controlling ladybird solanum, containing the expression inhibitor of the FTZ-F1 gene.

Preferably, the expression inhibitor is dsRNA.

More preferably, the dsRNA is the dsRNA shown in SEQ ID NO.2.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention obtains a FTZ-F1 gene of ladybug Solanum 28, which plays an important role in molt and pupation of ladybug Solanum 28, directly feeding dsFTZ-F1 finally leads to 28 Solanum Ladybugs die. The method has many advantages such as high specificity, convenient operation, high insecticidal efficiency, and environmental friendliness, and has a good application prospect.

Description of drawings

Figure 1 is the effect of different concentrations of dsFTZ-F1 on the mortality of the 1st instar larvae of Ladybug solani. Using data on larval mortality 10 days after the start of the experiment, survival curves were constructed using a Cox regression procedure. Different letters (such as a, b) indicate significant differences between the control curve and the treatment curve.

Figure 2 shows the phenotype difference between the normally developed dsGFP control group (B) and the dead dsFTZ-F1 treatment group (A) on the third day after the first instar larvae were fed with dsRNA.

Fig. 3 shows the changes of FTZ-F1 gene expression in ladybug Solanum solani on the second day after the 1st instar larva eats dsFTZ-F1 and dsGFP.

Figure 4 is the effect of feeding 100ng dsFTZ-F1 on the survival rate of the 4th instar larvae of Ladybug solani.

Figure 5 shows the phenotype difference between the normal pupated dsGFP control group (A) and the dead dsFTZ-F1 treatment group (B) on the 7th day after the 4th instar larvae were fed with dsRNA.

Fig. 6 shows the changes of FTZ-F1 gene expression in ladybug Solanum solani on the 2nd and 4th days after the 4th instar larvae fed on dsFTZ-F1 and dsGFP.

Detailed ways

The present invention will be further elaborated below in combination with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

The ladybugs used in the following examples were collected from Solanum nigrum on the campus of South China Agricultural University in June 2019, and then raised in an incubator with eggplant leaves (temperature 25±1°C, humidity 70%- 80%, photoperiod L:D=14:10).

RNA was extracted using TRIzol extraction method (Invitrogen, USA), reverse transcription reagent (PrimeScript ^TM RTreagent Kit with gDNA Eraser) was purchased from TAKARA Biotechnology Co., Ltd., dsRNA synthesis kit (MEGAscript ^TM T7) was purchased from Thermo Fisher Scientific, PCR reaction The kit used in the system (2×EasyTaq pcr Super Mix) was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the DNA purification and recovery kit (SteadyPureDNA) was purchased from TAKARA Biotechnology Co., Ltd.

法(Ct表示循环数)进行计算。应用SPSS 19.0软件采用单因素方差分析进行数据分析。The data processing method of the following examples: use Excel 2019 to carry out statistics on the survival rate of the ladybug solani, use SPSS 19.0 software to use Cox regression analysis to map, and analyze the difference between different concentrations using one-way analysis of variance. Analysis of the expression level changes of target genes after RNA interference, RT-qPCR data using

method (Ct represents the number of cycles) for calculation. Data analysis was performed using SPSS 19.0 software using one-way analysis of variance.

Example 1 Obtaining of growth and development-related gene FTZ-F1 dsRNA

The FTZ-F1 gene was obtained according to the previous transcriptome sequencing, and the nucleotide sequence of its gene fragment is shown in SEQ ID NO.1. The dsRNA of the gene FTZ-F1 (dsFTZ-F1) was further synthesized, and its sequence is shown in SEQ ID NO.2.

1. Experimental method

1. Total RNA extraction and cDNA first-strand synthesis of ladybug solanum.

Take 10 2nd instar larvae of Ladybug solani in a 2mL centrifuge tube, use the TRIzol method to extract total RNA, measure the concentration of RNA with NanoDropOne ^C , use the reverse transcription kit (PrimeScriptTM RT reagent Kit ^withgDNA Eraser, TAKARA) to synthesize first-strand cDNA.

2. Primer design

According to the obtained FTZ-F1 gene sequence (SEQ ID NO.1), the dsRNA primer P1 (Table 1) of the FTZ-F1 gene was designed, and the green fluorescent protein gene (GFP) was amplified from the GFP-containing plasmid preserved in the laboratory Obtain, dsRNA primer P2 of GFP gene (Table 1). RT-qPCR primer P3 of FTZ-F1 gene and RT-qPCR primer P4 of internal reference gene RPS18 were designed (Table 1).

Table 1: dsRNA synthesis and qPCR primers

3. The kit synthesizes dsRNAs of FTZ-F1 gene and GFP gene

Use primers P1 and P2 in Table 1 for PCR amplification. The reaction system for PCR amplification is 25 μL of 2×Easy Taq PCRSuper Mix, 1 μL of upstream primers, 1 μL of downstream primers, 1 μL of cDNA/GFP plasmid, and filled with dd H ₂ O to 50 μL. The reaction program of PCR amplification was pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 1min, 30 cycles; extension at 72°C for 10min. Store the amplified product at -20°C. The amplification results were detected by agarose gel electrophoresis.

Use the DNA purification and recovery kit (SteadyPure DNA, TAKARA) to recover and purify the two PCR products obtained above, as a template for in vitro transcription of dsRNA, the in vitro transcription system of dsRNA is 10x Reaction Buffer 5 μL, (ATP, GTP, CTP, UTP) solution Each 5 μL, Enzyme mix 5 μL, template 20 μL, ddH ₂ O make up to 50 μL. Place at 37°C for 4h. After the reaction, add 2.5 μL of TURBO DNase to remove residual template DNA, then purify dsRNA, and finally dissolve dsRNA with 50 μL ddH ₂ O to obtain purified dsFTZ-F1 and dsGFP, respectively, and verify the dsRNA bands by agarose gel electrophoresis.

2. Experimental results

Use the P1 primer to amplify the PCR amplification product with a size of 399bp. After sequencing and deleting the T7 promoter sequence, the nucleotide sequence with a size of 359bp is the target gene FTZ-F1. The nucleotide sequence is as shown in SEQ Shown in ID NO.2. Using the GFP-carrying plasmid as a template, the PCR product dsGFP with a size of 507 bp was amplified with the P2 primer. The target bands of dsGFP and dsFTZ-F1 were consistent with the sequencing results.

Example 2 Inhibitory Effect of dsFTZ-F1 on Ladybug Solanum

1. The lethal effect of dsFTZ-F1 on the 1st instar larvae of Ladybug solani

Ladybug dsFTZ-F1 feeding group: 10 1st instar larvae of ladybird solani were placed in a petri dish with filter paper and humidified cotton balls. Soak circular eggplant leaf discs with a diameter of 12 mm in ds FTZ-F1 (prepared in Example 1) solutions with a concentration of 100 ng/μL, 50 ng/μL, and 5 ng/μL for 1 min, air-dry them at room temperature for 20 min, and then feed the larvae, replacing them every 24 hours. Leaf disks once, after continuous feeding of dsFTZ-F1 soaked leaf disks for 2 days, larvae were fed with normal eggplant leaves.

Solanum solanum dsGFP feeding group: 10 1st instar larvae of solanum solani were placed in a petri dish with filter paper and humidified cotton balls. Use the dsGFP (prepared in Example 1) solution with a concentration of 100 ng/μL synthesized by the kit to soak a circular eggplant leaf disc with a diameter of 12 mm for 1 min, air-dry it for 20 min, then feed the larvae, replace the leaf disc every 24 hours, and continuously feed the dsGFP soaked 2 days later, the larvae were fed with normal eggplant leaves.

Each group is set with 5 repetitions, counts the number of dead ladybugs in each petri dish every 24h, and replaces new leaves, and the petri dish is placed in an artificial climate box (temperature 25 ± 1 ℃, humidity 70%) -80%, photoperiod L:D=14:10). The number of dead ladybugs in each petri dish of each group was counted, and the survival rate of ladybugs treated with dsGFP control group and different concentrations of dsFTZ-F1 was calculated.

2. The lethal effect of dsFTZ-F1 on the 4th instar larvae of Ladybug solani

Ladybug dsFTZ-F1 feeding group: put a 4th instar larva that was starved for 4 hours in a petri dish, drop 1 μL of 100ng/μL dsFTZ-F1 (prepared in Example 1) solution, wait until The larvae completely consumed the dsRNA solution, put fresh eggplant leaves and humidified cotton balls, and set up 10 experiments in total, with 3 repetitions in each group.

Ladybug dsGFP feeding group: put a 4th instar larva that has been starved for 4 hours in a petri dish, drop 1 μL of 100 ng/μL dsGFP (prepared in Example 1) solution, and wait until the larvae completely consume dsRNA solution, put fresh eggplant leaves and humidified cotton balls, set up 10 experiments in total, and set 3 repetitions in each group.

Every 24h counts the number of dead ladybugs in each petri dish, and replaces new blades, and the petri dish is placed in an artificial climate box (25 ± 1°C of temperature, 70%-80% of humidity, photoperiod L :D=14:10). Count the number of dead ladybugs in each petri dish of each group, and calculate the change in the survival rate of ladybugs in control and different treatment groups.

2. Experimental results

According to the statistical results, after two days of continuous feeding of the 1st instar larvae dsFTZ-F1, the survival rate of the 1st instar larvae decreased with the increase of the concentration of dsFTZ-F1. Trend (Figure 1), the feeding concentrations of the treatment groups were 5ng/μL, 50ng/μL and 100ng/μL, respectively, and the feeding concentration of the control group was 100ng/μL. According to the results in Figure 1, it was found that there were significant differences between the treatment groups with different concentrations and between the control groups (χ2=78.919, df=3, p<0.0001). Treatment group 50ng/μL (p<0.0001, Exp(B)=0.429) and 5ng/μL (p<0.0001, Exp(B)=0.027) and 100ng/μL (p<0.0001, Exp(B)=0.702) did not Significant difference; there was a significant difference between 5ng/μL (p<0.0001, Exp(B)=0.027) and 100ng/μL (p<0.0001, Exp(B)=0.702). The following conclusions can be drawn from the statistical results. When the concentration of the treatment group was 5ng/μL, 50ng/μL and 100ng/μL, the mortality rate was increased by 16.25 times, 21 times and 23 times compared with the control group.

Compared with feeding dsGFP, the phenotypic characteristics of ladybugs were significantly changed after feeding dsFTZ-F1. It was found that the 1st instar larvae started to feed dsRNA on the 3rd day, and the dsGFP control group's larvae normally entered the 2nd instar stage, while the larvae in the dsFTZ-F1 treatment group could not molt normally and died, and the phenotype characteristics were as follows: Figure 2 shows.

In addition, on the 7th day after the 4th instar larva was fed with dsFTZ-F1, the ladybug Solanum solani in the dsGFP control group entered the pupal stage normally, while the larvae in the treatment group gradually turned black, unable to pupate normally and all died ( Figure 4, Figure 5). It shows that eating dsFTZ-F1 can trigger a strong RNAi effect in the body of ladybug Solanum solani, resulting in the death of ladybug larvae.

Example 3 dsFTZ-F1 inhibits the expression of FTZ-F1 gene in ladybird Solanum solani

1. Experimental method

Collect 5ng/μL dsFTZ-F1 and dsGFP treatment of 1st instar larvae on the 2nd day and 100ngdsFTZ-F1 and dsGFP treatment on the 2nd day and 4th day 4th instar ladybug For larvae, 3 biological replicates were collected for each treatment. The RNA of Ladybug solani was extracted, reverse-transcribed into cDNA, and diluted 10 times as a template for RT-qPCR. RT-qPCR analysis was performed with P3 and P4 as primers. The RT-qPCR system (15 μL) contained 5.25 μL of ddH ₂ O, 7.5 μL of 2×SYBRGreen Master Mix (BIO-RAD Inc, Hercules, CA), 4 μM primers and 1.0 μL of cDNA first-strand template. The reaction conditions were 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, 39 cycles, with 3 technical replicates for each sample.

2. Experimental results

Taking dsGFP as a control, the relative expression changes of FTZ-F1 gene in the 1st and 4th instar larvae of Ladybug solani were analyzed after feeding dsFTZ-F1 (as shown in Figures 3 and 6). It can be seen from Figure 3 and Figure 6 that compared with the expression level of FTZ-F1 in ladybugs solanum spp. The expression level showed an obvious downward trend. On the second day after the first instar larvae were fed with dsFTZ-F1, the expression of FTZ-F1 gene decreased by 2.42 times compared with the control group (F _1,4 =70.743, p<0.0001); On the second day and the fourth day after dsFTZ-F1 started, the expression of FTZ-F1 gene decreased by 4.23 times compared with the control group (F _1,4 =527.282, p<0.0001) and 3.34 (F _1,4 = 294.139, p<0.0001), further illustrating that feeding dsFTZ-F1 can cause a strong RNAi effect in the ladybug Solanum solani, resulting in a significant decrease in the expression of the FTZ-F1 gene in the body, and the 1st instar larvae cannot molt. The 4th instar larvae could not successfully pupate, which led to the death of the ladybug Solanum.

The above-mentioned embodiment is a preferred implementation mode of the present invention, but the implementation mode of the present invention is not limited by the above-mentioned embodiment, any person familiar with the art can make Changes, modifications, substitutions, combinations, etc., should all be equivalent replacements, and are all included within the protection scope of the present invention.

sequence listing

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Claims (5)

1.FTZ-F1The application of any one of the genes and/or the expression inhibitor thereof in preventing the twenty-eight star gourd ladle insect pest and/or preparing a product for preventing the twenty-eight star gourd ladle insect pest is characterized in that the nucleotide sequence of the FTZ-F1 gene is shown as SEQ ID NO. 1.

2.FTZ-F1The application of any one of the gene and/or the expression inhibitor thereof in inhibiting the growth of the harmonia axyridis and/or preparing a product for inhibiting the growth of the harmonia axyridis is characterized in that the nucleotide sequence of the FTZ-F1 gene is shown as SEQ ID NO. 1.

3.FTZ-F1The application of any one of the gene and/or the expression inhibitor thereof in promoting the death of the harmonia axyridis and/or preparing a product for promoting the death of the harmonia axyridis is characterized in that the nucleotide sequence of the FTZ-F1 gene is shown as SEQ ID NO. 1.

4. The use according to any one of claims 1 to 3, wherein the expression inhibitor is dsRNA.

5. Prevention and treatment eggplantMethod for the production of a Dastaria ladybug, characterized in that feeding an exogenous dsRNA which silences/inhibits the dsRNAFTZ-F1And (3) expressing the gene, wherein the nucleotide sequence of the FTZ-F1 gene is shown as SEQ ID NO. 1.

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