CN116286815B - Application of wax synthesis gene PmFAR1 in Mealybug papaya in regulating Mealybug papaya - Google Patents

Abstract

The invention belongs to the field of agricultural biotechnology, relates to the technical field of prevention and control of Mealybug papaya, and specifically relates to the application of the waxy synthesis gene PmFAR1 of Mealybug papaya in regulating Mealybug papaya. In order to discover the wax synthesis gene of P. papaya that can be used to regulate P. papaya mealybug, the present invention uses RNA interference technology to reduce the expression level of P. papaya wax synthesis gene PmFAR1 and found that the death of P. papaya mealybug can be significantly improved. rate, significantly inhibited the growth and development of Mealybug papaya, significantly reduced the fecundity of Mealybug papaya and its affinity with the host, indicating that the PmFAR1 gene affects the affinity of Mealybug papaya with its host, especially in taking food. It plays an important role in colonization, survival and reproduction after feeding, suggesting that the wax synthesis gene PmFAR1 of P. papaya can be used in the regulation of P. papaya, providing a new way for the prevention and control of P. papaya.

Description

Application of wax synthesis gene PmFAR1 in Mealybug papaya in regulating Mealybug papaya

Technical field

The invention belongs to the field of agricultural biotechnology, relates to the technical field of prevention and control of Mealybug papaya, and specifically relates to the application of the waxy synthesis gene PmFAR1 of Mealybug papaya in regulating Mealybug papaya.

Background technique

Paracoccus marginatus Williams and Granara de Willink, also known as papaya mealybug, belongs to the order Hemiptera, the family Pseudococcidae, and the genus Paracoccus. It is one of the world's dangerous agricultural and forestry pests. , the harmful objects include cassava, fruit trees, vegetables, landscaping plants, etc. The damage is mainly caused by the sucking of nymphs and female adults, which in turn causes the stems to dry up, the leaves to become chlorotic, yellow, and fall off, and the fruit quality is reduced. In severe cases, Will cause the entire plant to die. Because of its short life history, strong fecundity, strong adaptability, easy development of drug resistance, and frequent disasters, the control of the papaya mealybug is very difficult. In the planting process of cassava, fruit trees, vegetables, landscaping plants, etc., currently we mainly rely on pesticides to prevent and control the damage caused by mealybugs. However, there are currently very few effective pesticides registered to prevent and control its damage, and due to the same The large number and high frequency of irrational use of pesticides has led to increasingly prominent problems such as the safety of agricultural products, pest resistance, and the safety of the ecological environment in the production areas, which has seriously restricted the healthy and sustainable development of cassava, fruit trees, vegetables, landscaping plants and other industries. Therefore, seeking green and efficient ways to prevent and control papaya mealybugs has become a major problem that needs to be solved urgently in the development of cassava, fruit trees, vegetables, landscaping plants and other industries.

Plant-mediated RNAi technology (RNA interference, RNAi) has become one of the hot spots in crop insect-resistant genetic engineering. RNAi technology is a phenomenon in which dsRNA (double-strand RNA) homologous to the target gene is introduced into living organisms to cause gene silencing. Compared with other genetic engineering research methods, RNAi technology has the advantages of specificity, safety and efficiency, and is widely used in the study of specific gene functions in biology. In recent years, the method of screening RNA target genes by using dsRNA in vitro feeding or injection to cause target gene expression and silencing has been widely used in the identification and functional analysis of key genes for insect growth and development.

Since the body wall of the mealybug has a thick wax layer, some drugs with low fat solubility cannot easily enter the target parts of the body of the mealybug due to their weak penetrating ability, resulting in the failure of chemical prevention and control. to the target requirements. The wax on the insect body surface can protect the insect body from water loss and the invasion of harmful external substances. The main component is hydrocarbons. Among them, the fatty acyl-CoA reductase encoded by the FAR gene is a key enzyme for the synthesis of hydrocarbons in insect epidermis and plays an important role in the reproduction and expansion of insect populations. Inhibiting FAR gene expression can inhibit wax synthesis and further inhibit the growth, development, reproduction and population growth of insects. Therefore, further digging into the wax synthesis genes of P. papaya that can regulate the feeding damage of P. papaya is expected to provide important scientific basis for the prevention and control of P. papaya. However, there have been no reports on the use of wax synthesis genes of Mealybug papaya for the control of Mealybug papaya.

Contents of the invention

In order to overcome the shortcomings of the above-mentioned existing technologies, the present invention uses RNA interference technology to inhibit the expression of the wax synthesis gene PmFAR1 of the mealybug P. papaya, thereby significantly increasing the mortality rate of the mealybug P. papaya, significantly inhibiting the normal development of the P. papaya mealybug, and significantly Reduce the fecundity of the papaya mealybug and its affinity with the host.

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

A first aspect of the present invention provides the application of a reagent that inhibits the expression of the wax synthesis gene PmFAR1 of Mealybug papaya and/or reduces the expression of the wax synthesis gene PmFAR1 of Mealybug papaya in the preparation of products for regulating Mealybug papaya, said The gene PmFAR1 has the nucleotide sequence shown in SEQ ID NO.3.

Preferably, the reagent is dsRNA that silences the wax synthesis gene PmFAR1 of Mealybug papaya.

Preferably, the regulation includes promoting the death of Mealybug papaya, inhibiting the growth and development of Mealybug papaya, reducing the fecundity of Mealybug papaya and its affinity with the host.

Based on the PmFAR1 transcript sequence information obtained by sequencing the transcriptome of P. papaya, the present invention clones P. papaya wax synthesis gene PmFAR1, whose nucleotide sequence is shown in SEQ ID NO. 3, and then uses the gene PmFAR1 as a target. , established an optimal silencing technology system for the wax synthesis gene PmFAR1 of the papaya mealybug, designed a dsRNA based on the wax synthesis gene PmFAR1 of the papaya mealybug, and then introduced it into papaya mealybug through feeding. Inside the body of the scale. The results showed that silencing or inhibiting the expression of the wax synthesis gene PmFAR1 of P. papaya mealybug can significantly increase the mortality rate of P. papaya mealybug after feeding on resistant and susceptible reference cassava varieties, and significantly inhibit the normal development and growth of P. papaya mealybug. Significantly reduced the fecundity of two-spotted spider mites and their affinity with the host, suggesting that the wax synthesis gene PmFAR1 of P. papaya can be used in the regulation of P. papaya.

More preferably, the dsRNA has the nucleotide sequence shown in SEQ ID NO.6.

More preferably, the synthetic primer for dsRNA includes an upstream primer as shown in SEQ ID NO. 4 and a downstream primer as shown in SEQ ID NO. 5.

The dsRNA synthesized based on the gene fragment of the wax synthesis gene PmFAR1 of Papaya mealybug is used to regulate Papaya mealybug. It has the advantages of species specificity, safety and high efficiency, and has good application in the green prevention and control of Papaya mealybug. The market application prospects and potential economic and ecological benefits can provide a new prevention and control approach for the green prevention and control of papaya mealybug.

Further, it also includes the encoding gene of the dsRNA, as well as the recombinant expression vector, transgenic cell line, recombinant bacteria, and expression cassette containing the dsRNA or its encoding gene.

Preferably, the types of products include but are not limited to pharmaceuticals, fertilizers, etc.

A second aspect of the present invention provides an agent for regulating Mealybug papaya, which agent inhibits the expression of the wax synthesis gene PmFAR1 of Mealybug papaya and/or reduces the expression level of the wax synthesis gene PmFAR1 of Mealybug papaya. The reagent serves as the main active ingredient, and the gene PmFAR1 has the nucleotide sequence shown in SEQ ID NO.3.

Preferably, the above-mentioned agent for regulating Mealybug papaya uses dsRNA that silences the wax synthesis gene PmFAR1 of Mealybug papaya as the main active ingredient, and the dsRNA has the nucleotide sequence shown in SEQ ID NO. 6, so The gene PmFAR1 has the nucleotide sequence shown in SEQ ID NO.3.

Preferably, the pharmaceutical agent also includes pesticide acceptable excipients.

More preferably, the auxiliary materials are selected from one or more of the following reagents: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, moisturizers, etc. Wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers Agent, plasticizer, surfactant, foaming agent, defoaming agent, thickener, inclusion agent, humectant, absorbent, diluent, flocculant and deflocculant, filter aid, release retardant.

The third aspect of the present invention provides a method for regulating the damage caused by Mealybug papaya to plants, specifically by: first inhibiting the expression of the wax synthesis gene PmFAR1 of Mealybug papaya and/or reducing the expression level of the wax synthesis gene PmFAR1 of Mealybug papaya. The reagent is sprayed on the plant leaves, and then the sprayed plant leaves are used to feed the Mealybug papaya, thereby achieving the purpose of regulating the damage of Mealybug papaya. The gene PmFAR1 has the nucleotide shown in SEQ ID NO.3 sequence.

Preferably, the above-mentioned method for regulating the damage caused by Mealybug papaya to plants is specifically: first spraying plant leaves with dsRNA that silences the wax synthesis gene PmFAR1 of Mealybug papaya, and the dsRNA has the nucleoside shown in SEQ ID NO.6 Acid sequence, the spraying concentration of the dsRNA is 500-1000ng/μL, and then the sprayed plant leaves are used to feed the mealybug papaya, thereby achieving the purpose of regulating the damage of the papaya mealybug. The gene PmFAR1 has the following SEQ The nucleotide sequence shown in ID NO.3.

Preferably, in the above-mentioned method for regulating damage caused by Mealybug papaya to plants, the plants include cassava. For example, the insect-susceptible reference cassava variety [South China 205 (SC205) (S)]. Of course, there are other host plants that can be damaged by the papaya mealybug.

Preferably, the spraying amount of dsRNA or other reagents is sprayed until the plant leaves are completely wet.

It should be noted that reagents other than dsRNA can also be used to act on plants through seed dressing or root irrigation, and the mode of action can be reasonably adjusted according to the characteristics of different reagents.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention discloses the application of PmFAR1, a waxy synthesis gene of P. papaya, in regulating P. papaya. After reducing the expression level of PmFAR1, a waxy synthesis gene of P. papaya, through RNA interference technology, the invention significantly improves P. papaya mealybug. The death rate of the scale significantly inhibited the growth and development of Mealybug papaya, significantly reduced the fecundity of Mealybug papaya and its affinity with the host, indicating that the gene PmFAR1 affects the affinity of Mealybug papaya with its host. , especially plays an important role in colonization, survival and reproduction after feeding, suggesting that the waxy synthesis gene PmFAR1 of P. papaya mealybug can be used in the regulation of P. papaya mealybug. The present invention has discovered for the first time that it can be used in P. papaya mealybug. The scale-regulated wax synthesis gene PmFAR1 provides a new way for the prevention and control of mealybugs, and has huge potential application prospects.

Description of the drawings

Figure 1 is a PCR amplification electrophoresis diagram of the wax synthesis gene PmFAR1 of Mealybug papaya (lane M is Trans2K DNAmarker; lane 1 is wax synthesis gene PmFAR1 of Mealybug papaya);

Figure 2 shows the relative expression of the wax synthesis gene PmFAR1 after Mealybug papaya feeds on cassava leaves treated with different concentrations of dsRNA;

Figure 3 shows the mortality rate of mealybug Mealybugs after feeding on cassava leaves treated with different concentrations of dsRNA;

Figure 4 shows the expression of the wax synthesis gene PmFAR1 after PmFAR1-silenced mealybugs feed on the leaves of resistant and susceptible cassava varieties;

Figure 5 shows the mortality rate of PmFAR1 silenced mealybugs after feeding on the leaves of resistant and susceptible cassava varieties;

Figure 6 shows the development period of PmFAR1-silenced P. papaya mealybug after feeding on the leaves of resistant and susceptible cassava varieties;

Figure 7 shows the number of eggs laid by a single female after the PmFAR1-silenced mealybug P. papaya feeds on the leaves of resistant and susceptible cassava varieties.

Detailed ways

The specific embodiments of the present invention will be further described below. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The test materials used in the following examples, unless otherwise specified, can be purchased through conventional commercial channels.

Example 1 Acquisition of wax synthesis gene PmFAR1 from Mealybug papaya

(1) Extract total RNA from Mealybugs papaya

1) Select the insect-susceptible reference cassava variety [South China 205 (SC205) (S)] obtained through insect resistance selection for indoor use and subculture 30 female adult mealybugs with the same development period and size in RNase- In a Free centrifuge tube, add 1mL of TRIzol, grind quickly with a grinding rod, immediately vortex vigorously to mix, and let stand at room temperature for 5 minutes;

2) Centrifuge (4°C, 12000×g) for 10 minutes, take the supernatant into a new EP tube;

3) Add 0.2 mL of chloroform to the EP tube, shake for 15 seconds, and let stand at 15-30°C for 3 minutes; centrifuge (4°C, 12000×g) for 15 minutes;

4) Take the colorless phase into a new EP tube, add an equal volume of isopropyl alcohol, mix upside down, and let stand at 15-30°C for 10 minutes;

5) Centrifuge (4°C, 12000 × g) for 10 minutes, aspirate the supernatant, add 1 mL of ice-cold 75% ethanol, shake gently and let stand for 20 minutes;

6) Centrifuge (4°C, 7500×g) for 5 min, aspirate the supernatant and dry it at room temperature, then add 30-50 μL ddH ₂ O to dissolve the precipitate, and use agarose gel electrophoresis and micro-UV spectrophotometer to detect RNA Purity (the ratio of OD ₂₆₀ /OD ₂₈₀ is 1.8-2.0) and integrity (the band is clear and complete).

(2) Design primers for the wax synthesis gene PmFAR1 of P. papaya mealybug

In view of the fact that there is no public reference genome, the transcriptome sequencing of P. papaya was entrusted to Guangzhou Genedenovo Co., Ltd. (https://www.genedenovo.com/), and the transcriptome of P. papaya was finally obtained with a size of 1.02GB. This information contains the coding region sequence of the PmFAR1 gene with a full length of 1590 bp (see SEQ ID NO. 3). Then, based on the coding region sequence of the PmFAR1 gene obtained from the transcriptome sequencing of P. papaya, Primer 5.0 software was used to design primers for amplifying the wax synthesis gene PmFAR1 of P. papaya:

PmFAR1-F: ATGATGCTGGTGGATAAG (SEQ ID NO. 1);

PmFAR1-R: GACAATAAAGGCAGGAAG (SEQ ID NO. 2).

(3) Reverse transcription to synthesize cDNA

cDNA was synthesized by reverse transcription according to the instructions of TaKaRa's PrimeScript ^TM II 1st Strand cDNA kit. The reaction system (10 μL) of reverse transcription is: Oligo dT 1 μL, dNTP 1 μL, RNA 1 μg, and RNase-FreeddH ₂ O. Make up to 10 μL. After the reaction system is prepared, place it at 65°C and incubate it for 5 minutes.

After incubation, add the following components to the above reaction system: 5×Primerscript buffer 5μL, RNase inhibitor RI 0.5μL, PrimerscriptRTase (reverse transcriptase) 1μL, RNase-Free ddH ₂ O 3.5μL. After slowly mixing, incubate at 50°C for 50 minutes and 95°C for 5 minutes. After the reaction is completed, place it in a -20°C refrigerator for later use.

(4) Amplification of the wax synthesis gene PmFAR1 of Mealybug papaya

The PCR reaction system (20 μL) is: cDNA 2 μL, PmFAR1-F 1 μL, PmFAR1-R 1 μL, PCR mixmaster 10 μL, ddH ₂ O 6 μL. The PCR reaction program is: pre-denaturation 95°C, 3min; denaturation 95°C, 30s; annealing 58°C, 30s; extension 72°C, 1min, 35cycles; extension 72°C, 10min.

(5) Confirm the cloning of the wax synthesis gene PmFAR1 of Mealybug papaya

The obtained PCR product was recovered through agarose gel, then connected to the pGEM-18T vector, and transformed into E. coli DH5α. After being identified as a positive clone by enzyme digestion and PCR, it was sequenced and then sequenced according to the obtained PmFAR1 sequence (see SEQ ID NO. .3), using DNAMAN Version 4.0 software and the PsFAR1 gene of Phenacoccus solenopsis (gene accession number: ANN23959.1) and PsFAR1 gene (gene accession number: ANN23959.1) published by the National Center for Biotechnology Information (NCBI). The full-length amino acid sequences of the PsFAR2 gene (gene accession number: ANN23960.1) of P.solenopsis) and the EpFAR1 gene (gene accession number: AGK27745.1) of Ericerus pela were compared. The comparison results showed that the PmFAR1 gene has a lipid The unique Rossmann folding core domain of the acyl-CoA reductase (FAR) family, and the sequence similarity to the three reported FAR genes exceeds 82%, confirming that the cloning has obtained the wax synthesis of Mealybugs papaya. Gene PmFAR1. The PCR amplification and electrophoresis results of the wax synthesis gene PmFAR1 of the Mealybug papaya are shown in Figure 1, indicating that the wax synthesis gene PmFAR1 of the Mealybug papaya was successfully amplified.

Sequence of PmFAR1 gene (SEQ ID NO.3):

ATGGAGGTAATAGATACTGATTCGAAAATAGGTCCGATGTCACAAATACAAGAATTTTTCAAAGATTCTTCGGTTTTCATAACCGGAGCTACCGGTTTCTTAGGCCACGTTTTGCTTTCCAAACTCCTCCGATCGTGCCCAGATATAAACAAAATTTACGTATTGTTACGAAAAAAAAGGTAAAACGGCGATAGAAAGATTCAAGGAGATGCTCGAAGATGAGGTCTTTTCAAGTAATAAAATCAGATTGCCCGGATTCTAT CGAAGGTGACACCCATTGTTGGAGATTGCATAAAACCTGGTTTAGGATTGAGCGAACTCGACAAAGAGCTGATCAAAAAAGAAGTCAACGTCGTGTTCCACGTCGCAGCGACGGTCCGATTCGACGCTCCTTTACGCCAGGCGGTCAACATGAACATACGATCAACGAGCGATCTGCTCGACATGGCGATGGATATGAAAAACTTGAAGGCTTTCGTTCACGTATCTACCGCCTTTTCGAATTGCACCGATAGAAAGGTTATCGA AGAGAAACTATACGATTCGCCTATCTCGTCCGAGAATCTAATGATGCTGGTGGATAAGCTGAGCGACGAAACTTTGGACAGGATTACACCTGGACTTCTCGGAACGTACCCGAACACCTACGTTTTATACGAAATGCATCGCCGAAGAAGTATGCCGAACCAAAGGCGCCAATTTACCTTTTGGTTGTTTTCCGACCAGCTATTGTCATTTCGTCAGCCCAGGAGCCTATCCCAGGTTGGATCAATAATGTATACGGGGCCAACGGG CGTAGTAGCCGCAGCAGCCGTAGGCTTACTCAGATCGCTTCAATCGGATAAAAAATGCAAAGCCAATGTGGTACCTTGCGATTACGTCGTGAACGCAGCCATAGCTGCCGCTTGGAGAGCATCGTCGAAAACTAAAAACGGCGAAATTATTCACGCAAATGGCAACGGAATTAACCAAAATTCTCTCAAATCAAAATCAGAAGACTCGAAAAATATCACCGTCTATAACTACAGCTGTGATTTACTGAAGAAGCCTTTCAACTGGAGG GAATTTACCGAATCGAACGAAAGACACGAGCCTAAAATGCCGTCTTCTTTATCGATATGGGCGTATAATTTAACTTTAAACAAGTATAAGTTTGTACATCGAGTATATTGTTTCTTTTTACATCTTCTTCCTGCCTTTATTGTCGATAATATTGCCAGATTAATCGGGAAAGAACCAAAGTTAATGGACGCGTATCAAAAACTACATAAATTAGCAGATGTACTATCCTACTTCAGCACCAAGGAATGGGACATGTCCACCGGAAA CGTAGTCAGCTTGTGGCAACAGCTATCTTACAAAGACAGGGAAATATTTAAGTTTGATATGAACGAACTAGATTGGATTACTTACTGGCACACTCACTGTGTGGGTATCAGAAGGTTCATCTTAAAAGAAGATCCGAAAACTATACCAGCAGCTCAAGTCAGACGAAGGAGGTTTTTATTCGCTCAATATCTAATAATCACCACGTTCGCCGCTCTATCTTTATACTTCTCGTACAATGTAATCAAACGTAGTAGGCTGT G.A.

Example 2 Study on the regulatory effect of Mealybug papaya wax synthesis gene PmFAR1 on Mealybug papaya

1. Analysis of silencing effect of wax synthesis gene PmFAR1 in Mealybug papaya

(1)Synthesis of dsRNA

1) Design and synthesize dsRNA primers for the wax synthesis gene PmFAR1 of Mealybug papaya

According to the sequence of the wax synthesis gene PmFAR1 of P. papaya, the primer design website E-RNAi (https://www.dkfz.de/signaling/e-rnai3/idseq.php) was used to design the synthesis of dsRNA and carry T7 initiation. Primer for sub:

dsPmFAR1-F: taatacgactcactatagggCGTTCACGTATCTACCGCCT (lowercase letters are T7 promoter, SEQ ID NO. 4);

dsPmFAR1-R: taatacgactcactatagggCGTGAATAATTTCGCCGTTT (lowercase letters are T7 promoter, SEQ ID NO. 5).

2) Synthesize dsRNA

MEGAscript RNAi Kit (Thermo Scientific, Wilmington, DE, USA) was used according to the kit's instructions (https://www.thermofisher.com/cn/zh/home/references/protocols/rnai-epigenetics-and-gene -regulation/rnai-protocol/megascript-rnai-kit.html) to synthesize dsRNA. The sequence of the resulting dsRNA is as follows:

The dsRNA sequence of the wax synthesis gene PmFAR1 of P. papaya mealybug (SEQ ID NO.6):

CGTTCACGTATCTACCGCCTTTTCGAATTGCACCGATAGAAAGGTTATCGAAGAGAAACTATACGATTCGCCTATCTCGTCCGAGAATCTAATGATGCTGGTGGATAAGCTGAGCGACGAAACTTTGGACAGGATTACACCTGGACTTCTCGGAACGTACCCGAACACCTACGTTTTATACGAAATGCATCGCCGAAGAAGTATGCCGAACCAAAGGCGCCAATTTACCTTTTGGTTGTTTTCCGACCAGCTATTGTCATTT CGTCAGCCCAGGAGCCTATCCCAGGTTGGATCAATAATGTATACGGGCCAACGGGCGTAGTAGCCGCAGCAGCCGTAGGCTTACTCAGATCGCTTCAATCGGATAAAAAATGCAAAGCCAATGTGGTACCTTGCGATTACGTCGTGAACGCAGCCATAGCTGCCGCTTGGAGAGCATCGTCGAAAACTAAAAACGGCGAAATTATTCACG.

(2) Expression analysis of wax synthesis gene PmFAR1 in Mealybugs papaya based on qPCR

1) Design qPCR primers for the wax synthesis gene PmFAR1 of P. papaya mealybug

Based on the gene sequence of the wax synthesis gene PmFAR1 of the papaya mealybug, primers for qPCR and internal reference gene Pmactin primers were designed using Primer 5.0 software:

Actin-qF:5’-CATCCTGGCTTTTGGATTTAG-3’ (SEQ ID NO.7);

Actin-qR:5’-TCCAAAGCAACATAGCACAAT-3’ (SEQ ID NO.8);

PmFAR1-qF: 5’-TATTCACGCAAATGGCAACG-3’ (SEQ ID NO. 9);

PmFAR1-qR: 5'-AGGCTTCTTCAGTAAATCACAGC-3' (SEQ ID NO. 10).

2) Reverse transcription to synthesize cDNA

The specific method was the same as step (3) in Example 1, and the cDNA of the wax synthesis gene PmFAR1 of P. papaya was synthesized by reverse transcription.

3) qPCR measurement and analysis of the expression level of wax synthesis gene PmFAR1 in Mealybug papaya

Use nuclease-free water to dilute the cDNA sample 5 times, and use the Actin gene of Mealybugs papaya as the internal reference. The qPCR reaction system (20 μL) is: 2×SYBR Premix E×TaqTM 10 μL, 0.5 μL each of the upstream and downstream primers (10 μmol/ L), cDNA 2μL, ddH ₂ O 7μL. The qPCR reaction conditions are: after pre-incubation at 95°C for 1 min, complete the following program in 40 cycles: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 20 s. Finally, the relative expression of genes was calculated according to the 2 ^-ΔΔCt method.

(3) Determine the optimal dsRNA concentration for silencing the wax synthesis gene PmFAR1 of P. papaya mealybug

Set the dsRNA treatment concentrations of 100ng/μL, 200ng/μL, 300ng/μL, 500ng/μL, and 1000ng/μL for the wax synthesis gene PmFAR1 of five mealybugs. Use clean water as a control and spray the susceptible insects after planting for 30 days. The reference cassava variety [South China 205 (SC205) (S)] plants were planted until the leaves were completely wet, and then female adults of the same development period and size that were subcultured indoors with SC205 (S) were inoculated into each treatment. The underside of the middle leaves of the plant (the 4th to 12th leaves under the top bud of the plant), and then observe and count the number of live mealybugs on papaya mealybugs 1d, 2d, 4d, 6d, and 8d after inoculation, and collect the live insects for use in papaya The total RNA of the mealybug was extracted, and then the qPCR measurement and analysis of the expression of the wax synthesis gene PmFAR1 of the mealybug was carried out according to the method of step (2). Each plant was inoculated with 9 leaves, and each leaf was inoculated with 30 insects. Each treatment was set up with 3 replicates.

(4)Test results

According to the relative expression level (Figure 2) and mortality rate (Figure 3) of the wax synthesis gene PmFAR1 of P. papaya after being treated with dsRNA of P. papaya wax synthesis gene PmFAR1, the effect of 500-1000ng/μL on P. papaya was determined. The mealybug wax synthesis gene PmFAR1 has the best silencing efficiency. Since there is no significant difference between 500ng/μL and 1000ng/μL in the silencing efficiency of the wax synthesis gene PmFAR1 of P. papaya mealybug, 500ng/μL PmFAR1dsRNA of P. papaya mealybug wax synthesis gene was determined to be the best gene silencing considering cost and effect. concentration.

2. The role of the wax synthesis gene PmFAR1 in Mealybug papaya in regulating Mealybug papaya

Select the insect-resistant reference cassava variety Huanan 9 (SC9) (R) and the insect-susceptible reference cassava variety Huanan 205 (SC205) (S) that have been identified for insect resistance and have been planted for 30 days. The female adult mealybugs of the same age and size were inoculated to the underside of the middle leaves of the cassava plant (4th to 12th under the top bud of the plant) after spraying 500ng/μL of the waxy synthesis gene PmFAR1dsRNA of the papaya mealybug. Leaves), observe and count the number of live mealybugs and the number of eggs laid by single females on 1d, 2d, 4d, 6d and 8d after inoculation, and collect the live insects for total RNA extraction of P. papaya, and then perform the papaya show qPCR measurement and analysis of the expression of wax synthesis gene PmFAR1 in mealybugs, and affinity analysis based on mortality, development period, and number of eggs laid by a single female (the higher the mortality rate, the longer the development period and the longer the development period of P. papaya) The lower the number of eggs laid by a single female, the lower the affinity with cassava). Each plant was inoculated with 9 leaves, and each leaf was inoculated with 30 insects. Each treatment was set up with 3 replicates.

The results showed that RNA interference technology can significantly inhibit the expression of the wax synthesis gene PmFAR1 of Mealybug papaya (Figure 4), significantly improve the mortality rate of Mealybug papaya after feeding on the resistant and susceptible reference cassava varieties (Figure 5), and significantly Inhibits the growth and development of Mealybug papaya (Figure 6), significantly reduces the fecundity of Mealybug papaya (Figure 7), and ultimately significantly reduces the affinity between Mealybug papaya and cassava (Figure 5, Figure 6, Figure 7) , which provides an important scientific basis for the widespread application of the wax synthesis gene PmFAR1 in regulating P. papaya mealybug.

Combining Example 1 and Example 2, it can be seen that after reducing the expression level of the wax synthesis gene PmFAR1 of P. papaya through RNA interference technology, the mortality rate of P. papaya can be significantly increased and the growth and development of P. papaya can be significantly inhibited. Significantly reduces the fecundity of Mealybug papaya, and plays an important role in affecting the affinity of two-spotted spider mites with their hosts, especially in colonization, survival and reproduction after feeding. At the same time, dsRNA synthesized based on the gene fragment of the wax synthesis gene PmFAR1 of the papaya mealybug is used to regulate the papaya mealybug. It has the advantages of species specificity, safety and high efficiency, and has many advantages in the green prevention and control of the papaya mealybug. With good market application prospects and potential economic and ecological benefits, it can provide a new prevention and control approach for the green prevention and control of papaya mealybug.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principle and spirit of the invention, and they still fall within the protection scope of the invention.

Claims (4)

1. The application of a reagent that inhibits the expression of the wax synthesis gene PmFAR1 of Mealybug papaya and/or reduces the expression of the wax synthesis gene PmFAR1 of Mealybug papaya in the preparation of products for regulating Mealybug papaya, characterized in that the gene The PmFAR1 nucleotide sequence is shown in SEQ ID NO.3; The reagent is dsRNA that silences the wax synthesis gene PmFAR1 of Mealybug papaya; The dsRNA nucleotide sequence is shown in SEQ ID NO.6; The synthetic primers for dsRNA include the upstream primer shown in SEQ ID NO.4 and the downstream primer shown in SEQ ID NO.5; The regulation includes promoting the death of Mealybug papaya, inhibiting the growth and development of Mealybug papaya, reducing the fecundity of Mealybug papaya and the affinity between Mealybug papaya and its host. 2. A medicament for regulating Mealybug papaya, characterized in that the medicament is a reagent that inhibits the expression of the wax synthesis gene PmFAR1 of Mealybug papaya and/or reduces the expression of the wax synthesis gene PmFAR1 of Mealybug papaya. As the main active ingredient; The medicament uses dsRNA that silences the wax synthesis gene PmFAR1 of Pawpaw mealybug as the main active ingredient. The dsRNA nucleotide sequence is as shown in SEQ ID NO.6, and the nucleotide sequence of the gene PmFAR1 is as SEQ ID NO.3. shown. 3. A method for regulating the damage caused by Mealybug papaya to plants, which is characterized by first utilizing the method of claim 1 to inhibit the expression of the wax synthesis gene PmFAR1 of Mealybug papaya and/or reduce the wax synthesis gene PmFAR1 of Mealybug papaya. The expressed reagent is sprayed on the plant leaves, and then the sprayed plant leaves are used to feed the mealybug P. papaya, thereby achieving the purpose of regulating the damage of the P. papaya mealybug. The nucleotide sequence of the gene PmFAR1 is as shown in SEQ ID NO.3 Show; The plant is cassava. 4. A method for regulating the damage caused by Mealybug papaya to plants according to claim 3, characterized in that first, dsRNA that silences the waxy synthesis gene PmFAR1 of Mealybug papaya is sprayed on the plant leaves, and the dsRNA nucleotides The sequence is shown in SEQ ID NO. 6. The spraying concentration of the dsRNA is 500-1000ng/μL, and then the sprayed plant leaves are used to feed the Mealybug papaya, thereby achieving the purpose of regulating the damage of Mealybug papaya, as described The nucleotide sequence of gene PmFAR1 is shown in SEQ ID NO.3; The plant is cassava.