CN116286815B - Application of papaya gecko waxy synthesis gene PmFAR1 in regulation and control of papaya gecko waxy synthesis gene - Google Patents
Application of papaya gecko waxy synthesis gene PmFAR1 in regulation and control of papaya gecko waxy synthesis gene Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural biology, relates to the technical field of prevention and control of geckos of papaya, and in particular relates to application of a geckos wax synthesis gene PmFAR1 of papaya in regulation and control of geckos of papaya. In order to excavate the papaya gecko waxy synthesis gene which can be used for regulating and controlling the papaya gecko, the invention discovers that after the expression quantity of the papaya gecko waxy synthesis gene PmFAR1 is reduced by an RNA interference technology, the death rate of the papaya gecko can be obviously improved, the growth and development of the papaya gecko can be obviously inhibited, the fertility of the papaya gecko and the affinity of the papaya gecko with a host are obviously reduced, the PmFAR1 gene plays an important role in influencing the affinity of the papaya gecko with the host, particularly in the aspects of colonization survival and reproduction after feeding, and the papaya gecko waxy synthesis gene PmFAR1 can be applied to regulating and controlling the papaya gecko, so that a new approach is provided for preventing and controlling the papaya gecko.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, relates to the technical field of prevention and control of geckos of papaya, and in particular relates to application of a geckos wax synthesis gene PmFAR1 of papaya in regulation and control of geckos of papaya.
Background
Papaya gecko (Paracoccusmarginatus Williams and Granara de Willink), also known as papaya gecko, belongs to the order of Hemiptera (Hemiptera), the family of gecko (pseudococidae), the genus gecko (Paracoccus), is one of the world dangerous agricultural and forestry pests, and the object of hazard comprises cassava, fruit trees, vegetables, garden greening plants and the like, is mainly caused by piercing and sucking of nymphs and female adults, and further causes stem withering, greening, yellowing and falling of leaves, and the quality of fruits is reduced, and the whole plant can die when serious. The papaya gecko has short life history, strong fertility, strong adaptability, easy generation of drug resistance and frequent disaster, so the prevention and treatment difficulty is great. In the planting process of cassava, fruit trees, vegetables, garden greening plants and the like, the occurrence hazard of the papaya gecko is mainly controlled by medicaments at present, but the effective medicaments for controlling the hazard of the papaya gecko are very few, and the problems of agricultural product safety, pest resistance, ecological environment safety in the producing area and the like are increasingly prominent due to the unreasonable administration of a large amount of the same medicaments, so that the healthy and sustainable development of industries such as cassava, fruit trees, vegetables, garden greening plants and the like is severely restricted. Therefore, the approach of seeking green and efficient prevention and control of the papaya gecko becomes a great difficulty to be solved in the industrial development of cassava, fruit trees, vegetables, garden greening plants and the like.
Plant-mediated RNAi technology (RNA interference) has become one of the hot spots in crop pest-resistant genetic engineering. RNAi technology is a phenomenon in which dsRNA (double-strand RNA) homologous to a target gene is introduced into a living organism to cause gene silencing. Compared with other genetic engineering research methods, RNAi technology has the advantages of specificity, safety, high efficiency and the like, and is widely applied to research of specific gene functions in biology. In recent years, methods for screening RNA target genes by using dsRNA in vitro feeding or injection to cause target gene expression and silencing have been widely used for identification and functional analysis of key genes for insect growth and development.
Because the body wall of the papaya gecko is provided with a thicker wax layer, some medicaments with low fat solubility are not easy to enter target positions in the papaya gecko due to weaker penetrability, so that the medicament prevention and control cannot meet the target requirements. The wax on the surface of the insect body can protect the insect body from water loss and invasion of external harmful substances, and the main component is hydrocarbon. Wherein fatty acyl-CoA reductase (FATTy acyl-CoA reductase) encoded by the FAR gene is a key enzyme for synthesizing hydrocarbon of insect cuticle, and plays an important role in the propagation expansion of insect population. The FAR gene expression can be inhibited to inhibit the synthesis of wax, and further inhibit the growth, development, reproduction and population growth of insects. Therefore, the waxy synthesis genes of the papaya elegans capable of regulating and controlling the feeding harm of the papaya elegans are deeply excavated, and an important scientific basis is hopeful for preventing and controlling the papaya elegans. However, no report on the use of the wax synthesis gene of the gecko in preventing and treating the gecko is available at present.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention inhibits the expression of the PmFAR1 of the wax synthesis gene of the elegans of the papaya through an RNA interference technology, thereby obviously improving the death rate of the elegans of the papaya, obviously inhibiting the normal development of the elegans of the papaya, and obviously reducing the fertility of the elegans of the papaya and the affinity of the elegans of the papaya with a host.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
the first aspect of the invention provides application of an agent for inhibiting the expression of a PmFAR1 waxy synthesis gene of gecko and/or reducing the expression level of the PmFAR1 waxy synthesis gene of gecko in preparing a product for regulating and controlling gecko, wherein the PmFAR1 gene has a nucleotide sequence shown as SEQ ID NO. 3.
Preferably, the agent is dsRNA for silencing the gecko waxy synthesis gene PmFAR1.
Preferably, the modulation comprises promoting death of the gecko, inhibiting growth and development of the gecko, reducing fertility of the gecko and affinity thereof for the host.
According to the sequence information of the PmFAR1 transcript obtained by sequencing the transcription group of the gecko, the nucleotide sequence of the PmFAR1 is shown as SEQ ID NO.3, then the gene PmFAR1 is used as a target, an optimal silencing technical system of the PmFAR1 of the gecko wax synthesis gene is established, and a dsRNA based on the PmFAR1 of the gecko wax synthesis gene is designed and then is introduced into the gecko through a feeding method. The result shows that after silencing or inhibiting the expression of the PmFAR1, the death rate of the gecko after eating resistance and sense reference cassava variety can be obviously improved, the normal development of the gecko is obviously inhibited, the fertility of the tetranychus urticae and the affinity of the gecko with a host are obviously reduced, and the PmFAR1 can be indicated to be applied to the regulation and control of the gecko.
More preferably, the dsRNA has a nucleotide sequence as shown in SEQ ID NO. 6.
More preferably, the synthetic primers of the dsRNA include 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 papaya gecko waxy synthesis gene PmFAR1 is used for regulating and controlling the papaya gecko, has the advantages of species specificity, safety, high efficiency and the like, has good market application prospect and potential economic and ecological benefits in green prevention and control of the papaya gecko, and can provide a new prevention and control way for the green prevention and control of the papaya gecko.
Further, the recombinant expression vector also comprises the coding gene of the dsRNA, and a recombinant expression vector, a transgenic cell line, a recombinant bacterium and an expression cassette containing the dsRNA or the coding gene thereof.
Preferably, the types of products include, but are not limited to, pharmaceutical agents, fertilizers, and the like.
In a second aspect, the invention provides an agent for regulating and controlling the gecko, which takes an agent for inhibiting the expression of a gecko wax synthesis gene PmFAR1 and/or reducing the expression level of the gecko wax synthesis gene PmFAR1 as a main active ingredient, wherein the gene PmFAR1 has a nucleotide sequence shown as SEQ ID NO. 3.
Preferably, the agent for controlling the gecko comprises dsRNA for silencing the gecko wax synthesis gene PmFAR1 as a main active ingredient, wherein the dsRNA has a nucleotide sequence shown as SEQ ID NO.6, and the gene PmFAR1 has a nucleotide sequence shown as SEQ ID NO. 3.
Preferably, the medicament further comprises an agropharmaceutically acceptable adjuvant.
More preferably, the auxiliary material is selected from one or more of the following agents: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filter aids, release retarders.
The third aspect of the invention provides a method for regulating and controlling the damage of papaya gecko to plants, which comprises the following steps: firstly, spraying plant leaves with a reagent for inhibiting the expression of a PmFAR1 gene and/or reducing the expression quantity of the PmFAR1 gene, and then feeding the sprayed plant leaves with the Paecilomyces varioti, so as to achieve the aim of regulating and controlling the damage of the Paecilomyces varioti, wherein the PmFAR1 gene has a nucleotide sequence shown as SEQ ID NO. 3.
Preferably, the method for controlling the damage of the papaya gecko to plants specifically comprises the following steps: firstly, dsRNA of a silencing gecko wax synthesis gene PmFAR1 is utilized to spray plant leaves, the dsRNA has a nucleotide sequence shown as SEQ ID NO.6, the spraying concentration of the dsRNA is 500-1000 ng/mu L, then the sprayed plant leaves are used for feeding the gecko, and the aim of regulating and controlling the hazard of the gecko is achieved, and the gene PmFAR1 has a nucleotide sequence shown as SEQ ID NO. 3.
Preferably, in the above method for controlling the damage of the plants of the gecko, the plants comprise cassava. For example, the insect is referred to the cassava variety [ south China 205 (SC 205) (S) ]. Of course, other host plants that may be compromised by the papaya gecko are also included.
Preferably, the dsRNA or other agent is sprayed in an amount such that the leaves of the plants are completely wetted.
It should be noted that other reagents except dsRNA can also be applied to plants by seed dressing or root irrigation, etc., and the action mode can be reasonably adjusted according to the characteristics of different reagents.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses an application of a papaya gecko waxy synthesis gene PmFAR1 in regulation and control of papaya gecko waxy synthesis gene PmFAR1, which obviously improves the death rate of the papaya gecko after reducing the expression level of the papaya gecko waxy synthesis gene PmFAR1 through an RNA interference technology, obviously inhibits the growth and development of the papaya gecko, obviously reduces the fertility of the papaya gecko and the affinity of the papaya gecko with a host, shows that the gene PmFAR1 plays an important role in influencing the affinity of the papaya gecko with the host, particularly in the aspects of colonization survival and reproduction after feeding, and prompts that the papaya gecko waxy synthesis gene PmFAR1 can be applied to regulation and control of the papaya gecko.
Drawings
FIG. 1 is a PCR amplification electrophoresis chart of a PmFAR1 gene of a PmFAR wax of Xiuxiu mealybugs (lane M is a Trans2K DNA marker; lane 1 is a PmFAR1 gene of a PmFAR wax of Xiuxiu mealybugs);
FIG. 2 shows the relative expression level of the waxy synthesis gene PmFAR1 of Gecko after eating cassava leaves treated with dsRNA of different concentrations;
FIG. 3 shows mortality of Gecko eating cassava leaves treated with different concentrations of dsRNA;
FIG. 4 shows the expression level of the waxy synthesis gene PmFAR1 after the PmFAR1 is silenced, the Pacific gecko feeds the leaves of the anti-cassava variety;
FIG. 5 shows mortality of PmFAR1 silenced Caesalpinia sinensis after feeding the leaves of the anti-cassava variety;
FIG. 6 shows the developmental stage of PmFAR1 silencing after feeding resistance to the cassava variety leaf;
FIG. 7 shows the amount of single female eggs laid after feeding resistance and cassava variety leaves by the papaya gecko with silencing of PmFAR1.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
Example 1 acquisition of the wax Synthesis Gene PmFAR1 from Xiuxiu Pachyos
(1) Extraction of total RNA of Chaenomeles speciosa
1) Selecting 30 female imago of the Chinese flowering quince gecko, which are subjected to secondary feeding and have consistent development duration and size, of a common tapioca variety [ south China 205 (SC 205) (S) ] which is obtained by indoor insect resistance screening, adding 1mL of TRIzol into a centrifuge tube of RNase-Free, quickly grinding by a grinding rod, immediately vortex, violently shaking and mixing, and standing at room temperature for 5min;
2) Centrifuging (4 ℃,12000 Xg) for 10min, and taking the supernatant into a new EP tube;
3) Adding 0.2mL of chloroform into the EP tube, shaking for 15s, and standing at 15-30 ℃ for 3min; centrifuging (12000 Xg) for 15min at 4deg.C;
4) Adding colorless phase into a new EP pipe, adding equal volume of isopropanol, mixing, standing at 15-30deg.C for 10min;
5) Centrifuging (4 ℃,12000 Xg) for 10min, absorbing and removing the supernatant, adding 1mL of ice-precooled 75% ethanol, slightly shaking, and standing for 20min;
6) Centrifuging (4deg.C, 7500 Xg) for 5min, sucking off supernatant, air drying at room temperature, and adding 30-50 μl ddH 2 O was dissolved and precipitated, and the purity (OD) of RNA was measured by agarose gel electrophoresis and a micro-UV spectrophotometer 260 /OD 280 The ratio of 1.8-2.0) and integrity (the band is clearly intact).
(2) Primer for designing papaya gecko waxy synthesis gene PmFAR1
In view of the unpublished reference genome, transcriptome sequencing of Gecko was delegated to Kttps (https:// www.genedenovo.com /) and finally, gecko transcript information of 1.02GB was obtained, which contained the coding region sequence of PmFAR1 gene of full length 1590bp (see SEQ ID NO. 3). Then, according to the coding region sequence of the PmFAR1 gene obtained by sequencing the transcription group of the gecko, a Primer 5.0 software is used for designing a Primer for amplifying the PmFAR1 of the gecko wax synthesis gene:
PmFAR1-F：ATGATGCTGGTGGATAAG(SEQ ID NO.1)；
PmFAR1-R：GACAATAAAGGCAGGAAG(SEQ ID NO.2)。
(3) Reverse transcription synthesis of cDNA
Reference is made to PrimeScript from TaKaRa TM II 1st Strand cDNA kit instructions for reverse transcription to cDNA. The reaction system (10. Mu.L) for reverse transcription was: oligodT 1. Mu.L, dNTP 1. Mu.L, RNA 1. Mu.g, RNase-Free ddH 2 O was made up to 10. Mu.L. After the reaction system was prepared, the mixture was incubated at 65℃for 5min.
After incubation, the following components were added to the reaction system: 5X Primerscript buffer. Mu.L of RNase inhibitor RI 0.5. Mu.L, primerstrTase 1. Mu.L, RNase-Free ddH 2 O3.5. Mu.L. Slowly mixing, incubating at 50deg.C for 50min, incubating at 95deg.C for 5min, and placing in a refrigerator at-20deg.C for use.
(4) Amplification of PmFAR1 gene synthesized by wax of Xiuqin papaya
The PCR reaction system (20. Mu.L) was: cDNA 2. Mu.L, pmFAR 1-F1. Mu.L, pmFAR 1-R1. Mu.L, PCR mixmaster 10. Mu.L, ddH 2 O6. Mu.L. The PCR reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 58 ℃ for 30s; extending at 72deg.C for 1min and 35cycles; extending at 72deg.C for 10min.
(5) Confirming cloning to obtain papaya Xiuqin waxy synthetic gene PmFAR1
The PCR product obtained was recovered by agarose gel, then ligated to pGEM-18T vector, transformed into E.coli DH 5. Alpha. And identified as positive clone by digestion and PCR, then sequenced, and based on the obtained PmFAR1 sequence (see SEQ ID NO. 3), the full-length amino acid sequence of the PmFAR1 gene (gene accession No. AN 23960.1) and Ericerus pela (gene accession No. AGK 27745.1) was compared using DNAMAN Version 4.0 software with the Ross sinensis folding (Ross fondand) core domain of the Hibiscus Lecanis PsFAR1 gene (gene accession No. ANN 23959.1) published by the national center of biotechnology information (National Center for Biotechnology Information, NCBI), and the three genes obtained by cloning were confirmed to be similar to the PmFAR gene of papaya powder by comparing the three genes with the three genes, namely, the PmFAR gene having a specific folding (Ross fond) core domain of the fatty acyl CoA reductase (PmFAR coenzyme A family. The PCR amplification electrophoresis result of the papaya gecko waxy synthesis gene PmFAR1 is shown in figure 1, which shows that the papaya gecko waxy synthesis gene PmFAR1 is obtained by successful amplification.
The sequence of the PmFAR1 gene (SEQ ID NO. 3):
ATGGAGGTAATAGATACTGATTCGAAAATAGGTCCGATGTCACAAATACAAGAATTTTTCAAAGATTCTTCGGTTTTCATAACCGGAGCTACCGGTTTCTTAGGCCACGTTTTGCTTTCCAAACTCCTCCGATCGTGCCCAGATATAAACAAAATTTACGTATTGTTACGAGAAAAAAAAGGTAAAACGGCGATAGAAAGATTCAAGGAGATGCTCGAAGATGAGGTCTTTCAAGTAATAAAATCAGATTGCCCGGATATTCTATCGAAGGTGACACCCATTGTTGGAGATTGCATAAAACCTGGTTTAGGATTGAGCGAACTCGACAAAGAGCTGATCAAAAAAGAAGTCAACGTCGTGTTCCACGTCGCAGCGACGGTCCGATTCGACGCTCCTTTACGCCAGGCGGTCAACATGAACATACGATCAACGAGCGATCTGCTCGACATGGCGATGGATATGAAAAACTTGAAGGCTTTCGTTCACGTATCTACCGCCTTTTCGAATTGCACCGATAGAAAGGTTATCGAAGAGAAACTATACGATTCGCCTATCTCGTCCGAGAATCTAATGATGCTGGTGGATAAGCTGAGCGACGAAACTTTGGACAGGATTACACCTGGACTTCTCGGAACGTACCCGAACACCTACGTTTATACGAAATGCATCGCCGAAGAAGTATGCCGAACCAAAGGCGCCAATTTACCTTTGGTTGTTTTCCGACCAGCTATTGTCATTTCGTCAGCCCAGGAGCCTATCCCAGGTTGGATCAATAATGTATACGGGCCAACGGGCGTAGTAGCCGCAGCAGCCGTAGGCTTACTCAGATCGCTTCAATCGGATAAAAAATGCAAAGCCAATGTGGTACCTTGCGATTACGTCGTGAACGCAGCCATAGCTGCCGCTTGGAGAGCATCGTCGAAAACTAAAAACGGCGAAATTATTCACGCAAATGGCAACGGAATTAACCAAAATTCTCTCAAATCAAAATCAGAAGACTCGAAAAATATCACCGTCTATAACTACAGCTGTGATTTACTGAAGAAGCCTTTCAACTGGAGGGAATTTACCGAATCGAACGAAAGACACGAGCCTAAAATGCCGTCTTCTTTATCGATATGGGCGTATAATTTAACTTTAAACAAGTATAAGTTTGTACATCGAGTATATTGTTTCTTTTTACATCTTCTTCCTGCCTTTATTGTCGATAATATTGCCAGATTAATCGGGAAAGAACCAAAGTTAATGGACGCGTATCAAAAACTACATAAATTAGCAGATGTACTATCCTACTTCAGCACCAAGGAATGGGACATGTCCACCGGAAACGTAGTCAGCTTGTGGCAACAGCTATCTTACAAAGACAGGGAAATATTTAAGTTTGATATGAACGAACTAGATTGGATTACTTACTGGCACACTCACTGTGTGGGTATCAGAAGGTTCATCTTAAAAGAAGATCCGAAAACTATACCAGCAGCTCAAGTCAGACGAAGGAGGTTTTTATTCGCTCAATATCTAATAATCACCACGTTCGCCGCTCTATCTTTATACTTCTCGTACAATGTAATCAAACGTAGTAGGCTGTGA。
EXAMPLE 2 investigation of the Regulation and control action of the wax-synthesizing Gene PmFAR1 of Xiuxiu Packo on Xiuxiu Packo
1. Silencing effect analysis of PmFAR1 gene synthesized by wax of gecko in elegans
(1) Synthesis of dsRNA
1) dsRNA primer for designing and synthesizing papaya gecko waxy synthesis gene PmFAR1
Based on the sequence of PmFAR1, a primer design website E-RNAi (https:// www.dkfz.de/signaling/E-RNAi3/idseq. Php) is used for designing primers for synthesizing dsRNA and carrying a T7 promoter:
dsPmFAR1-F: taatacgactcactatagggCGTTCACGTATCTACCGCCT (lower case T7 promoter, SEQ ID No. 4);
dsPmFAR1-R: taatacgactcactatagggCGTGAATAATTTCGCCGTTT (lower case T7 promoter, SEQ ID NO. 5).
2) Synthesis of dsRNA
dsRNA was synthesized using MEGAscript RNAi Kit (Thermo Scientific, wilmington, DE, USA) according to the protocol of the kit (https:// www.thermofisher.com/cn/zh/home/references/protocols/rnia-epigenetics-and-gene-regulation/rnia-protocol/mobility-rnia-kit. Html). The sequence of the resulting dsRNA is shown below:
dsRNA sequence (SEQ ID NO. 6) of PmFAR1 of gecko wax synthesis gene:
CGTTCACGTATCTACCGCCTTTTCGAATTGCACCGATAGAAAGGTTATCGAAGAGAAACTATACGATTCGCCTATCTCGTCCGAGAATCTAATGATGCTGGTGGATAAGCTGAGCGACGAAACTTTGGACAGGATTACACCTGGACTTCTCGGAACGTACCCGAACACCTACGTTTATACGAAATGCATCGCCGAAGAAGTATGCCGAACCAAAGGCGCCAATTTACCTTTGGTTGTTTTCCGACCAGCTATTGTCATTTCGTCAGCCCAGGAGCCTATCCCAGGTTGGATCAATAATGTATACGGGCCAACGGGCGTAGTAGCCGCAGCAGCCGTAGGCTTACTCAGATCGCTTCAATCGGATAAAAAATGCAAAGCCAATGTGGTACCTTGCGATTACGTCGTGAACGCAGCCATAGCTGCCGCTTGGAGAGCATCGTCGAAAACTAAAAACGGCGAAATTATTCACG。
(2) Expression analysis of papaya gecko waxy synthetic gene PmFAR1 based on qPCR
1) qPCR primer for designing papaya gecko waxy synthesis gene PmFAR1
According to the gene sequence of the wax synthesis gene PmFAR1 of the gecko of the Chinese flowering quince, primers for qPCR and primers for the internal reference gene Pmantin are designed by using Primer 5.0 software:
Actin-qF:5’-CATCCTGCGTTTGGATTTAG-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 synthesis of cDNA
The specific method is the same as the step (3) of the example 1, and cDNA of the wax synthesis gene PmFAR1 of the gecko is obtained through reverse transcription synthesis.
3) qPCR (quantitative polymerase chain reaction) determination analysis of expression quantity of PmFAR1 of wax synthesis gene of gecko
cDNA samples were diluted 5-fold with nucleic-free water, and the action gene of Xiuxiu Gecko was used as an internal control, and the qPCR reaction system (20. Mu.L) was: 2 XSYBR Premix E X Taq. Mu.L, 0.5. Mu.L (10. Mu. Mol/L) of each of the upstream and downstream primers, 2. Mu.L of cDNA, ddH 2 O7. Mu.L. The qPCR reaction conditions were: after a pre-incubation of 1min at 95℃the following procedure was completed in 40 cycles: denaturation at 95℃for 15s, annealing at 60℃for 15s, and extension at 72℃for 20s. Finally according to 2 -ΔΔCt The method calculates the relative expression amount of the genes.
(3) Determination of optimal dsRNA concentration of silencing PmFAR1 gene of gecko wax
Setting the dsRNA treatment concentration of five papaya gecko wax synthesis genes PmFAR1 of 100 ng/. Mu.L, 200 ng/. Mu.L, 300 ng/. Mu.L, 500 ng/. Mu.L and 1000 ng/. Mu.L, respectively spraying and planting the treated papaya gecko wax synthesis genes PmFAR1 for 30 days by taking clear water as a control, referring to the plant of the cassava variety [ south China 205 (SC 205) (S) ] until the leaves are completely wetted, respectively inoculating the indoor papaya gecko wax adults with consistent development calendar period and size of SC205 (S) subculture to the leaf backs of the middle leaves of each treated plant (4 th-12 th leaves under the top buds of the plant), observing the number of live papaya geckos after counting the grafted papaya geckos 1d, 2d, 4d, 6d and 8d, collecting total RNA extraction of the live papaya geckos for the papaya geckos, and then carrying out qPCR determination analysis on the expression quantity of the papaya gecko wax synthesis genes PmFAR1 according to the method of the step (2). 9 leaves of each plant are inoculated, 30 leaves are inoculated, and 3 replicates are arranged for each treatment.
(4) Test results
Papaya geelegans treated with dsRNA of PmFAR1 gene synthesized from gecko wax the relative expression level (figure 2) and death rate (figure 3) of the mealybugs wax synthesis gene PmFAR1, the silencing efficiency of 500-1000 ng/. Mu.L on the PmFAR1 of the wax synthesis gene of the gecko is determined to be the best. Because 500 ng/mu L and 1000 ng/mu L have no obvious difference on the silencing efficiency of the PmFAR1 of the wax synthesis gene of the gecko, the 500 ng/mu L of the PmFAR1dsRNA of the wax synthesis gene of the gecko is determined as the optimal gene silencing concentration by comprehensively considering the cost and the effect.
2. Application of papaya gecko waxy synthesis gene PmFAR1 in regulation and control of papaya gecko waxy synthesis gene
Selecting a disease-resistant reference cassava variety Huanan 9 (SC 9) (R) and a disease-sensitive reference cassava variety Huanan 205 (SC 205) (S) which are identified by insect resistance and planted for 30 days, respectively inoculating the female imago of the papaya gecko with consistent development duration and size to the leaf backs (4 th-12 th leaves under top buds) of the middle leaves of the disease-sensitive reference cassava variety by spraying 500 ng/mu L of the papaya gecko waxy synthetic gene PmFAR1dsRNA according to the method of the step (3), respectively observing and counting the live number of the live papaya gecko with the mite 1d, 2d, 4d, 6d and 8d and the single female spawning number, collecting total RNA extraction of the live worms for the papaya gecko, then carrying out PCR (polymerase chain reaction) determination analysis of the expression quantity of the papaya gecko waxy synthetic gene PmFAR1, and carrying out qPCR (the high death rate of papaya gecko, the longer development duration and the lower single female spawning number and the lower affinity with the cassava). 9 leaves of each plant are inoculated, 30 leaves are inoculated, and 3 replicates are arranged for each treatment.
The result shows that the expression of the PmFAR1 of the wax synthesis gene of the gecko can be obviously inhibited by an RNA interference technology (figure 4), the feeding resistance and the death rate of the gecko after the reference cassava variety are obviously improved (figure 5), the growth and development of the gecko are obviously inhibited (figure 6), the fertility of the gecko is obviously reduced (figure 7), and finally the affinity between the gecko and the cassava is obviously reduced (figures 5, 6 and 7), thereby providing an important scientific basis for the wide application of the PmFAR1 of the wax synthesis gene of the gecko in regulating and controlling the gecko.
According to the comprehensive embodiment 1 and embodiment 2, after the expression level of the wax synthesis gene PmFAR1 of the elegans carinata is reduced by an RNA interference technology, the death rate of the elegans carinata can be obviously improved, the growth and development of the elegans carinata can be obviously inhibited, the fertility of the elegans carinata can be obviously reduced, and the compatibility of the tetranychus urticae with hosts is influenced, and the method plays an important role in the aspects of colonization survival and reproduction after feeding. Meanwhile, dsRNA synthesized based on the gene fragment of the papaya gecko waxy synthesis gene PmFAR1 is used for regulating and controlling the papaya gecko, has the advantages of species specificity, safety, high efficiency and the like, has good market application prospect and potential economic and ecological benefits in green prevention and control of the papaya gecko, and can provide a new prevention and control way for the green prevention and control of the papaya gecko.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to 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, and yet fall within the scope of the invention.
Claims (4)
1. The application of a reagent for inhibiting the expression of a PmFAR1 gene and/or reducing the expression level of the PmFAR1 gene in the wax synthesis of the gecko in the preparation of a product for regulating and controlling the gecko in the gecko is characterized in that the nucleotide sequence of the PmFAR1 gene is shown as SEQ ID NO. 3;
the reagent is dsRNA for silencing a gecko waxy synthesis gene PmFAR 1;
the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 6;
the synthesis primer of the dsRNA comprises an upstream primer shown as SEQ ID NO.4 and a downstream primer shown as SEQ ID NO. 5;
the regulation and control comprises the steps of promoting death of the papaya gecko, inhibiting growth and development of the papaya gecko, reducing fertility of the papaya gecko and affinity of the papaya gecko with a host.
2. A medicament for regulating and controlling papaya gecko is characterized in that, the agent takes an agent for inhibiting the expression of the PmFAR1 of the wax synthesis gene of the gecko and/or reducing the expression quantity of the PmFAR1 of the wax synthesis gene of the gecko as a main active ingredient;
the medicament takes dsRNA for silencing a gecko waxy synthesis gene PmFAR1 as a main active ingredient, the nucleotide sequence of the dsRNA is shown as SEQ ID NO.6, and the nucleotide sequence of the gene PmFAR1 is shown as SEQ ID NO. 3.
3. A method for regulating and controlling the damage of the mealybugs on plants is characterized in that the reagent for inhibiting the expression of the PmFAR1 of the mealybugs wax synthesis gene and/or reducing the expression quantity of the PmFAR1 of the mealybugs wax synthesis gene is utilized to spray plant leaves, and then the sprayed plant leaves are used for feeding the mealybugs on the plants, so that the aim of regulating and controlling the damage of the mealybugs on the papaya is achieved, and the nucleotide sequence of the gene PmFAR1 is shown as SEQ ID NO. 3;
the plant is cassava.
4. The method for regulating and controlling the damage of the meadow geckos to plants according to claim 3, wherein dsRNA of a silencing meadow geckos wax synthesis gene PmFAR1 is utilized to spray plant leaves, the nucleotide sequence of the dsRNA is shown as SEQ ID NO.6, the spraying concentration of the dsRNA is 500-1000 ng/mu L, and the sprayed plant leaves are used for feeding the meadow geckos, so that the aim of regulating and controlling the damage of the meadow geckos is fulfilled, and the nucleotide sequence of the gene PmFAR1 is shown as SEQ ID NO. 3;
the plant is cassava.
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| CN114921467A (en) * | 2022-04-25 | 2022-08-19 | 中国热带农业科学院三亚研究院 | dsRNA (double-stranded ribonucleic acid) based on myzus persicae effect factor MpC002 gene and application thereof in prevention and treatment of myzus persicae |
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