CN117701567A - Application of miR-8545 inhibitor in preparation of plutella xylostella Bt insecticidal protein Cry1Ac resistance treatment medicament - Google Patents
Application of miR-8545 inhibitor in preparation of plutella xylostella Bt insecticidal protein Cry1Ac resistance treatment medicament Download PDFInfo
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Abstract
The invention discloses application of a miR-8545 inhibitor in preparation of a plutella xylostella Bt insecticidal protein Cry1Ac resistance treatment medicament, wherein the miR-8545 inhibitor is used for reducing the expression level of miR-8545 in plutella xylostella larvae, and is an antisense complementary non-coding RNA sequence of miR-8545, and the nucleic acid sequence of the miR-8545 inhibitor is shown as SEQ ID No. 5. According to the invention, miR-8545 is used as a new target for treating the resistance of the plutella xylostella to the Bt insecticidal protein Cry1Ac, and a miR-8545 inhibitor capable of reducing the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac is developed, and miRNA small molecules are directly targeted and inhibited, so that the protein synthesis which causes the resistance of the plutella xylostella to the Bt insecticidal protein Cry1Ac is inhibited, and the process is a brand-new treatment scheme from the aspects of safety, specificity and effectiveness, so that the process has a wide application prospect.
Description
Technical Field
The invention relates to the fields of toxicology and molecular biology, in particular to application of a miR-8545 inhibitor in preparation of a plutella xylostella Bt insecticidal protein Cry1Ac resistance treatment drug.
Background
Plutella xylostella Plutella xylostella (L.), belonging to the family Lepidoptera (Lepidotera) and Plutellidae, is a worldwide cruciferous vegetable pest, and has more than 40 host plants, which severely restrict cruciferous vegetable production. The whole development of the plutella xylostella can be divided into 4 stages (egg stage, larva stage, pupa stage and adult stage), wherein the leaves of the plant are damaged in the larva stage, and when serious, the plant only leaves residual main pulse, so that the yield and quality of the vegetable are greatly reduced, and great economic loss is caused. It is reported that the economic loss caused by plutella xylostella is up to $40-50 hundred million each year worldwide, and the economic loss caused by plutella xylostella is up to $7.7 hundred million in China. The reason why plutella xylostella is so serious is that it is extremely liable to develop resistance to various chemical pesticides. It is reported that the plutella xylostella has almost all chemical pesticides with different degrees of resistance at present, so that chemical control of plutella xylostella in fields becomes difficult, and therefore, finding a new control method is particularly important.
Bacillus thuringiensis (Bacillus thuringiensis, bt) is a gram-positive baculous bacterium that produces multiple insecticidal crystal proteins during the spore formation stage, and the produced crystal proteins are capable of killing different target pests efficiently and individually. In addition, bt becomes the microbial pesticide with the largest world consumption due to the characteristics of broad insecticidal spectrum, high efficiency, specificity, no harm to human and livestock environment and the like. Meanwhile, various Bt insecticidal protein genes have been successfully cloned and transferred into various important commercial crops for pest control, and the planting area has exceeded 1.9 hundred million hectares. However, the massive and unreasonable application of Bt has led to 13 pests developing resistance to Bt formulations or Bt crops in the field, severely affecting the popularization and use of Bt biopesticides and transgenic Bt crops. Plutella xylostella is the first pest found in the field to develop resistance to Bt biologicals, thus making it an important model for revealing the molecular mechanism of insect Bt resistance. Therefore, the research of the Bt resistance mechanism of the plutella xylostella and the identification of the key genes provide theoretical support for Bt resistance monitoring, prediction and forecast, and have important theoretical and practical significance in the aspects of sustainable popularization and application of Bt preparations and Bt-transformed crops, increase of economic income of farmers and the like.
MicroRNA (miRNA) is a class of endogenous non-coding single stranded RNA molecules of about 18-24 nucleotides in length that are capable of binding to specific sites of a target gene (referred to as "seed" regions) to direct messenger RNA (mRNA) degradation or translational inhibition. In humans, more than 2500 mature miRNAs are registered in the miRBase database, with about 60% of the genes regulated by miRNAs. In insects, more and more researches indicate that miRNAs are involved in regulating biological functions of metamorphosis development, reproduction, neurodegeneration, drug resistance and the like of insects. In addition, miRNA is a key factor of an epigenetic regulation channel, and can respond to external stimulus in time and regulate and control the expression of downstream genes. At present, miRNAs are involved in a variety of important physiological and pathological processes of life bodies, including growth and development, cell proliferation and differentiation, regulation of immune activities, and vital activities such as insect pesticide resistance.
After the insect develops drug resistance to the pesticide, the expression level of miRNA in the body is changed remarkably, and the expression level of the related resistance gene in the body is regulated to change, so that the resistance of the insect to the pesticide is mediated. Currently, agonists and inhibitors of in vitro chemical synthesis of mirnas are an important way to study the role of differentially expressed mirnas in insect pesticide resistance. Meanwhile, the production cost of the miRNA agonist and inhibitor is reduced year by year, so that the application cost is obviously reduced. Naturally, chemical synthesis of functional miRNA agonists or inhibitors has become an important means for controlling and delaying insect pesticide resistance. Therefore, in the process of treating the resistance of the plutella xylostella to the Bt insecticidal protein Cry1Ac, the development of an effective functional miRNA agonist or inhibitor for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac is particularly urgent, and for this reason, the research is carried out.
Disclosure of Invention
Aiming at the problem of medicament deficiency in the existing diamond back moth Bt insecticidal protein Cry1Ac resistance treatment process, the invention prepares a specific, safe, green and efficient diamond back moth Bt insecticidal protein Cry1Ac resistance treatment medicament by chemically synthesizing a specific miRNA agonist and inhibitor.
According to the detection analysis of intestinal transcriptome and RT-qPCR in Bt insecticidal protein Cry1Ac sensitive and resistant populations in a laboratory of the inventor, the expression level of miR-8545 gene is obviously increased in 4 plutella xylostella Bt insecticidal protein Cry1Ac resistant populations. Based on this result, the inventors designed and utilized chemical synthesis of agonists and inhibitors of miR-8545, wherein the miR-8545 agonist (mimic) nucleic acid sequence is shown in SEQ ID NO. 4, and the miR-8545 inhibitor (inhibitor) nucleic acid sequence is shown in SEQ ID NO. 5.
On the basis, the relation between miR-8545 and the resistance of the diamond back moth Bt insecticidal protein Cry1Ac is studied. Research results show that microinjection of the miR-8545 agonist into the haemolymph of the plutella xylostella 3-instar larvae significantly increases the expression level of miR-8545, and toxicity bioassay results after 72 hours show that the injection of the miR-8545 agonist significantly reduces the death rate of the larvae. Microinjection of the miR-8545 inhibitor into the haemolymph of the plutella xylostella 3-instar larvae significantly reduces the expression level of miR-8545, and a toxicity biological assay result after 72 hours shows that microinjection of the miR-8545 inhibitor significantly increases the death rate of the larvae. The result shows that the synthesized miR-8545 inhibitor can be used for preparing a medicament for treating the Cry1Ac resistance of the plutella xylostella Bt insecticidal protein.
In the invention for preparing a plutella xylostella Bt insecticidal protein Cry1Ac resistance treatment medicament based on a miR-8545 inhibitor, a specific antisense complementary non-coding RNA sequence is designed based on 20 nucleotide sequences of miR-8545, and the sequence can specifically target the miR-8545 sequence in the plutella xylostella, so that the miR-8545 sequence in the plutella xylostella is consumed, the expression level of miR-8545 is reduced to slow down the resistance of the plutella xylostella to Bt insecticidal protein Cry1Ac, and the method has the characteristics of specificity, safety, greenness and high efficiency.
The technical scheme provided by the invention is as follows: a miRNA inhibitor for managing the resistance of plutella xylostella Bt insecticidal protein Cry1Ac, wherein the miRNA inhibitor for managing the resistance of plutella xylostella Bt insecticidal protein Cry1Ac is for reducing the expression level of miRNA.
Further, the miRNA inhibitor comprises an antisense complementary non-coding RNA sequence of miR-8545 sequence; preferably, the antisense, complementary non-coding RNA sequence is 18-24 bases in length, more preferably 20 bases in length.
Preferably, the nucleic acid sequence of the antisense complementary non-coding RNA sequence of the miRNA inhibitor is shown as SEQ ID NO. 5.
Meanwhile, the invention also provides application of the miRNA inhibitor for treating the resistance of the diamond back moth Bt insecticidal protein Cry1Ac in preparing a medicament for treating the resistance of the diamond back moth to the Bt insecticidal protein Cry1 Ac.
The application is further realized by applying the miRNA inhibitor for treating the resistance of the diamond back moth Bt insecticidal protein Cry1Ac to a planting place or a field or spraying the miRNA inhibitor on the surface of the diamond back moth.
The invention also provides a method for preparing the plutella xylostella sensitive to the Bt insecticidal protein Cry1Ac, which is obtained by injecting the miRNA inhibitor for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac into the plutella xylostella.
The invention also provides a method for controlling and assisting in controlling plutella xylostella, which comprises the step of applying the miRNA inhibitor for controlling the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac while controlling the plutella xylostella by applying the Bt insecticidal protein Cry1Ac at a planting site or a field.
Has the following advantages and effects:
the miRNA inhibitor for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac can reduce the expression level of miR-8545 in the plutella xylostella, improve the sensitivity of the plutella xylostella to the Bt insecticidal protein Cry1Ac, reduce the resistance of the plutella xylostella to the Bt insecticidal protein Cry1Ac, and can be used in combination with other pesticides to provide a new thought and theoretical basis for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1 Ac.
(1) The specificity is strong. According to the invention, 20 nucleotide sequences of the in-vivo specific miR-8545 of the plutella xylostella are used as target sequences, and the sequence can specifically target the in-vivo miR-8545 sequence of the plutella xylostella by designing the antisense and complementary non-coding RNA sequence of the miR-8545 sequence, so that the sequence has no influence on other organisms and has very strong specificity.
(2) The safety is high. The invention uses 20 nucleotide sequences of the in-vivo specificity miR-8545 of plutella xylostella as target sequences, and designs the specificity antisense complementary non-coding RNA sequence of the miR-8545 sequence, so that the sequence can not be combined with genes in human beings and other animals, and is harmless to the human beings and other animals, so that the safety is high.
(3) Green and high efficiency. The traditional chemical pesticides have the defects of drifting toxicity, pesticide residues and the like, and the miR-8545 inhibitor is a novel biological substance, and does not cause drifting toxicity and pesticide residues when being used, so that the novel biological substance is a green product. In addition, the miR-8545 inhibitor can rapidly interact with an in-vivo miR-8545 sequence after entering the inside of the plutella xylostella, and can rapidly and efficiently reduce the in-vivo miR-8545 expression quantity of the plutella xylostella, so that the plutella xylostella has high efficiency.
In conclusion, the miR-8545 inhibitor obtained by the invention can specifically, safely, environmentally and efficiently inhibit the expression level of miR-8545 in the plutella xylostella, reduce the resistance of the plutella xylostella to Bt insecticidal protein Cry1Ac, and has important significance for the treatment of Bt resistance of plutella xylostella in the field. The plutella xylostella provided by the invention has the characteristics of strong specificity, high safety, green and high efficiency and the like on the Bt insecticidal protein Cry1Ac resistance treatment medicament, so that the plutella xylostella has a wide application prospect.
Drawings
FIG. 1 is a first strand cDNA synthesis of the specific reverse transcription stem-loop primer used in the present invention on the midgut miR-8545 sequence of Plutella xylostella larvae, wherein the specific reverse transcription stem-loop primer (miR-8545-R) used in amplification is underlined in italics, and the direction of the primer is indicated by the arrow.
FIG. 2 is a diagram showing the amplification of the midgut miR-8545 sequence of plutella xylostella larvae by using specific primer pairs, wherein primers used in amplification are respectively a forward primer (qmiR-8545-F) and a reverse primer (qmiR-8545-R) and the directions of upstream and downstream primers are marked by arrows.
FIG. 3 is a melting curve obtained by amplifying the midgut miR-8545 sequence of plutella xylostella larvae using a specific primer pair.
FIG. 4 is the results of a real-time fluorescent quantitative PCR assay for detecting the resistance of the diamond back moth Bt insecticidal protein Cry1Ac of example 1. Wherein A is a 4-instar larva midgut cDNA negative control sample of the Bt-sensitive plutella xylostella population DBM1Ac-S of example 1; b is a 4-instar larva midgut cDNA test sample of the plutella xylostella population SZ-R resistant to the Bt insecticidal protein Cry1Ac in example 1; c is a 4-year-old larva midgut cDNA test sample of the plutella xylostella population SH-R resistant to the Btk preparation in example 1; d is a 4-instar larva midgut cDNA test sample of the group DBM1Ac-R of the near isogenic line plutella xylostella population which generates resistance to the Bt insecticidal protein Cry1Ac in the example 1; e is a 4-instar larva midgut cDNA test sample of the Plutella xylostella population NIL-R resistant to the Bt insecticidal protein Cry1Ac of example 1.
FIG. 5 is a graph showing the variation of miR-8545 expression level in vivo at different time points after 3-year-old larvae of Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella DBM1Ac-S are injected with miR-8545 agonist. Wherein A is miR-8545 expression level change after 3-instar larvae of Bt insecticidal protein Cry1Ac sensitive plutella xylostella population DBM1Ac-S in example 2 are injected with Buffer; b is miR-8545 expression level change after 3-year larva of Bt insecticidal protein Cry1Ac sensitive plutella xylostella population DBM1Ac-S in example 2 is injected with PolyA sequence; c is miR-8545 expression level change of 3-instar larvae of Bt insecticidal protein Cry1Ac sensitive plutella xylostella population DBM1Ac-S in example 2 after miR-8545 agonist injection.
FIG. 6 is a change in mortality after injection of miR-8545 agonist into 3-year-old larvae of Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella DBM1Ac-S and treatment with Bt insecticidal protein Cry1 Ac. Wherein a is mortality after 3-instar larvae of Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella DBM1Ac-S in example 2 are injected with Buffer solution and treated with Bt insecticidal protein Cry1 Ac; b is mortality after in vivo injection of PolyA sequences and treatment with Bt insecticidal protein Cry1Ac after 3-instar larvae of Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella DBM1Ac-S in example 2; c is mortality after in vivo injection of miR-8545 agonist and treatment with Bt insecticidal protein Cry1Ac after 3-instar larvae of Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella DBM1Ac-S in example 2.
FIG. 7 shows the variation of miR-8545 expression level in vivo at different time points after 3-year-old larvae of the plutella xylostella population NIL-R, which are resistant to Bt insecticidal protein Cry1Ac, are injected with miR-8545 inhibitor. Wherein A is miR-8545 expression level change after 3-instar larvae of a plutella xylostella population NIL-R with resistance generated by Bt insecticidal protein Cry1Ac in the embodiment 2 are injected with Buffer; b is miR-8545 expression level change after 3-year larva of a diamondback moth population NIL-R with resistance generated by Bt insecticidal protein Cry1Ac in the example 2 is injected with a PolyA sequence; c is miR-8545 expression level change after 3-year larva of a diamondback moth population NIL-R with resistance generated by Bt insecticidal protein Cry1Ac in the embodiment 2 is injected with miR-8545 inhibitor.
FIG. 8 is a change in mortality after injection of miR-8545 inhibitor into 3-instar larvae of the plutella xylostella population NIL-R, which developed resistance by the Bt insecticidal protein Cry1Ac, and treatment with the Bt insecticidal protein Cry1 Ac. Wherein a is mortality after 3-instar larvae of the plutella xylostella population NIL-R, which were resistant to the Bt insecticidal protein Cry1Ac of example 2, were injected with Buffer and treated with Bt insecticidal protein Cry1 Ac; b is mortality after injection of PolyA sequence and treatment with Bt insecticidal protein Cry1Ac to 3-instar larvae of the plutella xylostella population NIL-R that developed resistance in example 2; c is mortality after 3-instar larvae of the plutella xylostella population NIL-R, which developed resistance by the Bt insecticidal protein Cry1Ac in example 2, were injected with an inhibitor of miR-8545 and treated with the Bt insecticidal protein Cry1 Ac.
Detailed Description
The invention will be further described and illustrated with reference to the following specific examples, but the scope of the invention is not limited thereto. Material sample preparation prior to experiment:
(1) Plutella xylostella Bt insecticidal protein sensitive population (DBM 1 Ac-S): the plutella xylostella population is bred in an insect group breeding room of a vegetable and flower research institute of China academy of agricultural science by using an insect-free cabbage seedling and radish seedling breeding method for subculture until now, is never contacted with any pesticide and is sensitive to Bt preparations.
(2) A diamondback moth Bt insecticidal protein Cry1Ac resistant population (DBM 1 Ac-R): the plutella xylostella population is bred in an insect group breeding room of vegetable and flower research institute of China academy of agricultural science by using an insect-free cabbage seedling and radish seedling breeding method. Meanwhile, during the secondary feeding period, bt Cry1Ac protoxins are used for resistance selection and maintenance in the 3-year-old larval stage.
(3) A diamond back moth Bt insecticidal protein Cry1Ac resistant near isogenic line population (NIL-R): the population utilizes two populations of DBM1Ac-S and DBM1Ac-R to carry out continuous 6-generation hybridization and backcross, and Bt Cry1Ac protoxin is continuously used for selecting after the subsequent populations, so that a Bt Cry1Ac high-resistance plutella xylostella near-isogenic line NIL-R population corresponding to the DBM1Ac-S is successfully established. The population is then bred in an insect breeding room of the vegetable and flower institute of China agricultural sciences by using an insect-free cabbage seedling and radish seedling breeding method. During the subculture, the Bt Cry1Ac protoxins were used for resistance selection and maintenance in the 3-instar larval stage.
(4) Shenzhen seed selection group (SZ-R): the population is collected in cabbage fields in Shenzhen area of Guangdong province in 2003, and then is bred in insect breeding chambers of insect groups of vegetable and flower research institute of China academy of agricultural sciences by using an insect-free cabbage seedling and radish seedling breeding method, and simultaneously, bt Cry protoxins are used for carrying out resistance screening and maintenance in 3-year larva period during the secondary breeding period.
(5) Shanghai selected population (SH-R): the population is collected in cabbage fields in suburban areas of Shanghai in 2005, and is then bred in insect breeding chambers of insect groups of vegetable and flower research institute of China academy of agricultural sciences by using insect-free cabbage seedlings and radish seedlings, and during the secondary breeding period, btk preparation (16000 IU/mg wettable powder) is used for resistance screening and maintenance in 3-year larval stage.
The 5 populations used above: the method comprises the steps of a plutella xylostella Bt insecticidal protein sensitive population (DBM 1 Ac-S), a plutella xylostella Bt insecticidal protein Cry1Ac resistant population (DBM 1 Ac-R), a plutella xylostella Bt insecticidal protein Cry1Ac resistant near isogenic line population (NIL-R), a Shenzhen obsolete population (SZ-R) and an Shanghai obsolete population (SH-R).
The five plutella xylostella populations are all kept in isolation in an insect breeding room, pupas are firstly placed in an adult breeding cage (the size is determined by the pupa amount), and the periphery of the cage is surrounded by 80 meshes; after the adults emerge, hanging one absorbent cotton ball soaked with 10% of honey water in the cage to supplement nutrition for the adults; and finally, carrying out secondary breeding by using the larvae after the fresh insect-free cabbage seedlings are inoculated. The raising temperature is 25+/-1 ℃, the relative humidity is about 65%, and the photoperiod is illumination: dark = 16h:8h.
Example 1
And detecting miR-8545 expression levels in the cDNA samples of the midgut of 4-year-old larvae of the Bt insecticidal protein Cry1Ac sensitive population DBM1Ac-S and 4 Bt insecticidal protein Cry1Ac resistant populations by using a real-time fluorescent quantitative PCR technology.
1. miR-8545 specific reverse transcription stem-loop primer 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCATCGA-3' is designed by taking the midgut miR-8545 sequence of plutella xylostella as a target sequence (the sequence is shown as SEQ ID NO. 1), and then miR-8545 first-strand cDNA is synthesized by reverse transcription by taking the midgut total RNA of plutella xylostella as a template.
The sequence of the specific primer reverse transcription miR-8545 is shown in figure 1, and the length of a first-strand cDNA template synthesized by reverse transcription is 64bp.
2. The first chain cDNA sequence synthesized by reverse transcription of the midgut miR-8545 of plutella xylostella is taken as a target sequence, and a specific fluorescent quantitative primer is designed according to the principle of real-time fluorescent quantitative PCR design, and is as follows:
forward primer (qmiR-8545-F): 5'-CGGGTGCAGGGCCTGG-3' (SEQ ID NO. 2)
Reverse primer (qmiR-8545-R): 5'-AGTGCAGGGTCCGAGGTATT-3' (SEQ ID NO. 3).
The sequence of the miR-8545 fragment amplified by the specific primer is shown in figure 2, and the amplified band size is 55bp, so that the requirement of miRNA real-time fluorescent quantitative PCR reaction is met. Meanwhile, the specific primer of the plutella xylostella U6 microRNA amplifies the internal reference gene, the amplified band size is 61bp, and the requirements of real-time fluorescent quantitative PCR reaction are met. The specificity of the two amplified fragments is confirmed after sequencing verification and sequence alignment.
3. Extracting intestinal RNA samples in 5 populations, reversely transcribing the intestinal RNA samples into cDNA templates by using specific stem-loop primers, and reacting in a real-time fluorescence quantitative PCR reaction system comprising the specific primers in the steps 1 and 2;
(1) The extraction steps of the plutella xylostella midgut RNA are as follows:
(1) 5 populations of four-instar larvae were taken, dissected on ice for midgut using dissecting forceps, and cleaned using 0.7% nacl solution.
(2) The midgut tissue is placed in a 1.5mL RNase-free centrifuge tube, l mL of Trizol reagent is added into the centrifuge tube, then the mixture is placed in a tissue grinder for full grinding, after grinding, the liquid is poured into a new 1.5mL RNase-free centrifuge tube, and the mixture is placed on ice for 5min.
(3) To the centrifuge tube, 300. Mu.l of chloroform was added, vigorously shaken for 15s, placed on ice for 5min, and centrifuged at 12000rpm for 10min at 4 ℃.
(4) The supernatant was pipetted into a new 1.5mL RNase-free centrifuge tube, 400. Mu.l of isopropanol was added, mixed gently upside down, left on ice for 10min and centrifuged at 12000rpm for 10min at 4 ℃.
(5) The supernatant was discarded, and the precipitate was washed with 1ml of 75% ethanol. Centrifugation is carried out at 8000rpm for 5min at 4 ℃.
(6) After the RNA was naturally dried, 50. Mu.l of water was added for dissolution and stored at-80 ℃. Determination of OD 260 /OD 280 And (3) detecting whether RNA is degraded by electrophoresis, and selecting proper conditions and reagents to carry out reverse transcription experiments or preserving at-80 ℃ for later use.
(2) cDNA template synthesized by reverse transcription
Taking the following example of the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) kit of Nanjinouzan biotechnology Co., ltd, the experimental procedure is as follows:
(1) before use, the components of the kit are thawed according to the instruction, vibrated and centrifugally mixed.
(2) Removal of genomic DNA reaction:
TABLE 1
(3) Reverse transcription reaction (performed on ice):
TABLE 2
(3) Real-time fluorescent quantitative PCR reaction:
taking the synthesized first-strand cDNA as a template, and then adopting a SYBR GreenI chimeric fluorescence method to carry out miRNA real-time fluorescence quantitative PCR:
(1) 20 μl of the fluorescent quantitative PCR reaction system consisted of:
TABLE 3 Table 3
| Reagent(s) | System of |
| 2×miRNA Universal SYBR qPCR Master Mix | 10μl |
| 10 mu M forward primer | 0.4μl |
| 10 mu M reverse primer | 0.4μl |
| cDNA template | 1μl |
| RNase-free ddH 2 O | To 20 μl |
* The product is a kit of miRNA Universal SYBR qPCR Master Mix of the biological technology Co-Ltd.
(2) The fluorescent quantitative PCR reaction conditions are as follows:
pre-denaturation at 95℃for 5min, denaturation at 95℃for 10sec, annealing at 60℃for 30sec,40 cycles.
After completion of the PCR process, the reaction was performed at 1.6℃s -1 The temperature rise rate of (2) is increased from 72 ℃ to 95 ℃ for analysis and verification of a melting curve, and the specific process is as follows: 95 ℃ for 15s, then 60 ℃ for 1min, according to 0.15 ℃ s -1 The temperature rise rate of (2) was increased from 60℃to 95℃and finally 95℃for 1s.
The single standard melting curve of the fluorescence quantification obtained finally is shown in FIG. 3 according to the result of the fluorescence quantification reaction.
(4) Real-time fluorescent quantitative PCR reaction process:
the system and conditions of the real-time fluorescent quantitative PCR reaction process are as described in the step (3).
4. Detection of
The invention uses ABIQuantum studio 3 real-time fluorescent quantitative PCR instrument for detection.
5. Analysis of results
When the ABIQuantum studio 3 real-time fluorescence quantitative PCR instrument performs result analysis, the baseline setting takes 3-15 cycles of fluorescence signals. For comparison, the threshold values of all amplification curves of the sample to be tested (4 samples of 4-instar larvae midgut cDNA of 4-instar larvae of the 4-instar population of the Bt insecticidal protein Cry1Ac resistant plutella xylostella) and the negative control (4-instar larvae midgut cDNA of 4-instar larvae of the Bt insecticidal protein Cry1Ac sensitive plutella xylostella population DBM1 Ac-S) were set to 0.096612 (at the middle of the logarithmic phase of the amplification curve, and the other instrument base lines and threshold values were adjusted accordingly according to the instrument).
The final real-time fluorescent quantitative PCR reaction detection results are shown in FIG. 4, wherein A is a 4-instar larva midgut cDNA negative control sample of the plutella xylostella population DBM1Ac-S sensitive to Bt insecticidal proteins; b is a 4-instar larva midgut cDNA positive control sample of the plutella xylostella population SZ-R which generates resistance to the Bt insecticidal protein Cry1 Ac; c is a 4-instar larva midgut cDNA sample to be detected of a plutella xylostella population SH-R which generates resistance to Btk; d is a 4-instar larva midgut cDNA sample to be tested for generating resistance to Bt insecticidal protein Cry1 Ac; e is a 4-instar larva midgut cDNA sample to be tested of a plutella xylostella near isogenic line population NIL-R which generates resistance to the Bt insecticidal protein Cry1 Ac.
Under the specific wavelength condition, according to the judgment result of the respective delta Ct values among the anti-sensitive samples of fluorescence detection, a typical S-shaped amplification curve is generated by the two samples, wherein the delta Ct value of the DBM1Ac-S sample is 7.09+/-0.26 (SEM), the delta Ct value of the SZ-R sample is 5.99+/-0.02 (SEM), the delta Ct value of the SH-R sample is 5.80+/-0.06 (SEM), the delta Ct value of the DBM1Ac-R sample is 5.59+/-0.02 (SEM), and the delta Ct value of the NIL-R sample is 5.36+/-0.07 (SEM). Through analysis, the expression level of miR-8545 in the 4 Bt insecticidal protein Cry1Ac resistant plutella xylostella populations is obviously increased relative to DBM1Ac-S negative control.
Example 2
Based on the detection result in the example 1, an agonist and an inhibitor of miR-8545 are synthesized, and the effect of miR-8545 on the resistance of the diamond back moth Bt insecticidal protein Cry1Ac and the application of the miR-8545 inhibitor are clarified.
1. On the basis of a midgut miR-8545 sequence of plutella xylostella, an agonist of miR-8545 is chemically synthesized, the agonist is injected into 3-year-old larvae of a plutella xylostella Bt insecticidal protein Cry1Ac sensitive population DBM1Ac-S by using a microinjection system, and the variation of miR-8545 expression levels in plutella xylostella at different time points after the agonist is injected is detected by using a real-time fluorescent quantitative PCR technology in example 1.
The variation of the expression level of miR-8545 in vivo after 3-year-old larvae of the Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella DBM1Ac-S are injected with miR-8545 agonists is shown in a graph 5, wherein A is the variation of the expression level of miR-8545 after 3-year-old larvae of the Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella population DBM1Ac-S are injected with Buffer; b is miR-8545 expression quantity change after 3-year larva of Bt insecticidal protein Cry1Ac sensitive plutella xylostella population DBM1Ac-S is injected with PolyA sequence; c is miR-8545 expression level of Bt insecticidal protein Cry1Ac sensitive plutella xylostella population DBM1 Ac-S3-year-old larvae injected with miR-8545 agonist. Compared with a Buffer control group and a PolyA sequence injection group, the miR-8545 expression quantity in the miR-8545 agonist injection group is obviously increased. From the above, the injection of miR-8545 agonist can significantly improve the expression level of miR-8545 in plutella xylostella larvae.
2. And detecting the sensitivity change of the plutella xylostella larvae to the Bt insecticidal protein Cry1Ac after the injection of the miR-8545 agonist by using a virulence bioassay technology.
The final virulence bioassay results are shown in fig. 6, wherein a is mortality after 3-instar larvae of the Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella population DBM1Ac-S are injected with Buffer and treated with Bt insecticidal protein Cry1 Ac; b is the mortality of 3-instar larvae of Bt insecticidal protein Cry1Ac sensitive plutella xylostella population DBM1Ac-S after being injected with PolyA sequences and treated by Bt insecticidal protein Cry1 Ac; and C is the mortality of 3-instar larvae of the Bt insecticidal protein Cry1 Ac-sensitive plutella xylostella population DBM1Ac-S after being injected with miR-8545 agonist and being treated by the Bt insecticidal protein Cry1 Ac. Compared with a Buffer control group and a PolyA sequence injection group, the miR-8545 agonist injection group has the advantage that the mortality of plutella xylostella larvae after being treated by the Bt insecticidal protein Cry1Ac for 72 hours is obviously reduced. From this, it can be seen that the injection of miR-8545 agonist can significantly improve the resistance of larvae to Bt insecticidal protein Cry1 Ac.
3. The inhibitor of miR-8545 is chemically synthesized, and is injected into 3-instar larvae of a group NIL-R of resistant plutella xylostella generated by Bt insecticidal protein Cry1Ac by using a microinjection system, and the variation of the expression level of miR-8545 in the plutella xylostella at different time points after the inhibitor of miR-8545 is injected is detected by using a real-time fluorescent quantitative PCR technology in example 1.
The final real-time fluorescence quantitative PCR detection result is shown in FIG. 7, wherein A is miR-8545 expression quantity change after 3-year-old larvae of a plutella xylostella population NIL-R with resistance generated by Bt insecticidal protein Cry1Ac are injected with Buffer; b is miR-8545 expression level change after 3-year larva of a diamondback moth population NIL-R with Bt insecticidal protein Cry1Ac for generating resistance is injected with a PolyA sequence; and C is miR-8545 expression quantity change after 3-instar larvae of a plutella xylostella population NIL-R with Bt insecticidal protein Cry1Ac generating resistance are infused with miR-8545 inhibitor. Compared with the Buffer control group and the PolyA sequence injection group, the miR-8545 inhibitor injection group has obviously reduced expression quantity. From the results, the miR-8545 inhibitor can be injected to obviously reduce the expression level of miR-8545 in plutella xylostella.
4. And detecting the sensitivity change of the plutella xylostella larvae to the Bt insecticidal protein Cry1Ac after injection of the miR-8545 inhibitor by using a virulence bioassay technology.
The final virulence bioassay results are shown in fig. 8, wherein a is mortality of 3-instar larvae of the plutella xylostella population NIL-R, which have developed resistance to the Bt insecticidal protein Cry1Ac, injected with Buffer and treated with Bt insecticidal protein Cry1 Ac; b is 3-instar larvae of a plutella xylostella population NIL-R with resistance generated by Bt insecticidal protein Cry1Ac, and the mortality after treatment by Bt insecticidal protein Cry1Ac is utilized; and C is 3-instar larvae of a plutella xylostella population NIL-R with resistance generated by the Bt insecticidal protein Cry1Ac, and miR-8545 inhibitor is injected and mortality is achieved after treatment by the Bt insecticidal protein Cry1 Ac. Compared with a Buffer control group and a PolyA sequence injection group, the mortality of plutella xylostella larvae in the miR-8545 inhibitor injection group after being treated by Bt insecticidal protein Cry1Ac is obviously increased. From this, it can be seen that the injection of the miR-8545 inhibitor can significantly reduce the resistance of plutella xylostella larvae to Bt insecticidal protein Cry1 Ac.
The results are summarized below: in the miR-8545 agonist treatment group, the expression level of miR-8545 in the plutella xylostella larva is obviously increased, and a toxicity biological assay result shows that the injection of the miR-8545 agonist leads to the enhancement of the resistance of the plutella xylostella larva to the Bt insecticidal protein Cry1 Ac. In contrast, in the miR-8545 inhibitor treatment group, the expression level of miR-8545 in the plutella xylostella larva is obviously reduced, and a toxicity biological assay result shows that the resistance of the plutella xylostella larva to the Bt insecticidal protein Cry1Ac is obviously reduced by injecting the miR-8545 inhibitor. These results show that the miR-8545 inhibitor has the function of inhibiting the resistance of plutella xylostella larvae to Bt insecticidal protein Cry1Ac, can be used as an inhibitor for inhibiting the resistance of plutella xylostella larvae to Bt insecticidal protein Cry1Ac, can be prepared into patent medicine field application, and provides a new thought and theoretical basis for the research of the resistance treatment of plutella xylostella larvae Bt insecticidal protein Cry1 Ac.
Claims (8)
1. A miRNA inhibitor for treating the resistance of a plutella xylostella Bt insecticidal protein Cry1Ac, which is characterized in that the miRNA inhibitor for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac is used for reducing the expression level of miRNA.
2. The miRNA inhibitor for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac according to claim 1, wherein the miRNA inhibitor for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac comprises an antisense complementary non-coding RNA sequence consisting of a miR-8545 sequence.
3. A miRNA inhibitor for the management of diamond back moth Bt insecticidal protein Cry1Ac resistance according to claim 2, characterized in that said antisense complementary non-coding RNA sequence is 18-24 bases in length, preferably 20 bases in length.
4. A miRNA inhibitor for the management of diamond back moth Bt insecticidal protein Cry1Ac resistance according to claim 3, wherein the antisense complement non-coding RNA sequence has a nucleic acid sequence as shown in SEQ ID No. 5.
5. Use of a miRNA inhibitor for managing the resistance of the diamond back moth Bt insecticidal protein Cry1Ac according to any one of claims 1 to 4 for the preparation of a medicament for the management of the resistance of diamond back moth to Bt insecticidal protein Cry1 Ac.
6. The use according to claim 5, characterized in that it is achieved by applying said miRNA inhibitor for managing the resistance of the diamond back moth Bt insecticidal protein Cry1Ac to the planting site or field, or to the diamond back moth body surface.
7. A method for preparing plutella xylostella sensitive to Bt insecticidal protein Cry1Ac, which is characterized in that miRNA inhibitor for treating the resistance of plutella xylostella Bt insecticidal protein Cry1Ac is injected into plutella xylostella.
8. The auxiliary method for preventing and controlling the plutella xylostella is characterized in that the miRNA inhibitor for treating the resistance of the plutella xylostella Bt insecticidal protein Cry1Ac is applied at the same time of applying the Bt insecticidal protein Cry1Ac at a planting site or in a field.
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