CN113142211B - Application of small molecule compound ZK-PI-9 in preparation of trehalase inhibitor - Google Patents
Application of small molecule compound ZK-PI-9 in preparation of trehalase inhibitor Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
- A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
- A01N37/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system having at least one carboxylic group or a thio analogue, or a derivative thereof, and one oxygen or sulfur atom attached to the same aromatic ring system
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- Life Sciences & Earth Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an application of a small molecular compound ZK-PI-9 in preparation of a trehalase inhibitor. The trehalase inhibitor ZK-PI-9 can cause slow growth, malformation and even death of insects, so that the activity of trehalase of the insects is reduced, and the trehalase inhibitor ZK-PI-9 has potential as a biological pesticide. The invention can be applied to research and preparation of novel biopesticides with trehalase as a target.
Description
Technical Field
The invention relates to the technical field of insect control, in particular to application of a small molecular compound ZK-PI-9 in preparation of a trehalase inhibitor.
Background
China is a country with frequent biological disasters in agricultural production, thousands of tons of grains are lost due to insect pests on average every year, and the damage of the insect pests to crops is reflected not only in the aspect of reducing the yield of agricultural products, but also in the aspect of weakening the quality of the agricultural products. It is estimated that the potential food loss from world pests is around 45%. The use of pesticides to control pests is therefore unavoidable and important in agricultural production. However, long-term dependence on pesticides brings about serious 3R problems (drug resistance, residue and pest rampant), which not only does not achieve the purpose of pest control, but also causes a series of problems of environmental pollution, reduced biological diversity and the like. The method becomes an important means for green prevention and control of pests by regulating the growth and development of the pests, and is key to find a target gene capable of playing a regulating role and develop a proper biological pesticide.
Trehalose is a non-reducing disaccharide, the most important carbohydrate in insect haemolymph, and the energy released by hydrolysis is the main source of energy required for flight, development, molting and stress reactions. The trehalose can be used as an energy storage substance to participate in energy metabolism of organisms, and can also be used as a signal molecule to regulate glycolysis process and a structural substance to participate in the formation of insect body walls and regulate the growth and development of insects. Under various adverse conditions, the trehalose is used as a signal molecule energy source and a cell wall glycolipid component to protect organisms from environmental stresses such as high temperature, cold, dryness, hunger and the like, so that the vitality and the activity of insects are improved.
Trehalase is the only enzyme known in all organisms to break down trehalose and is also the first enzyme in the insect's in vivo chitin synthesis pathway. Inhibition of trehalase has been found to result in abnormal insect development, stunted growth, decreased flight ability and fertility, and increased mortality. And because trehalase is indispensable in insects and trehalase inhibition has no toxic effect on higher mammals, it has become one of important targets for pest control, and effective novel trehalase inhibitor products with practical pesticide significance are being developed.
A small molecule compound ZK-PI-9, the Chinese name of N- (4-chloro-2-methoxy-5-methylphenyl) -3-hydroxy-2-naphthamide, the molecular formula: c19H16ClNO3Relative molecular mass: 341.8 CAS registry number 5165-81-1, the structural formula is shown in formula I.
Disclosure of Invention
The invention aims to provide a novel trehalase inhibitor ZK-PI-9 (molecular formula: C)19H16ClNO3Relative molecular mass: 341.8) which can regulate the protein related to the trehalose metabolism of insects, so that the expression of trehalase in mRNA level is reduced, and the insects grow slowly, are malformed and even die.
Application of a small molecular compound ZK-PI-9 in preparation of trehalase inhibitors. Preferably, the trehalase is insect soluble Tre1 or membrane-bound trehalase Tre 2.
The invention also provides application of the small molecular compound ZK-PI-9 in preparing pesticides. Preferably, the target to be killed by the insecticide is an insect.
The invention also provides a pesticide, and the active ingredient of the pesticide is a micromolecular compound ZK-PI-9. Preferably, the concentration of the small molecule compound ZK-PI-9 is not less than 2X 10-3mmol/ml. Preferably, the target of killing by the insecticide is an insect such as Spodoptera frugiperda (Spodoptera frugiperda). The small molecular compound ZK-PI-9 acts on trehalase to play a role of a trehalase inhibitor, the trehalase is the most important carbohydrate substance in insect haemolymph and has an important role, and the trehalase widely exists in insect bodies, so that the pesticide taking the small molecular compound ZK-PI-9 as an active ingredient has a broad spectrum of insect killing effects.
The invention has the following beneficial effects: the trehalase inhibitor ZK-PI-9 can cause slow growth, deformity and even death of insects, so that the activity of trehalase of the insects is reduced, and the trehalase inhibitor has the potential of being used as a biological pesticide. The invention can be applied to research and preparation of novel biopesticides with trehalase as a target.
Drawings
FIG. 1 Spodoptera frugiperda injection 2X 10-3Changes in pupa weight (A) and pupa length (B) after mmol/ml trehalase inhibitor ZK-PI-9: denotes P<0.05, significant difference, indicates P<0.01, the difference is extremely obvious, the same is true below;
FIG. 2 Spodoptera frugiperda injection 2X 10-3Pupation, eclosion and death change after mmol/ml trehalase inhibitor ZK-PI-9, wherein A: spodoptera frugiperda injection 2X 10-3Pupation rate, eclosion rate and mortality rate after mmol/ml trehalase inhibitor ZK-PI-9, B: failure to pupate and death.
FIG. 3 Spodoptera frugiperda injection 2X 10-3Change of glucose content of mmol/ml trehalase inhibitor ZK-PI-948 h.
FIG. 4 Spodoptera frugiperda injection 2X 10-3Change of total glycogen content of trehalase inhibitor ZK-PI-948 h in mmol/ml.
FIG. 5 Spodoptera frugiperda injection 2X 10-3Change of trehalose content of trehalose inhibitor ZK-PI-948 h in mmol/ml.
FIG. 6 Spodoptera frugiperda injection 2X 10-3mmol/ml trehalase inhibitor ZK-PI-948 h trehalase activity change: the trehalase includes soluble trehalase (A) and membrane-bound trehalase (B).
FIG. 7 is an evolutionary developmental tree constructed by Spodoptera frugiperda Tre1, Tre2 amino acid sequence and other insect Tres.
FIG. 8 Spodoptera frugiperda injection 2X 10-3Expression level of trehalase inhibitor ZK-PI-948 h trehalase gene mmol/ml: the trehalase comprises soluble trehalase (A) and membrane-bound trehalase (B); RPL10 was used as an internal reference gene.
Detailed Description
Spodoptera frugiperda is an important devastating agricultural pest native to America and is listed in the blacklist of ten plant pests in the world as reported annually by the '2017 world plant status quo'. The Spodoptera frugiperda invades China across the border in Yunnan in two years, has high feeding impurity, high propagation and migration capacities and wide suitable growing area, and poses great threat to the development of the corn planting industry in China. The present invention is described in detail in the following examples, which take Spodoptera frugiperda as an example.
The trehalase inhibitor ZK-PI-9 has effective concentration of 2 × 10-3mmol/ml, using 2% DMSO as a solvent, trehalase inhibitor was supplied by the university of agriculture, China PMDD laboratory.
Example 1
Spodoptera frugiperda injection 2X 10-3Change of phenotype and development period after mmol/ml ZK-PI-948 h.
1. Experimental materials: spodoptera frugiperda (Spodoptera frugiperda) was from the laboratory rearing population and the source of the test insects was from the Hangzhou population of the Zhejiang agricultural institute. The raising conditions of Spodoptera frugiperda are as follows: the temperature is 26 +/-1 ℃, the illumination ratio is 16L to 8D, and the relative humidity is 60 +/-10%. The larvae were fed in a feed box with artificial feed (table 1) on one end, and the adults were nourished with 10% hydromel simulated nectar.
TABLE 1 Spodoptera frugiperda Young fodder formulation
Raw materials | Dosage of |
Bean curd | 111.0g |
Yeast extract | 33.8g |
Wheat germ | 52.8g |
Ascorbic acid | 3.4g |
Sorbic acid (hexadienoic acid) | 1.1g |
P-hydroxybenzoic acid methyl ester | 2.1 |
10% Formaldehyde | 8.4ml |
Water (Mixer) | 490.0ml |
Water (agar solution) | 338.0ml |
Agarose (agarose) | 13.5g |
2. Spodoptera frugiperda microinjection: 2% DMSO as solvent is prepared at a concentration of 2 × 10-3mmol/ml ZK-PI-9, the volume of validamycin injection pumped out by each injector is firstly quantified by a standard capillary before injection, and the volume of the validamycin injection is adjusted to the injection amount by adjusting the nitrogen pressure. And selecting Spodoptera frugiperda larvae of day 1 with 3 ages as injection objects. Paralyzing spodoptera frugiperda larvae on ice, placing the spodoptera frugiperda larvae on filter paper of a microinjection instrument objective table by using a tip small writing brush to enable the abdomen of the spodoptera frugiperda larvae to face upwards, injecting spodoptera frugiperda larvae into the chest between a second pair of feet and a third pair of feet (the epidermis is thin and is easy to prick into the chest), wherein the injection amount is 300 nl/head, and placing the spodoptera frugiperda larvae into a feed box after injection.
3. Observation of phenotype and developmental process of spodoptera frugiperda 48h after injection: the larvae were aged daily from the third day after the trehalase inhibitor injection, and their life cycles were counted, and the results are shown in table 2. The body length of the spodoptera frugiperda larvae after injection was measured daily using a ruler as a tool for measuring body length, and compared to the control group, the results are shown in table 3. 30-60 worms are observed in the treatment process, the molting condition of the larvae is checked every day, the number of normal, malformed and dead larvae is recorded, and the malformed and dead larvae are photographed.
The results in tables 2 and 3 show that after the trehalase inhibitor ZK-PI-9 is injected, the life cycles of 4L and 5L of Spodoptera frugiperda are longer than that of a control group, and the average body length of other ages is similar to that of the control group, which indicates that the trehalase inhibitor ZK-PI-9 can influence the development process of Spodoptera frugiperda.
TABLE 2 injection 2X 10-3mmol/ml ZK-PI-9 Spodoptera frugiperda developmental calendar
TABLE 3 injection 2X 10-3mmol/ml ZK-PI-9 Spodoptera frugiperda larva length of each instar
Unit: cm of | 4L | 5L | 6L |
CK | 1.64±0.031 | 2.24±0.026 | 2.73±0.039 |
ZK-PI-9 | 1.65±0.015 | 2.27±0.034 | 2.75±0.087 |
4. After Spodoptera frugiperda is injected, the weight of the larvae is weighed when the larvae are 6L, the weight of pupae and the length of pupae are weighed when the larvae become pupae, the results are shown in figure 1, and the pupation rate is recorded at the same time. Recording the emergence rate when the adult insects emerge, considering the adult insects to be malformed if the adult insects cannot shed or have no normal wings when the adult insects emerge, considering the adult insects to be died if the adult insects still do not appear after 15 days, and photographing and recording all malformed individuals by using a camera. And finally, counting the pupation rate, the eclosion rate and the death rate of the spodoptera frugiperda after injection, wherein the results are shown in a figure 2.
The results in FIG. 1 show that the pupa length of Spodoptera frugiperda injected with trehalase inhibitor ZK-PI-9 is significantly shorter than that of control group CK, and the pupa weight is smaller than that of control group CK.
The results in FIG. 2 show that the pupation rate and the emergence rate of Spodoptera frugiperda injected with trehalase inhibitor ZK-PI-9 are both lower than those of the control group CK, and the mortality rate is higher than that of the control group CK. The trehalase inhibitor ZK-PI-9 is shown to affect the phenotypic change of spodoptera frugiperda.
Example 2
Spodoptera frugiperda injection 2X 10-3Spodoptera frugiperda sugar content after mmol/ml ZK-PI-948 h.
1. By 2X 10-3mmol/ml ZK-PI-9 was injected into Spodoptera frugiperda larvae of day 1 at 3 ages, the method was the same as example 1, and 21 Spodoptera frugiperda larvae at 48h after injection were randomly selected and divided into 3 replicates, each of which had 7 replicates, for detecting the trehalase activity and the change in sugar content. During sampling, the required spodoptera frugiperda larvae are cleaned by distilled water, water on the larvae is absorbed by filter paper and then put into an EP (ethylene propylene glycol) tube, and the larvae is put into a refrigerator at the temperature of minus 80 ℃ for freezing storage. Subsequent quantitative experiments were performed.
2. Spodoptera frugiperda 2X 10-3Determination of the glucose content 48h after mmol/ml of ZK-PI-9 injection: taking 7 spodoptera frugiperda larvae into a centrifuge tube, adding 2 sterilized small steel balls, putting into a tissue disruptor for disruption, and carrying out the following procedures: 55Hz, 120 s; pouring out the small steel balls after the crushing is finished, and adding 200 mu l PBS for ultrasonic crushing for 30 min; after disruption, 800. mu.l PBS was added, and the mixture was centrifuged at 1000 Xg and 4 ℃ for 20 min. 150. mu.l of 20800 Xg centrifuged supernatant and 20800 Xg precipitated suspension were taken into an EP tube to prepare samples. Glucose standards were prepared according to the system of Table 4. The standard and sample were measured for glucose content in nmol/. mu.g protein using a glucose assay kit (Sigma) and absorbance at 540nm using a microplate reader, and the results are shown in FIG. 3.
TABLE 4 preparation of glucose marker
Glucose Standard solution (ul) | PBS(μl) |
0 | 50 |
1 | 49 |
2 | 48 |
3 | 47 |
4 | 46 |
5 | 45 |
3. Spodoptera frugiperda 2X 10-3Determination of the Total glycogen content 48h after mmol/ml of ZK-PI-9 injection: adding 160 μ L1000 Xg supernatant sample into 32 μ L0.1U/L amyloglucosidase, water bathing at 40 deg.C for 4h, adding 150 μ L sample into new EP tube, adding 300 μ L glucose analytical reagent, and mixing to obtain sample. Glucose standards were prepared according to the system of Table 4. The total glycogen content was measured by measuring the absorbance at 540nm with a microplate reader using a glucose assay kit, and the total glycogen content unit was nmol/μ g protein, and the results are shown in FIG. 4.
4. Spodoptera frugiperda 2X 10-3Determination of trehalose content 48h after mmol/ml ZK-PI-9 injection: 30. mu.l of 1000 Xg supernatant, 20800 Xg supernatant, and 20800 Xg precipitate suspension were each put into an EP tube to prepare a sample. Standard curves were prepared by 2 Xgradient dilution with 1.6mM trehalose standard solution to 0.8mM, 0.4mM, 0.2mM, 0.1mM, and 0.05 mM. Processing the sample and standard curve by anthrone method, measuring 630nm absorbance by enzyme labeling instrument, and performing trehaloseThe results of the assay, in which the total glycogen content was expressed in nmol/. mu.g protein, are shown in FIG. 5.
According to the illustrations of FIGS. 3, 4 and 5, 2X 10 injections were made-3The glucose content and the trehalose content of the ZK-PI-9 Spodoptera frugiperda in mmol/ml are higher than those of a control group CK, and the total glycogen content in vivo is obviously higher than that of the control group CK. The influence of trehalase inhibitor ZK-PI-9 on the sugar content in Spodoptera frugiperda is shown.
Example 3
Spodoptera frugiperda injection 2X 10-3Trehalase activity changed after mmol/ml ZK-PI-948 h.
FIG. 6 shows an injection of 2X 10-3The activity of soluble trehalase in the Spodoptera frugiperda with mmol/ml ZK-PI-9 is lower than that of a control group CK, and the activity of membrane-bound trehalase is extremely obviously lower than that of the control group CK. Show 2X 10-3The ZK-PI-9 inhibited by the trehalase in mmol/ml can effectively inhibit the trehalase activity of spodoptera frugiperda.
Example 4
Spodoptera frugiperda injection 2X 10-3mmol/ml ZK-PI-948 h soluble trehalase gene TRE1, membrane-bound trehalase gene TRE 2.
1. By 2X 10-3mmol/ml ZK-PI-9 was injected into 3 st day 1 Spodoptera frugiperda larvae as in example 1. 9 spodoptera frugiperda larvae which are 48 hours after injection are randomly sampled by the method of example 2, are divided into 3 parallels, and each parallels has 3 heads and is used for detecting the expression quantity of trehalose metabolism key genes TRE1 and TRE 2.
2. Extracting RNA from Spodoptera frugiperda by Trizol method, and collecting total amount of 1 μ g RNAThe RT reagent Kit With gDNA Eraser reverse transcription Kit is used for cDNA single-strand synthesis. Mu.l of the reverse transcript was taken for template for qRT-PCR. The qRT-PCR reaction system is 20 μ l, which is as follows: mu.l of each of the forward and reverse primers, 1. mu.l of cDNA, 10. mu.l of SYBR Premix ExTaq (TBgreen), and sterile ultrapure water to make up to 20. mu.l, the primer sequences were as follows:
SfTRE1-F:5’-TCAGATGAAGGTGAACTCGAAGA-3’,
SfTRE1-R:5’-GGAATGATGAATCCGTGGGTA-3’,
SfTRE2-F:5’-CTGCTGCTGTCGGAGATGA-3’,
SfTRE2-R:5’-TAGGAGGGGAGGCTGTGAT-3’,
qRT-RPL10-F:5’-GATGACATGGAATGGATG-3’,
qRT-RPL10-R:5’-GATGACATGGAATGGATG-3’。
the sequence rpl of the trehalase gene TRE1(GenBank number: ABE27189.1) and TRE2(GenBank number: ACF94698.1) of Spodoptera frugiperda is a known sequence. The results of the evolutionary development trees constructed by Sf-Tre1(ABE27189.1) and Sf-Tre2(ACF94698.1) based on amino acid sequences and other insect tres are shown in fig. 7, wherein the sources of the Tre protein species and their accession numbers: spodoptera litura: Sl-Tre1(ADA63846.1), Sl-Tre2(ADA 63845.1); spodoptera exigua: Se-Tre1(ABY86218.1), Se-Tre2(ABU 95354.1); yellow wild borer heirtia vitessoides: Hv-Tre1(AZM68711.1), Hv-Tre2(AYO 46921.1); bamboo worm Omphisa fuscidigeras: Of-Tre1(ABO 20846.1); Of-Tre2(ABO 20845.1); antheraea pernyi: Ap-Tre1A (ARD05073.1), Ap-Tre2(ARD 05075.1).
The specific PCR procedure was: pre-denaturation at 95 ℃ for 3 min; then denaturation is carried out for 5s at 95 ℃, and annealing and extension are carried out for 25s at 55-62.5 ℃ (cycle is carried out for 40 times); finally, drawing a 65-95 ℃ dissolution curve. Data analysis was performed using the 2- Δ T method and ANOVA in STATISTICA 6.0.0, and the results are shown in FIG. 8.
FIG. 8 shows an injection of 2X 10-3The expression levels of Spodoptera frugiperda soluble trehalase gene TRE1 and membrane-bound trehalase gene TRE2 of ZK-PI-9 in mmol/mlThe reduction shows that ZK-PI-9 has no obvious influence on the trehalose gene of spodoptera frugiperda.
Sequence listing
<110> university of teachers in Hangzhou
Application of <120> small molecular compound ZK-PI-9 in preparation of trehalase inhibitor
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tcagatgaag gtgaactcga aga 23
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggaatgatga atccgtgggt a 21
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctgctgctgt cggagatga 19
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
taggagggga ggctgtgat 19
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gatgacatgg aatggatg 18
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gatgacatgg aatggatg 18
Claims (2)
2. the application of small molecular compound ZK-PI-9 in preparing pesticide,
the small molecular compound ZK-PI-9 can kill insects by inhibiting the activity of soluble trehalase TRE1 or membrane-bound trehalase TRE2 of insects,
the structural formula of the small molecular compound ZK-PI-9 is shown as the formula I:
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