A cytochrome CYP6AE28 of Cnaphalocrocis medinalis and its application

Abstract

The present invention provides a cytochrome CYP6AE28 of Cnaphalocrocis medinalis and its application, and the nucleotide sequence of CYP6AE28 is as shown in SEQ ID NO.1. The CYP6AE28 of the present invention and its encoded protein are of great significance for analyzing the insecticide resistance mechanism of Cnaphalocrocis medinalis, controlling its resistance, and ensuring the green and safe production of rice.

Description

Technical Field

[0001] The present invention relates to the technical field of biological genetic engineering, and particularly relates to a cytochrome CYP6AE28 of Cnaphalocrocis medinalis and its application. Background Art

[0002] The rice leaf folder (Cnaphalocrocis medinalis Guenee), belonging to the order Lepidoptera, family Pyralidae, also known as the scraping green worm and bract leaf worm, is one of the most destructive pests among the three major rice borers. The rice leaf folder has a relatively wide host range. It not only feeds on and damages rice at all growth stages, but also harms crops such as corn (Zea mays L.), wheat (Triticum aestivum L.), sorghum (Sorghum bicolor L.), sugarcane (Saccharum officinarum L.), wild rice (Zizania latifolia L.), etc., as well as gramineous weeds such as barnyard grass (Echinochloa crusgalli L.), goosegrass (Eleusine indica L.), and green foxtail (Setaria viridis L.). The damage symptoms of the rice leaf folder are extremely obvious. Newly hatched larvae often gather in the heart leaves and eat the mesophyll inside the leaf sheaths. Around the third instar, they gradually move down to the middle of the leaves to form a longitudinally rolled cylindrical single-leaf bract, and a few also form a 3- to 5-leaf multi-leaf bract. They eat the mesophyll inside the bract, leaving only the epidermis, showing white stripes. When it is severe, there are numerous insect bracts, and the field is full of white leaves. In recent years, the damage has become increasingly serious. During the period from 2015 to 2020 alone, the annual occurrence area of the rice leaf folder reached 19 million hectares 2 , resulting in a yield loss of more than 7 million tons, equivalent to 3.6% of the total rice area. Currently, chemical control is still the main means to control the rice leaf folder, but problems such as affecting the paddy field ecosystem, the increase in drug resistance, and the resurgence of the rice leaf folder have emerged; therefore, it is particularly necessary to study green control methods. Due to the overlapping and alternating growth cycles of the rice leaf folder, the contact amount of the rice leaf folder with pesticides is generally increased, resulting in an increase in drug resistance. Previous studies have found that the resistance levels of the rice leaf folder to 10 pesticides have increased significantly in the field population. The field populations tested from 2019 to 2021 showed sensitive or moderate resistance levels (RR = 1.3–22.1) to chlorantraniliprole, while the resistance level of chlorantraniliprole in 2022 was as high as 64.9–113.7 times. We have also observed in Sichuan Province that the resistance of the rice leaf folder to chlorantraniliprole is significantly higher than that of other pesticides. Currently, as an anthranilic diamide compound, the drug resistance of chlorantraniliprole has become a major problem in agricultural production.

[0003] Due to the unscientific use of pesticides, populations of Cnaphalocrocis medinalis in some areas have developed varying levels of resistance, and the development of insect resistance is the result of the combined action of multiple factors. Among them, the resistance mechanisms of Cnaphalocrocis medinalis mainly include the reduction of the sensitivity of the drug action site, the enhancement of the ability of insects to metabolize pesticides in vivo, and the unique physical protection mechanisms of insects, etc. (1) Insects may develop tolerance to pesticides through metabolic detoxification mechanisms. Such insects have specific metabolic enzymes in their bodies that can decompose or transform pesticides and reduce their toxic effects. Detoxifying enzyme families such as carboxylesterase, glutathione transferase, and mixed-function oxidase play an essential role in metabolic resistance. (2) Secondly, insects may reduce the absorption of pesticides through reduced epidermal permeability. These insects have a special epidermal structure that can reduce the penetration of pesticide molecules and their entry into the body. This mechanism makes it difficult for pesticides to exert their toxic effects, thus increasing the tolerance of insects to pesticides. (3) Avoid contact with pesticides through behavioral adaptation. They may change their feeding habits, activity time, or habitat selection, etc., to reduce the chance of contact with pesticides. (4) Target resistance is the resistance caused by the reduced sensitivity of the target receptor in insects to pesticides. Common pesticide action targets are: nicotinic acetylcholine receptor (nAChR), γ-aminobutyric acid receptor (GABA), insect sodium channel, acetylcholinesterase (ACHE). Currently, the in-depth exploration of the resistance mechanism of Cnaphalocrocis medinalis mainly focuses on the ability to metabolize pesticides, and there are few reports on target resistance research.

[0004] Cytochrome P450, also known as multifunctional oxidase or P450 or CYP, is an oxidase system present in most organisms, with rich species diversity and functions. In the life activities of insects, cytochrome P450 plays an important role. Its functions mainly include catalyzing important substances such as pheromones, synthesizing and degrading fatty acids, mainly to maintain the normal physiological activities of organisms; secondly, cytochrome P450 catalyzes plant secondary substances and mutagen precursors and participates in the detoxification metabolism of foreign compounds such as pesticides. In insects, the cytochrome P450 gene family in insects is rich and diverse, including 48 families such as CYP4, CYP6, CYP9, and CYP12. It is particularly noteworthy that the three gene families CYP6, CYP9, and CYP12 only exist in insects, and most of the resistance-related genes are contained in these three gene families. For example, CYP4D4v2, CYP4G2, and CYP6A38 were found to participate in the detoxification metabolism of houseflies resistant to cypermethrin. Under the stress of imidacloprid and nitroiminoimidazole, the expression levels of CYP6AY1 and CYP6ER1 in brown planthoppers and white-backed planthoppers were also significantly up-regulated. In summary, with the development of biological technologies, how current insect cytochrome P450 regulates insect detoxification metabolism is a hot topic of exploration. Therefore, it is particularly necessary to deeply explore the genes regulating detoxification metabolism and participate in the resistance mechanism of Cnaphalocrocis medinalis, which can provide strong theoretical support and practical guidance for the management of agricultural pest resistance and integrated prevention and control, and lay a solid foundation for the safety and sustainable development of future agricultural production. Summary of the Invention

[0005] In view of the increasingly serious resistance situation of the rice pest Cnaphalocrocis medinalis in China, which has become difficult to meet the urgent needs of current green rice production safety, the present invention is committed to exploring key factors that can regulate detoxification metabolism genes, and thus provides a cytochrome CYP6AE28 of Cnaphalocrocis medinalis and its application, which provides a new strategy for the management of agricultural pest resistance and also provides strong guarantee for the green production safety of China's rice industry.

[0006] To achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0007] In the first aspect, the present invention provides a cytochrome CYP6AE28 of Cnaphalocrocis medinalis, and the nucleotide sequence of CYP6AE28 is as shown in SEQ ID NO.1.

[0008] In the second aspect, the present invention provides the amino acid encoding the cytochrome CYP6AE28 of Cnaphalocrocis medinalis described in the first aspect, and the amino acid sequence is as shown in SEQ ID NO.2.

[0009] Thirdly, the present invention provides dsRNA for interfering with the expression of cytochrome CYP6AE28 of the rice leaf folder, and the nucleotide primers of the dsRNA are as shown in SEQ ID NO.3.

[0010] Fourthly, the present invention provides a preparation method of the dsRNA described in the third aspect, comprising the following steps:

[0011] Step 1, extracting the total RNA of the rice leaf folder and reverse transcribing it into cDNA;

[0012] Step 2, using the cDNA as a template to perform gene cloning to obtain a CYP6AE28 gene fragment;

[0013] Step 3, ligating the CYP6AE28 gene fragment onto a vector and transforming through competent cells to obtain a CYP6AE28 gene plasmid;

[0014] Step 4, performing PCR amplification using the full-length sequence of the CYP6AE28 gene plasmid as a template, and purifying the amplification product to obtain the dsRNA.

[0015] Preferably, in the step 2, the primer sequences used for gene cloning are respectively as shown in SEQ ID NO:38 and SEQ ID NO:39.

[0016] Preferably, in the step 4, the primer sequences used for PCR amplification are respectively as shown in SEQ ID NO:42 and SEQ ID NO:43.

[0017] Fifthly, the present invention provides the application of the cytochrome CYP6AE28 of the rice leaf folder described in the first aspect and / or the amino acid described in the second aspect and / or the dsRNA described in the third aspect in the preparation of products for controlling the rice leaf folder.

[0018] Sixthly, the present invention provides the application of the cytochrome CYP6AE28 of the rice leaf folder described in the first aspect and / or the amino acid described in the second aspect and / or the dsRNA described in the third aspect in reducing the insecticide resistance of the rice leaf folder.

[0019] Seventhly, the present invention provides an insecticide, comprising: the dsRNA described in the third aspect and chlorantraniliprole.

[0020] Eighthly, the present invention provides a method for controlling the rice leaf folder, feeding the rice leaf folder with the dsRNA described in the third aspect, and then applying chlorantraniliprole.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention discovers a key factor CYP6AE28 that can regulate detoxification metabolism genes. This CYP6AE28 and its encoded protein are of great significance for analyzing the resistance mechanism of Cnaphalocrocis medinalis, controlling its resistance, and ensuring the green and safe production of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present invention, form a part of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the drawings:

[0024] Figure 1 It is the determination result of the carboxylesterase activity of Cnaphalocrocis medinalis in Example 2 of the present invention. Among them, a and b represent that there are significant differences in enzyme activity at the P = 0.05 level among different treatments; Sus represents the susceptible strain; 22CP represents the resistant population; TPP is triphenyl phosphate (carboxylesterase inhibitor).

[0025] Figure 2 It is the determination result of the glutathione transferase activity of Cnaphalocrocis medinalis in Example 2 of the present invention. Among them, a, b, and c represent that there are significant differences in enzyme activity at the P = 0.05 level among different treatments; Sus represents the susceptible strain; 22CP represents the resistant population; DEM is piperonyl butoxide (glutathione transferase inhibitor).

[0026] Figure 3 It is the determination result of the cytochrome P450 enzyme activity of Cnaphalocrocis medinalis in Example 2 of the present invention. Among them, a, b, and c represent that there are significant differences in enzyme activity at the P = 0.05 level among different treatments; Sus represents the susceptible strain; 22CP represents the resistant population; PBO is piperonyl butoxide (cytochrome P450 enzyme inhibitor).

[0027] Figure 4 It is the mRNA expression levels of 15 P450 genes in Rongxian and Naxi in Example 3 of the present invention. Among them, Figure A represents the Rongxian population, and Figure B represents the Naxi population; different lowercase letters indicate significant differences at the P = 0.05 level.

[0028] Figure 5 It is the mRNA expression levels of 15 resistance-related P450 genes in Qianwei and Cuiping in Example 3 of the present invention. Among them, Figure A represents the Qianwei population, and Figure B represents the Cuiping population. Different lowercase letters indicate significant differences at the P = 0.05 level.

[0029] Figure 6 It is the mRNA expression levels of 15 resistance-related P450 genes in Fushun and Hejiang in Example 3 of the present invention. Among them, Figure A represents the Fushun population, and Figure B represents the Hejiang population. Different lowercase letters indicate significant differences at the P = 0.05 level.

[0030] Figure 7 It is the expression level of cytochrome P450 gene in the susceptible strain under the stress of chlorantraniliprole in Example 3 of the present invention. Among them, Figure A represents 24 h of chlorantraniliprole stress, and Figure B represents 48 h of chlorantraniliprole stress; different lowercase letters indicate significant differences at the P = 0.05 level.

[0031] Figure 8 It is the expression level of cytochrome P450 gene in the resistant population under the stress of chlorantraniliprole in Example 3 of the present invention. Among them, Figure A represents 24 h of chlorantraniliprole stress; Figure B represents 48 h of chlorantraniliprole stress; the same lowercase letters indicate significant differences at the P = 0.05 level.

[0032] Figure 9 It is the plasmid construction map of CYP6AE28 in Example 4 of the present invention.

[0033] Figure 10 It is the expression level and mortality rate after CYP6AE28 interference in Example 5 of the present invention. Among them, Figure A represents the expression level after CYP6AE28 interference, and Figure B represents the mortality rate under the conditions of 0 ng / head and 1 ng / head of chlorantraniliprole after injecting dsGFP and dsCYP6AE28; different lowercase letters indicate significant differences at the P = 0.05 level. Detailed implementation manners

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, which are explanations of the present invention rather than limitations.

[0035] For the molecular biology experimental methods not specifically described in the following examples, they are all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the kits and product manuals.

[0036] CYP6AE28 nucleotide sequence (SEQ ID NO.1):

[0037] ATGTTACCAATATATTTAATACTTTGCGTTGCCGTTATCTACTGTCTATATCGGTTAGCGACACTAAA

[0038] ATACAATTATTGGAAGAAGCGGAACGTCCCTCACCCCCCTCCGACACCCGTTCTTGGCAACTATGGA

[0039] CCTTACCTTCTCATGCAGAAATACTTCGGGCAGGTCGCTCAGGAAATCTGCCAGAAGTACCCGAACG

[0040] CACCCTACATCGGCGTTTACTACGGGACCGAACCGACCCTCATCGTGCAAGATCCCGAGCTCATCAA

[0041] GATCATCACGACAAAGGACTTCTACTACTTTAACAGCAGAGAGATTTCCGAGCATACACATAAAGA

[0042] AGTCACTACTCAAAACCTTTTCTTTACCTATGGAGACCAATGGAAGGCGGTCCGGCAGAACATGACT

[0043] CCACTATTCTCGTCCGCAAAAATGAGGAACATGTTCCCCCTCATTGCGAAGTGTTCGGTCTCATTCCA

[0044] GGAGCACGTCGACCAGGAAACCAGCTCGAGCGACGTCATAGACGTCAAATCTCTCATGGCGAGGTA

[0045] CACTATGGCTTGTATAGTTTCCTGCGCGTTTGGAGCAGAGGTGGACACTCTGACCCCGGAGGGCAAA

[0046] GACAGCCAATTCGTTAAGATCGGCAAGCTGATATTTTTGAACTCGTATCTTAGGGGAGGACTGACTG

[0047] TGTTTAGAGCTATCTGGCCGACCTTATTTTACAAGTTGGGGCTGAAGAACTTCCCTGATGAGATGAT

[0048] CAACTTTTTCAAACGCTTTGTGACGAACGTTTTTGAGGCGCGCAAGTACACACCGACGCGTAGAAAT

[0049] GACTTTGTTGATCTCCTTCTGAATTTGAGGCAGCAGAAATCTATCGTTGCTGACAGTTTGTCAAACGG

[0050] GAAGACTGGTAGGGAGGATAAAGTGGAATTGCCTGTAACAGATGATTTGTTAGTGTCTCAGTGTGCG

[0051] TTGTTCTTTGCCGCTGGTTTCGAGACGTCGTCGACAGCTACGTCGTTTTTGCTGTATGAACTGTCGAA

[0052] GAAGCCCGAAATCCAAGAGAGAGTGCTGCAGGAAGTGGATGCGTTCTTGGCCAAGCACGACAATAA

[0053] GCTGACTTACGACTGCGTGACCGAGCTGCCGTACACGCAGGCTTGTATCGACGAATCTCTTCGGCTG

[0054] CATCCAGCTCTTGGCGTGATCACACGCGAGGTCATGGAGGAGTATACTTTACCTGATGGCCTGGTTC

[0055] TGGAAAAGGGTGTGCGTATCCATATACCAGTGTATGGCCTTCACTACAACCCTGATCACTTCTCCGA

[0056] CCCTGAAGAGTTCCGTCCTGAGAGATTCGAAGGCAGCAACCGGAATGACATAAAATCCCACACTTA

[0057] CATACCGTTTGGAGATGGCTCTAGGATCTGTATTGGTCTGAGGTTTGCAAAGATGCAGATGCTAGCG

[0058] GGCCTGATATCGCTGCTGAAGAAGTATCGAGTGGAAATGACGAAGGATACGCCCACTAAACTAGAC

[0059] TACGATCCTCGGGCCCTCGTCACCATCCCTCTGCAGTCGGTCAACCTGAAGCTGGTACCTCGGGAAG

[0060] GATGGGAGAAGAGGATCTTTGTCCAGTGA

[0061] CYP6AE28 amino acid sequence (SEQ ID NO.2):

[0062] MLPIYLILCVAVIYCLYRLATLKYNYWKKRNVPHPPPTPVLGNYGPYLLMQKYFGQVAQEICQKYPNAPY

[0063] IGVYYGTEPTLIVQDPELIKIITTKDFYYFNSREISEHTHKEVTTQNLFFTYGDQWKAVRQNMTPLFSSAK

[0064] MRNMFPLIAKCSVSFQEHVDQETSSSDVIDVKSLMARYTMACIVSCAFGAEVDTLTPEGKDSQFVKIGKL

[0065] IFLNSYLRGGLTVFRAIWPTLFYKLGLKNFPDEMINFFKRFVTNVFEARKYTPTRRNDFVDLLLNLRQQKS

[0066] IVADSLSNGKTGREDKVELPVTDDLLVSQCALFFAAGFETSSTATSFLLYELSKKPEIQERVLQEVDAFLAK

[0067] HDNKLTYDCVTELPYTQACIDESLRLHPALGVITREVMEEYTLPDGLVLEKGVRIHIPVYGLHYNPDHFSD

[0068] PEEFRPERFEGSNRNDIKSHTYIPFGDGSRICIGLRFAKMQMLAGLISLLKKYRVEMTKDTPTKLDYDPRA

[0069] LVTIPLQSVNLKLVPREGWEKRIFVQ

[0070] Example 1

[0071] Determination of the resistance level of the Cnaphalocrocis medinalis population in Sichuan Province is as follows:

[0072] 1.1 Bioassay of Cnaphalocrocis medinalis: The method of Zheng et al. (Zheng XS, Ren XB and Su JY, Insecticide susceptibility of Cnaphalocro-cis medinalis (Lepidoptera: Pyralidae) in China. J Econ Entomol 104: 653–658 (2011)), namely the rice seedling immersion method, was used to determine the susceptibility of newly hatched larvae of Cnaphalocrocis medinalis to insecticides. The rice seedlings were pretreated. Rice seedlings of uniform size and about 3 weeks old (about 25 cm) were selected and immersed in the prepared pesticide suspension. After waiting for 1 minute, they were fished out and used when there was no obvious moisture on the leaves. A petri dish with a diameter of 7 cm was taken and lined with three pieces of filter paper. 2 mL of sterile and enzyme-free water was added to keep the filter paper moist. The treated rice leaves were cut into 5-cm-long segments, and a dozen rice leaves were placed on the petri dish. At the same time, a plastic bottle containing newly hatched larvae was taken and hung. A brush was taken, and the silk thread spit out by the larvae about to slide down was gently picked up (pay attention not to touch the larvae), and the larvae were transferred to the prepared rice seedlings. The treated larvae were placed in a constant temperature incubator, and the temperature was set at 25 °C, the relative humidity was 60%, and the light-dark ratio was 14H:10D. After 72 hours of cultivation, the results were observed and recorded. Those larvae that could not move normally and coordinately after being touched by the brush were regarded as dead. The results were summarized in Table 1.

[0073] Table 1 Resistance results of Cnaphalocrocis medinalis populations in different rice-growing areas of Sichuan in 2022 to four insecticides

[0074]

[0075]

[0076] The results in Table 1 showed that the field populations of Cnaphalocrocis medinalis in 6 places in Sichuan in 2022 had a relatively high level of resistance to chlorantraniliprole, at a low to medium level of resistance (RR was 7.34–19.94 times); they were sensitive to abamectin and indoxacarb (RR was 3.58–1.16 times; 2.38–0.63 times). In summary, we found that a certain degree of resistance to chlorantraniliprole had occurred in the rice-growing areas of Sichuan.

[0077] Example 2

[0078] Determination of the activity of detoxification and metabolic enzymes is as follows:

[0079] The increase in detoxifying enzyme activity can accelerate the metabolic process of pesticides in organisms, leading to the rapid decomposition of pesticides in the body, so that they cannot effectively reach and act on their targets, and thus drug resistance is generated. The detoxifying enzyme families involved are diverse, mainly including GSTs, P450s, esterases, etc. Therefore, based on the research results of Example 1, we measured the activities of carboxylesterase, glutathione transferase and cytochrome oxidase (P450s), and evaluated the effects of corresponding synergists on the activities of these enzymes. The detailed steps are as follows;

[0080] 2.1 Determination of synergistic effect: A certain dose of synergist (1.6 μg / head for DEM and PBO, 3.2 μg / head for TPP) was dropped onto the pronotum of the third-instar larvae of Cnaphalocrocis medinalis. After 1 h, the healthy and surviving larvae were transferred onto rice seedlings, and then bioassay was carried out (the operation was the same as 1.1).

[0081] 2.2 Determination of carboxylesterase activity Carboxylesterase: Using 5 - 7 mg of third-instar larvae as a replicate, add 1200 μL of homogenate (0.04 mol / L, pH 7.0) and homogenize in an ice bath. After centrifuging at 11000 r / min for 30 min, transfer the supernatant to a new 1.5 ml centrifuge tube. Then centrifuge again at 4 °C and 11628 r / min for 10 min, and transfer the supernatant to a new 1.5 ml centrifuge tube. Finally, centrifuge at 4 °C and 11000 r / min for 5 min to obtain the crude enzyme solution for the final enzyme activity determination. For the enzyme activity of carboxylesterase (CarE) in a 96-well microplate reader, add 75 μL of enzyme solution and 100 μL of 0.3 mM α-NA (α-naphthyl acetate: physostigmine = 1:1, freshly prepared) to each well in sequence, and incubate at 30 °C for 10 min. Then add 25 μL of the color reagent (mix SDS and fast blue B salt evenly according to a volume ratio of 5:2, freshly prepared) to terminate the reaction (react at 30 °C for 10 min), and measure the OD with a microplate reader at a measurement wavelength of 600 nm each.

[0082] After subtracting the corresponding control OD value from the measured OD value, the corresponding amount is obtained through the standard curve. The value obtained by querying is divided by the actual protein content in the enzyme solution and the reaction time (10 min) to obtain the specific enzyme activity nmol / (mg·min). The results are as Figure 1 shown.

[0083] 2.3 Determination of glutathione S-transferase activity: The glutathione S-transferase activity was determined according to the methods of Habig and Jakoby. Five to seven milligrams of third-instar larvae were taken as one replicate, and homogenized in an ice bath using 1600 μL of phosphate buffer. Subsequently, centrifugation was performed multiple times at 4°C and 11,000 r / min to separate the supernatant, which was stored at low temperature for later use. In a transparent 96-well plate, 10 μL of crude enzyme solution (diluted 10-fold with 0.1 M PBS at pH 7.6), 100 μL of 1.2 mM CDNB, and 100 μL of 6 mM GST were added. 0.1 M PBS at pH 7.6 was used as a control. A control group was set up, using 0.1 M PBS at pH 7.6 as a control, and a blank control was set up, with the sample control replaced by PBS buffer. Each sample was measured in triplicate. To ensure the accuracy of the results, each sample was measured three times. The OD 340 value was substituted into the corresponding formula to calculate the specific activity value of glutathione S-transferase. The results are as Figure 2 shown.

[0084] 2.4 Determination of mixed-function oxidase activity: Referring to the method of Aitio, we determined the activity of mixed-function oxidase. Five to seven individuals were selected as one sample, and after adding phosphate buffer at a concentration of 0.1 mol / L and pH 7.6, they were ground on ice. After thorough grinding, centrifugation was performed multiple times (centrifuged at 11,000 r / min for 15 min) to take the supernatant, which was placed at 4°C for later use. Using 7-ethylcoumarin (ECOD) as a substrate, 80 μL of 0.5 mM ECOD + 50 μL of enzyme solution were added to a black microplate, and incubated (30°C, 3 min). Subsequently, it was shaken to mix evenly, and then 10 μL of NADPH was added to start the reaction. The changing OD value was recorded at an emission wavelength of 585 nm, an excitation wavelength of 530 nm, and a temperature of 30°C, with an interval of 45 s. The change values of the curve within 15 min were statistically analyzed, and the cytochrome P450 7-hydroxylase activity was calculated using a 7-hydroxycoumarin standard curve. The results are as Figure 3 shown.

[0085] The results of the three detoxification metabolic enzyme synergists are shown in Table 2.

[0086] Table 2 Test results of three detoxification metabolic enzyme synergists

[0087]

[0088]

[0089] Note: Synergistic ratio = LC50 value of the test insect strain against chlorantraniliprole / LC50 value of chlorantraniliprole after using the synergist.

[0090] The research results show that when we treated the sensitive strain and resistant population with PBO, the synergistic ratios were found to be 1.48 times and 1.91 times respectively. Therefore, cytochrome P450 monooxygenase plays a crucial role in the process of the rice leaf folder developing resistance to chlorantraniliprole. In addition, through the determination of the activity of cytochrome P450 monooxygenase, the results are as Figures 1 - 3 shown, among which, Figure 1 It shows that after treating the indoor sensitive strain (SUS-Lab) and the resistant field population (22-CP) with the enzyme inhibitor TPP respectively, compared with before treatment, the carboxylesterase activity of 22-CP did not decrease significantly; in addition, there was no significant difference in carboxylesterase between the SUS-Lab strain and the original population of 22-CP. Therefore, during the adaptation process of the rice leaf folder in the field population (22-CP) to chlorantraniliprole, carboxylesterase may not be involved or may be indirectly involved in the detoxification process. Figure 2 It shows that there were also significant differences in the glutathione transferase enzyme activities between the SUS-Lab strain and the original population of the 22-CP strain, which were 0.2344 and 0.3149 mmol / (min·mg pro -1 ). Therefore, during the adaptation process of the rice leaf folder in the field population (22-CP) to chlorantraniliprole, glutathione transferase may play an important role. Figure 3 It shows that compared with before treatment, after treating the indoor sensitive strain (Sus-Lab) and the resistant field population (22-CP) with the cytochrome P450 enzyme inhibitor PBO respectively, the cytochrome P450 enzyme activities of the two populations decreased, which were 5.5798 and 14.454 pmol / (min·mg pro -1 , showing a synergistic effect; in addition, there were also significant differences in the cytochrome P450 enzyme activities between the SUS-Lab strain and the original population of the 22-CP strain, which were 9.8820 and 19.0389 pmol / (min·mg pro -1 . Therefore, we speculate that the increase in the activity of cytochrome P450 monooxygenase can bring a strong metabolic resistance ability to the rice leaf folder.

[0091] Example 3

[0092] Screen genes related to the resistance of the rice leaf folder population, specifically as follows:

[0093] Currently, there is an increasing exploration of the relationship between the cytochrome P450s family and insecticide resistance. In this study, based on previous bioassay results, we found that the rice leaf folder (Cnaphalocrocis medinalis) in various regions of Sichuan has developed medium to low levels of resistance to chlorantraniliprole. To deeply explore its resistance mechanism, we used qPCR technology to measure the expression levels of multiple P450 genes in order to identify the key resistance genes. The detailed steps are as follows;

[0094] 3.1 Total RNA Extraction and Detection

[0095] 1. Take several 3rd instar larvae of the rice leaf folder, quickly freeze them in liquid nitrogen in a mortar pre-cooled with liquid nitrogen, and grind them with a pestle. During this process, continuously add liquid nitrogen until the tissue is ground into a powder.

[0096] 2. Place the ground sample in a sterile and enzyme-free centrifuge tube, add an appropriate amount of RNA isolater, let it stand for a while, and continue the experiment after the sample is completely dissolved.

[0097] 3. Centrifuge the above lysate at 11200 r / min at 4°C for 5 min, and take the supernatant.

[0098] 4. Transfer the supernatant to a new centrifuge tube, and add 1 / 5 volume of chloroform. Vigorously shake for 10 s. After forming an emulsion, let it stand for 5 min and then centrifuge at 11200 r / min at 4°C for 20 min.

[0099] 5. Centrifuge at 11200 r / min at 4°C for 20 min.

[0100] 6. Carefully take out the supernatant. At this time, the solution is layered; the upper layer is a colorless aqueous phase, the white middle layer, and the red organic lower layer. Carefully take out the colorless upper aqueous phase to a new centrifuge tube.

[0101] 7. Add pre-cooled isopropanol (equal volume), invert several times up and down, and let it stand at 4°C for 15 min.

[0102] 8. Centrifuge at 11200 r / min at 4°C for 15 min. At this time, a white precipitate can be seen.

[0103] 9. Carefully remove the supernatant in the centrifuge tube, add 1 mL of 75% ethanol (prepared with RNase free ddH2O). Gently flick the bottom of the tube and let it stand at room temperature for 5 min.

[0104] 10. Centrifuge at 11200 rpm at 4°C for 5 min, remove the supernatant, and dry it in a sterile and enzyme-free environment for 5 - 10 min

[0105] 11. Then add 30 μL of RNase free ddH2O, noting not to over-dry.

[0106] 12. Detect after complete melting and store the sample at -80 °C.

[0107] 3.2 First-strand cDNA synthesis and gDNA removal

[0108] According to the instruction manual of Plus All-in-one 1st Strand cDNA SuperMix (Gdna Purge) kit from Proteintech Group, Inc. :

[0109] (1) Vigorously mix all components of the reagent and prepare the system shown in Table 3 below:

[0110] Table 3 Reaction system

[0111]

[0112] (2) React at 37 °C for 5 min, incubate at 85 °C for 30 s to terminate the reaction.

[0113] 3.3 qPCR primer design

[0114] Use the NCBI online web page to design P450 genes related to detoxification metabolism of Cnaphalocrocis medinalis for recording the melting curve and recording Ct. The internal reference gene in this article is the housekeeping gene CmActin of Cnaphalocrocis medinalis (GenBank accession number: JN029806), and 3 biological replicates are performed. The primer sequences are shown in Table 4 below.

[0115] Table 4 qPCR primer information for differentially expressed genes

[0116]

[0117]

[0118] 3.4 Fluorescent quantitative PCR

[0119] (1) According to the instruction manual of qPCR SYBR Green Master Mix kit, the system is as follows in Table 5:

[0120] Table 5 Reaction system

[0121]

[0122]

[0123] (2) Pre-denature at 95 °C for 5 min, then enter 39 cycles of 95 °C for 10 s, 56 °C for 20 s, and 72 °C for 20 s, and finally once in the melting curve stage.

[0124] (3) Use 2 –ΔΔCT to calculate the relative expression level.

[0125] The mRNA expression levels of multiple P450 genes were measured using qPCR technology, and the results are as Figures 4 - 8 shown. The results indicate that in the chlorantraniliprole-resistant population, the expression level of the CYP6AE28 gene is significantly higher than that of other P450 genes. In addition, it was further found that under the stress of chlorantraniliprole, the expression level of the CYP6AE28 gene also showed a significant upward trend.

[0126] Example 4

[0127] Verification of the function of CYP6AE28 in Cnaphalocrocis medinalis mediating chlorantraniliprole resistance is as follows:

[0128] Based on the above research, we found that cytochrome P450 enzymes play an important role in the resistance of Cnaphalocrocis medinalis to chlorantraniliprole. Therefore, to further clarify the function of this gene, we obtained the full-length CYP6AE28 using gene cloning technology and simultaneously verified it using gene interference technology. The detailed steps are as follows;

[0129] 4.1 Total RNA extraction and detection

[0130] 1. Take several 3rd instar larvae of Cnaphalocrocis medinalis, quickly freeze them in liquid nitrogen in a pre-cooled mortar, and grind them with a pestle. During this process, continuously add liquid nitrogen until the tissue is ground into a powder.

[0131] 2. Place the ground sample in a sterile and enzyme-free centrifuge tube, add an appropriate amount of RNA isolater, let it stand for a while, and continue the experiment after the sample is completely dissolved.

[0132] 3. Centrifuge the above lysate at 11200 r / min at 4°C for 5 min and take the supernatant.

[0133] 4. Transfer the supernatant to a new centrifuge tube and add 1 / 5 volume of chloroform. Vigorously shake for 10 s, let it stand for 5 min after forming an emulsion, and then centrifuge at 11200 r / min at 4°C for 20 min.

[0134] 5. Centrifuge at 11200 r / min at 4°C for 20 min.

[0135] 6. Carefully take out the supernatant. At this time, the solution is layered; the upper layer is a colorless aqueous phase, the white middle layer and the red organic lower layer. Carefully take out the colorless upper aqueous phase to a new centrifuge tube.

[0136] 7. Add pre-cooled isopropanol (equal volume), invert it several times up and down, and let it stand at 4°C for 15 min.

[0137] Centrifuge at 11,200 r / min at 4 °C for 15 min, and a white precipitate can be seen at this time.

[0138] 9. Carefully remove the supernatant in the centrifuge tube and add 1 mL of 75% ethanol (prepared with RNase-free ddH2O). Flick the bottom of the tube and let it stand at room temperature for 5 min.

[0139] 10. Centrifuge at 11,200 rpm at 4 °C for 5 min, remove the supernatant, and dry it in a sterile and enzyme-free environment for 5 - 10 min

[0140] 11. Then add 30 μL of RNase-free ddH2O, taking care not to over-dry.

[0141] 12. Detect after complete melting and store the sample at -80 °C.

[0142] 4.2 First-strand cDNA synthesis and gDNA removal

[0143] According to the instruction manual of the Plus All-in-one 1st Strand cDNA SuperMix (Gdna Purge) kit from Novoprotein Scientific Inc. :

[0144] (1) Vigorously shake and mix all components of the reagent to prepare the system shown in Table 6 below:

[0145] Table 6 Reaction system

[0146]

[0147] (2) React at 37 °C for 5 min, incubate at 85 °C for 30 s to terminate the reaction.

[0148] 4.3 Full-length primer design

[0149] As shown in Table 7:

[0150] Table 7 Full-length cloning primers for CYP6AE28

[0151]

[0152] 4.4 PCR amplification

[0153] According to the instruction manual of the 2×Hieff PCR Master Mix kit from Yeasen Biotech Co., Ltd., establish the system as shown in Table 8:

[0154] Table 8 Reaction system

[0155]

[0156] Reaction conditions: pre-denaturation at 98°C for 3 min; 98°C for 10 s; 55 - 65°C for 30 s; 72°C for 5 min; 38 cycles, extension at 72°C for 5 min.

[0157] 4.5 Recovery and purification of the target fragment

[0158] According to the instruction manual of Gel Extraction Kit from Beijing Noblelide Technology Co., Ltd. The target fragment was recovered as follows:

[0159] 1. DNA adsorption equilibrium treatment: Add 200 μL of buffer CBS to the Gel Recovery Column, centrifuge at 12000 r / min for 1 min, pour out the waste liquid in the collection tube, add 200 μL of ddH2O to the Gel Recovery Column, centrifuge at 12000 r / min for 1 min, and pour out the waste liquid in the collection tube. Put the Gel Recovery Column back into the collection tube.

[0160] 2. Cut out the gel containing the target fragment from the agarose gel, estimate the weight or accurately weigh it. Add 100 μL of Binding Solution for every 100 mg of 1% agarose gel.

[0161] 3. Incubate in a water bath at 50 - 60°C for 5 - 10 min, gently invert the mixture intermittently every 2 - 3 min until the gel block is completely melted.

[0162] 4. Transfer the above mixture to the adsorption column with a 2 mL collection tube, let it stand at room temperature for 2 min, centrifuge at 12000 r / min for 1 min, and pour out the waste liquid in the collection tube.

[0163] 5. Put the collection column back into the collection tube, add 500 μL of WA Solution, centrifuge at 12000 r / min for 1 min, and pour out the waste liquid in the collection tube.

[0164] 6. Put the collection column back into the collection tube, add 500 μL of Wash Solution, centrifuge at 12000 r / min for 1 min, and pour out the waste liquid in the collection tube.

[0165] 7. Repeat step 6 once.

[0166] 8. Put the adsorption column back into the collection tube, centrifuge at 12000 r / min for 1 min, open the lid of the adsorption column, and let it stand at room temperature for 5 - 10 min or at 50°C for 2 - 5 min to completely remove the Wash Solution.

[0167] 9. Place the adsorption column into a clean 1.5 mL collection tube. Add 30 - 50 μL of Elution buffer to the center of the membrane while it is suspended. Cover the lid and incubate at 37°C for 2 min. Centrifuge at 12,000 r / min for 1 min. The liquid in the centrifuge tube is the solution containing the target gene.

[0168] 4.6 Ligation and transformation of the target fragment

[0169] 1. According to the -Blunt Simple Cloning Kit of Beijing TransGen Biotech Co., Ltd., perform ligation and transformation of the target fragment. The specific steps are as follows, and the reaction system is shown in Table 9:

[0170] Table 9 Reaction system

[0171]

[0172] 2. Mix the above system with a pipette tip and let it stand for 5 min for the reaction.

[0173] 3. Take 10 μL of the above system and add it to 100 μL of competent cells. Gently mix them and incubate at room temperature for 5 min.

[0174] 4. Add 300 - 500 μL of LB or SOC medium (without antibiotics), incubate at 37°C, and shake culture at 180 r / min for 10 min.

[0175] 5. Take 200 μL of the bacterial solution and spread it on a plate (LB or SOC solid medium containing ampicillin resistance), and screen for recombinant transformants after overnight culture (37°C).

[0176] 4.7 Identification of recombinant plasmids

[0177] 1. Colony / bacterial solution PCR identification: Use a sterile pipette tip or toothpick to pick a single colony into the Colony PCR Mix, mix well, and directly perform PCR reaction by adding primers.

[0178] 2. Sequencing analysis: The optional sequencing primer sequences (5’-3’) are as follows:

[0179] M13F: TGTAAAACGACGGCCAGT (SEQ ID NO.40),

[0180] M13R: CAGGAAACAGCTATGACC (SEQ ID NO.41).

[0181] 4.8 Gene sequence and protein analysis

[0182] In this paper, DNAMAN 5.0 was used to reveal the similarities and differences among sequences. Compute pI / Mw (http: / / web.expasy.org / compute_pi / ) was used to predict the relative molecular mass and isoelectric point of proteins; TMHMM Server (http: / / www.cbs.dtu.dk / services / TMHMM / ) was used to predict transmembrane helical structures; meanwhile, NetPhos 2.0 (http: / / www.cbs.dtu.dk / services / NetPhos / ) and DictyOGlyc (http: / / www.cbs.dtu.dk / services / DictyOGyc) were used to identify phosphorylation and glycosylation modification sites of proteins; online analysis with SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / secpred_sopma.pl) revealed the secondary structure of proteins. The phylogenetic tree was constructed using MEGA 7.0 software. Finally, through the online tool I-TASSER (http: / / zhanglab.ccmb.med.umich.edu / I-TASSER) and SYBYL-X2.1.1 software, we predicted the structures of proteins and simulated the docking process of proteins with drug molecules respectively. The construction of recombinant plasmids was as Figure 9 shown.

[0183] 4.9 RNAi

[0184] 4.9.1 Primer design of interfering RNA is shown in Table 10:

[0185] Table 10 Primer sequences of T7 promoter of P450 gene

[0186]

[0187] Note: The underlined part is the specific sequence of the T7 promoter (TAATACGACTCACTATAGGG).

[0188] 4.9.2 Total RNA extraction, detection and reverse transcription

[0189] The reverse transcription is the same as 3.1 above.

[0190] 4.9.3 Reverse transcription and PCR amplification

[0191] The reverse transcription is the same as 3.2 above, and the PCR amplification is the same as 4.4 above.

[0192] 4.9.4 Fragment recovery and purification

[0193] The same as 4.3 above.

[0194] 4.9.5 In Vitro Transcription Reaction

[0195] After thawing the reagents on ice, the transcription system is as shown in Table 11 below.

[0196] Table 11 Transcription System

[0197]

[0198] Add the above system to a 200 μL PCR tube and gently pipette to mix well. Incubate at 37 °C for 2 h or overnight.

[0199] 4.9.6 Removal of DNA and ssRNA

[0200] According to the Magnetic Residual DNA Sample Preparation Kit of Yeasen Biotech Co., Ltd. Remove DNA and ssRNA from the above system. The reaction system is shown in Table 12:

[0201] Table 12 Reaction System

[0202]

[0203] The reaction conditions are 37 °C for 30 min.

[0204] 4.9.7 Purification of dsRNA with Magnetic Beads

[0205] The dsRNA product can be purified by various methods such as magnetic bead method, column method, phenol / chloroform extraction, and gel extraction and recovery. In this experiment, the magnetic bead purification technology is selected.

[0206] 1. Transfer 0.5 - 5 μL of the RNA sample to a clean RNase-free mL centrifuge tube.

[0207] 2. According to the sample volume, add BeyoMagTM RNA Clean Magnetic Beads according to the dosage in the following table (make sure to mix well before use). Generally, the dosage of magnetic beads is 2 times the sample volume. Gently vortex or pipette 10 times and incubate at room temperature for 5 min. Then place the centrifuge tube in the magnetic beads on the magnetic rack, incubate at room temperature for 30 s, and carefully remove the supernatant.

[0208] 3. Keep the centrifuge tube in the magnetic rack all the time. Open the centrifuge tube cap and add 200 μL of freshly prepared 80% ethanol (v / v) solution to wash the magnetic beads. Incubate at room temperature for 30 s and carefully remove the supernatant.

[0209] 4. Repeat step 3 once.

[0210] 5. Keep the centrifuge tube in the magnetic stand at all times, open the centrifuge tube cap, and dry it at room temperature until the magnetic beads just start to crack (about 5 - 10 min).

[0211] 6. Take the centrifuge tube out of the magnetic stand, add an appropriate amount of ultrapure water (10 - 30 μL), gently vortex or pipette gently to mix well.

[0212] 7. Incubate at room temperature for 5 min.

[0213] 8. After briefly centrifuging the centrifuge tube at low speed, place it in the magnetic stand for 5 min to separate. After the solution becomes clear, carefully aspirate the supernatant into a clean centrifuge tube, and the purification of RNA is completed. The nucleotide sequence of the obtained dsRNA is shown in SEQ ID NO.3;

[0214] ATGGAGACCAATGGAAGGCGGTCCGGCAGAACATGACTCCACTATTCTCGTCCGCAAAAATGAGGA

[0215] ACATGTTCCCCCTCATTGCGAAGTGTTCGGTCTCATTCCAGGAGCACGTCGACCAGGAAACCAGCTC

[0216] GAGCGACGTCATAGACGTCAAATCTCTCATGGCGAGGTACACTATGGCTTGTATAGTTTCCTGCGCG

[0217] TTTGGAGCAGAGGTGGACACTCTGACCCCGGAGGGCAAAGACAGCCAATTCGTTAAGATCGGCAAGCTGATATTTTTGAACTCGTATCTTAGGGGAGGACTGACTGTGTT.

[0218] Example 5

[0219] RNA interference experiment, using the dsRNA obtained in Example 4, specifically as follows:

[0220] 1. Dosage and larval age during interference injection

[0221] During the RNA interference process, the microinjection method was adopted. However, since the most suitable dsRNA injection concentration and the appropriate larval age are still uncertain, we need to explore these conditions through a series of experiments. ddH2O added with an appropriate amount of Allura Red was used as the experimental control group, and larvae at the third and fourth instars were injected with doses of 50 nL, 100 nL, and 150 nL respectively. By observing the mortality rate of the larvae after injection, we selected the dose with a lower mortality rate as the injection dose for the subsequent experiments.

[0222] 2. Determination of interference time effect

[0223] Healthy fourth-instar larvae were selected, and the injection volume for each larva was 50 nL (concentration: 2000 μg / μL). In addition, dsGFP was used as the control group for the experiment. The injected third-instar larvae of Cnaphalocrocis medinalis were placed in a petri dish containing fresh rice leaves and reared under the conditions of a constant temperature of 26 ± 1 °C, a humidity of 70% - 80%, and a photoperiod of 14L:10d. We observed and recorded the feeding situation, appearance phenotype, and survival status of these larvae every 24 h. At 24 h and 48 h respectively, 6 healthy surviving larvae were selected, RNA was extracted, and then the expression levels of related interfering genes were measured to verify the interference efficiency.

[0224] According to Figure 10 The interference efficiency results in Figure A of showed that after 24 h of RNAi interference injection, the expression level of cytochrome CYP6AE28 decreased significantly by 54% compared with the control, and the interference efficiency was the highest (86%) after 48 h. Therefore, healthy surviving larvae were selected for insecticide sensitivity determination 48 h after microinjection.

[0225] 3. Determination of the sensitivity of test insects to interference

[0226] Healthy surviving larvae were selected for insecticide sensitivity determination 48 h after microinjection. The drop method was used for treatment, with 15 larvae in each treatment and 3 biological replicates; the results were observed 72 h later. The results are as shown in Figure B of Figure 10 which showed that the larvae after CYP6AE28 interference injection had significantly increased sensitivity to the LC 25 dose of chlorantraniliprole, and their mortality rate increased to 62.22%.

[0227] In summary, in order to verify whether CYP6AE28 is involved in the detoxification metabolism of Cnaphalocrocis medinalis to chlorantraniliprole, this study conducted an interference experiment on CYP6AE28. The results showed that interfering with CYP6AE28 could significantly increase the sensitivity of Cnaphalocrocis medinalis larvae to chlorantraniliprole. Therefore, CYP6AE28 plays an important role in the detoxification metabolism of Cnaphalocrocis medinalis to chlorantraniliprole.

[0228] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. A rice leaf folder cytochrome CYP6AE28 gene, characterized in that: The nucleotide sequence of the CYP6AE28 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the rice leaf folder cytochrome CYP6AE28 gene according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A dsRNA for interfering with the expression of the cytochrome CYP6AE28 gene of the rice leaf folder, characterized in that: The nucleotide sequence of the dsRNA is shown in SEQ ID NO.

3.

4. The method for preparing dsRNA according to claim 3, characterized in that: The steps include: Step 1, extracting total RNA from rice leaf folder and reverse transcribing it into cDNA; Step 2, using the cDNA as a template to clone the gene to obtain a CYP6AE28 gene fragment, wherein the nucleotide sequence of the CYP6AE28 gene is shown in SEQ ID NO.1; Step 3, connecting the CYP6AE28 gene fragment to the vector, and transforming the competent cells to obtain the CYP6AE28 gene plasmid; Step 4, using the full-length sequence of the CYP6AE28 gene plasmid as a template, performing PCR amplification, and purifying the amplified product to obtain the dsRNA.

5. The preparation method according to claim 4, characterized in that: In step 2, the primer sequences used for gene cloning are shown in SEQ ID NO: 38 and SEQ ID NO: 39 respectively.

6. The preparation method according to claim 4, characterized in that: In step 4, the primer sequences used for PCR amplification are shown as SEQ ID NO:42 and SEQ ID NO:43 respectively.

7. Use of the dsRNA according to claim 3 in preparing a product for controlling rice leaf folder.

8. Use of the dsRNA according to claim 3 in reducing the resistance of rice leaf folder to chlorantraniliprole.

9. An insecticide, characterized in that: Comprising: the dsRNA according to claim 3 and chlorantraniliprole.

10. A method for controlling rice leaf folder, characterized in that: The dsRNA according to claim 3 is used to feed rice leaf folders, and then chlorantraniliprole is applied.

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