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    • 专利摘要:
    The present invention relates to an application of an AMEP412 protein from Bacillus subtilis in preventing and controlling whitefly. In the present invention, an insecticidal activity of the AMEP412 protein against whitefly and an application thereof in preventing and controlling whitefly are discovered. 5 The AMEP412 protein can kill whitefly at a low concentration, and has the stability against thermal treatment and natural degradation. The AMEP412 protein can be taken into the intestine by whitefly, but does not inhibit the protease activity in the intestine. In addition, spraying the AMEP412 protein in the greenhouse has also achieved a good control effect against whitefly. In the present invention, a novel material is accumulated for the biological control of whitefly. 1 2
    公开号:NL2026309A
    申请号:NL2026309
    申请日:2020-08-20
    公开日:2021-07-30
    发明作者:Liu Quan；Yin Kuide；Xiao Lijie
    申请人:Univ Heilongjiang Bayi Agricultural；
    IPC主号:
    专利说明:

    -1- Insecticidal Activity of AMEP412 Protein Against Whitefly and Application Thereof in Preventing and Controlling Whitefly Technical Field The present invention belongs to the technical field of plant protection and biological control, and relates to an insecticidal activity of AMEP412 protein on whitefly and an application thereof in preventing and controlling whitefly.
    Background Whitefly (Bemisia tabaci) is a major threat to several crops such as vegetables, cotton, and omamentals.
    Whitefly damages field crops by sucking sap of plant stems and leaves and transmitting plant viruses, causing great losses to agricultural production and the national economy.
    The main strategy for controlling powdery mildew is using chemical pesticides.
    However, whitefly has developed resistance to pesticides that are used excessively and frequently.
    In this connection, the exploration of novel pesticides should become a new research focus.
    Compared with chemical pesticides, proteins with insecticidal activity have a relatively low probability of resistance to insects due to their different modes of action, which has become a hotspot in pesticide research.
    The application of insecticidal protein in pest control has been studied and reported frequently.
    The most famous example is the Cry toxin of Bacillus thuringiensis (Bt) bacteria.
    The transgenic Bt cotton producing Cry toxins has been developed and can effectively control the larvae of lepidopteran pests.
    The widespread cultivation of transgenic Bt cotton has greatly reduced the use of chemical pesticides.
    However, Cry toxin has no effect on whitefly.
    In recent years, some researchers have focused on screening insecticidal proteins from plants that are rarely infested by whitefly.
    Das ef a!. reported in 2009
    -2- that mannose-binding lectins extracted from leaves of Allium sativum (garlic) can effectively defense against whitefly. As reported by Jin et al. in 2012. the Agglutinin of Pinellia ternata was successfully expressed in chloroplasts and proved to have the ability to resist whitefly. In 2016, Shukla ef al. identified an insecticidal protein (Tmal2) from an edible fern and transgenic cotton lines that express Tmal2 showed certain resistance to whitefly. Although these plant-derived insecticidal proteins have a certain potential for controlling whitefly, they are not widely used at present due to the low extraction rate and limited application of transgenes.
    In the previous studies, the present inventors isolated and identified an AMEP412 protein from Bacillus subtilis BU412, which can stimulate the defense response of plants, improve the disease resistance of plants, and also has an antagonistic effect on Strepromyces scabies, making it an ideal candidate for development as a biological pesticide. In the present invention, the aim is to provide a novel application of the AMEP412 protein.
    Summary The objective of the present invention is to provide a novel application of an AMEP412 protein from Bacillus subtilis, to overcome the problem of insufficient insecticidal protein against whitefly, and to achieve a green prevention and control of whitefly.
    The present invention is realized through the following technical solutions: an application of an AMEP412 protein from Bacillus subtilis in preventing and controlling whitefly.
    The amino acid sequence of the above-mentioned AMEP412 protein from Bacillus subtilis is shown as SEQ ID NO:1.
    -3- Further, when the AMEP412 protein is fed to adult whiteflies, a concentration of the AMEP412 protein required to reach a median lethal dose is 15.57 pg/mL, and a concentration of the AMEP412 protein required to reach a 90% lethality rate is 63.27 pg/mL. Further, the AMEP412 protein can be ingested by adult whiteflies and enters the body, and is concentrated in the intestine. Further, the AMEP412 protein has a thermal stability.
    Further, the AMEP412 protein has a stability against natural degradation.
    Further, when the AMEP412 protein is sprayed in a greenhouse at a concentration of 100 ug/mL, a lethality rate of whiteflies is 56% after spraying for 2 days.
    Firstly, different concentration gradients of AMEP412 protein were artificially fed to the adult whiteflies, and the half-lethal rate (LC50) and the concentration of the AMEP412 protein required for 90% lethality rate were calculated. Subsequently, the AMEP412 protein was subjected to a thermal treatment and a natural degradation treatment, and an insecticidal activity of a treated protein sample against the adult whiteflies was tested to determine the stability of the AMEP412 protein. Subsequently, the AMEP412 protein was labeled by FITC. After feeding the adult whitefly, a fluorescence microscope was used to observe the fluorescence distribution in the body of the adult whitefly, and the localization of the AMEP412 protein in the body of the adult whitefly was determined. In addition, the inhibitory effect of the AMEP412 protein on trypsin was tested to rule out the possibility of killing whiteflies by inhibiting the protease activity in the intestine of whitefly.
    -4- Advantages of using the above technical solutions: In the present invention, an insecticidal activity of an AMEP412 protein against whitefly and an application thereof in preventing and controlling whitefly are discovered. The AMEP412 protein can kill whitefly at a low concentration, and has the stability against thermal treatment and natural degradation. The AMEP412 protein can be taken into the intestine by whitefly, but does not inhibit the protease activity in the intestine. In addition, spraying the AMEP412 protein in the greenhouse has also achieved a good control effect against whitefly. In the present invention, a novel material is accumulated for the biological control of whitefly.
    Brief Description of the Drawings FIG. 1 shows lethality rates of different concentrations of AMEP412 proteins against adult whiteflies, wherein the same letter indicates that the difference is not significant, while different letters indicate that the difference between treatments is significant (a=5%): FIG. 2 shows lethality rates of AMEP412 protein to adult whiteflies after thermal treatment and natural degradation, wherein CK indicates that no AMEP412 protein added, Untreated indicates AMEP412 protein without treatment, and the same letter indicates that the difference is not significant, while different letters indicate that the difference between treatments is significant (0=5%); and FIG. 3 shows a fluorescent localization of FITC-labeled AMEP412 protein in adult whiteflies, wherein the bright spots in the abdominal cavity are excited green fluorescence, indicating that the AMEP412 protein is located in the intestine.
    Detailed Description of the Embodiments
    -5- The present invention is further described by the following embodiments. It should be understood that these embodiments are merely for illustrative purposes and are not intended to limit the scope of the present invention. The source of biological materials in the present invention:
    1. The used Bacillus subtilis BU412 was deposited at the China Center for Type Culture Collection, referred to as CCTCC, on March 30, 2016, with a deposit number of CCTCC M
    2016142.
    2. The AMEP412 protein: Patent application number 201810928176.1, patent entitled “Novel function of AMEP412 protein from Bacillus subtilis and identification of antimicrobial peptides”, with an application date of 20180814, a publication date of December 28, 2018, and a publication number of CN 109096379A. Embodiment 1 In this embodiment, the insecticidal efficacy of artificial feeding of AMEP412 protein from Bacillus subtilis to whiteflies is described. The liquid artificial food used for feeding was made by addmg 5% veast extract and 30% sucrose dissolved in distilled water, sterilizing, and adding AMEP412 protein of different concentrations (1, 5, 10, 20, 40 and 80 pg/mL), followed by mixing for subsequent use. The artificial food without AMEP412 protein was used as a control. The adult whiteflies of 1-2 days old were driven from plant leaves to 50 mL test tubes, each containing at least 50 adult whiteflies. Each of the caps of the test tubes was covered with two stretched sealing films. The artificial food (100 pL) was added between the two films, and then the cap covered the test tube in reverse to keep the double-layer film structure inside the test tube, and placed vertically upward. Each experiment was repeated three times. The mortality was determined after 2 days
    -6- of feeding. and the mortality was recorded by counting the dead adult whiteflies at the bottom of the test tube. One-way analysis of variance (ANOVA) was used to analyze the data, and the Tukey's-HSD (“honestly significant difference” or “honest significant difference”) test (a=5%) was used for comparison. The median lethal concentration (LC350) and 90% lethal concentration were calculated by probit analysis of the SPSS program.
    The mortality of whiteflies was calculated after 2 days of feeding, as shown in FIG. 1. As the concentration of AMEP412 protein increased, the mortality of whiteflies increased gradually, showing a positive correlation. When the concentration of AMEP412 protein was 0, 1, 5, 10,
    20. 40 and 80 mg/mL, the corresponding mortality of whiteflies was 7.2%, 17.4%, 31.0%,
    47.9%, 65.5%, 82.7% and 95.6%, respectively. The concentration of AMEP412 protein required to reach a median lethal dose is 15.57 pg/mL (Table 1). In addition, the concentration of the AMEP412 protein required to reach a 90% lethality rate is 63.27 pg/mL. These results indicate that AMEP412 protein has insecticidal activity against adult whiteflies, with a low required median lethal concentration, indicating that the AMEP412 protein has a high insecticidal activity.
    Table 1 Calculation results of the median lethal concentration of AMEP412 protein Median lethal 95% lower 95% upper Slope (SE) Chi-square
    P concentration confidence confidence + standard (degrees of value (LC50) (ug/mL) limit (ng/mL) limit (pg/mL) error freedom)
    15.57 10.86 21.00 2.10+0.28 0.83(3) 0.84 Embodiment 2 In this embodiment, the stability experiments of AMEP412 protein from Bacillus subtilis are described.
    27- To test the heat resistance of AMEP412 protein, the protein samples were treated at 95°C for 15 minutes and 30 minutes, respectively. Subsequently, the artificial food was prepared with the cooled protein samples according to the method in embodiment 1, and the final concentration was adjusted to 60 pg/mL to test the insecticidal effect against whiteflies. In addition, untreated protein samples were used as controls, and each treatment was repeated three times. To test the stability of AMEP412 protein to natural degradation, the protein samples were placed in a 1.5 mL centrifuge tube and left to stand for 24 hours and 48 hours at room temperature of 25°C, respectively. After that, the artificial food was prepared with the protein samples according to the method in embodiment 1, and the final concentration was adjusted to 60 pg/mL to test the insecticidal effect against whiteflies. In addition, untreated protein samples were used as controls, and each treatment was repeated three times. The above lethality rate data of whiteflies was analyzed by one-way analysis of variance and compared using Tukey's HSD test (a = 5%).
    The results showed (FIG. 2) that compared with the AMEP412 protein without heat treatment, the lethality rate of AMEP412 protein treated at 95°C for 15 minutes and 30 minutes against whiteflies decreased by 0.57% and 0.49%, respectively, with no significant difference, indicating that the AMEP412 protein has a good thermal stability. Compared with the AMEP412 protein without natural placement treatment, after the AMEP412 protein was naturally placed at 25°C for 24 hours, the mortality of whiteflies decreased by 2.73%, with no significant difference. However, after the AMEP412 protein was placed naturally at 25°C for 48 hours, the lethality rate against whiteflies decreased by 16.38%. Although a significant difference is achieved, the lethality rate is still 70%, so AMEP412 protein is considered to have good stability against natural degradation. These results indicate that AMEP412 has stability to thermal treatment and natural degradation, which is beneficial for application in high temperature environments and prolongation of its action time.
    -8- Embodiment 3 In this embodiment, the fluorescent localization of AMEP412 protein from Bacillus subtilis is described.
    The AMEP412 protein was incubated with fluorescein FITC, and the FITC that was not bound to AMEP412 protein was removed according to the difference in molecular volume by molecular sieve chromatography to obtain FITC-labeled AMEP412 protein. Subsequently, the FITC-labeled AMEP412 protein was fed to the adult whiteflies. After the dead adult whiteflies were cleaned with distilled water, an Olympus BX60 fluorescence microscope was used to excite green fluorescence at a wavelength of 495 nm for fluorescence microscopic observation. The results showed (FIG. 3) that the green fluorescence was concentrated in the intestine of the adult whitefly, indicating that AMEP412 protein can be ingested by adult whitefly and localized in the intestine. This result suggests that the AMEP412 protein exerts its insecticidal action in the intestine.
    Embodiment 4 In this embodiment, the effect of AMEP412 protein from Bacillus subtilis on trypsin activity is described.
    The action mechanism of some insecticidal proteins is to act as protease inhibitors, by inhibiting the activity of serine proteases (such as trypsin) in the insects’ intestines, so that the insects cannot digest the food they ingest and cause death. In order to test whether the AMEP412 protein from Bacillus subtilis can achieve insecticidal effect as a protease inhibitor, the effect of AMEP412 protein on trypsin activity was tested in this embodiment.
    The activity test of trypsin was carried out on the basis of detecting the catalvtic ability of
    -9- trypsin to catalyze the substrate N-a-benzoy1-DL-argmine-p-nitroaniline (BAPNA) to produce products.
    BAPNA has a special absorption at 405 nm.
    The effect of AMEP412 protein on trypsin activity can be determined by measuring changes in absorbance. 0.2 mL trypsin solution (0.1 mg/mL), 0.2 mL AMEP412 protein (0.1 mg/mL) and 1.6 mL buffer (0.2 M Tris HCI pH 7.8) were incubated in a reaction cup for 3 minutes.
    The enzymatic reaction 1s initiated by adding 1mL of substrate (1 mg/mL BAPNA). Then, the solution was immediately mixed and the increase in absorbance at 405 nm was recorded every minute for 5 minutes.
    A solution without AMEP protein was used as a control.
    Each treatment was repeated three times.
    The inhibition rate was calculated as follows: Inhibition rate (%)=(AMEP412 treatment AA405 nm/min - control AA405 nm/min:)*100/control AA405 nm/min.
    The results showed (Table 2) that the inhibitory rate of AMEP412 protein on trypsin was 2.97%, with no significant difference.
    The results indicate that AMEP412 protein has no inhibitory effect on trypsin activity, suggesting that the insecticidal activity of AMEP412 is not a mechanism of action as a protease inhibitor.
    Table 2 Effect of AMEP412 protein on trypsin activity AA405 nm/min AA405 nm/min Inhibition rate (%) (AMEP412 treatment) (Control) Meanz*standard error 0.0927+0.0017 0.0900+0.0022 2.97 £227 Embodiment 5 In this embodiment, the prevention and control effect of AMEP412 protein from Bacillus subtilis on whitefly in greenhouse is described.
    -10- In the greenhouse, tomato plants were planted to a height of about 50 cm.
    Three tomato plants were treated as one treatment, covered with gauze nets, and at least 200 adult whiteflies of 1- 2 days old were inoculated into each net.
    The purified elicitor AMEP412 protein sample was diluted to 100 pg/mL for spray treatment, with buffer as a control.
    Each treatment was repeated 3 times.
    The mortality of whiteflies was counted 2 days after spraying.
    The results showed (Table 3) that after spraying two days of 100 ug/mL AMEP412 protein, the mortality of whiteflies was 56.2%, with a very significant difference (a= 1%). Although compared with the feeding experiment, the mortality has decreased, but considering the influence of various factors in the actual environment, the inhibition rate of more than 50% was ideal.
    Therefore. the AMEP412 protein from Bacillus subtilis has a good prevention and control effect on whitefly in greenhouse.
    Table 3 The prevention and control effect of AMEP412 protein on whitefly in greenhouse Treatment Mortality (%) CK 1.23+0.02B AMEP412 56.2+4 37A
    In the present invention, the insecticidal activity of the AMEP412 protein from Bacillus subtilis against adult whiteflies is discovered, the stability of the AMEP412 protein to thermal treatment and natural degradation is further confirmed, the localization of the AMEP412 protein in the body of whitefly is detected, and the lysis effect of the AMEP412 protein on insect cells is confirmed.
    Furthermore, in the present invention, the prevention and control effect of the protein on whitefly in the greenhouse is detected, and new material and experience are accumulated for biological control.
    权利要求:
    Claims (7)
    [1]
    -11 - Conclusions I. Use of a AMEP412-Ciwit of Bacillus subtilis in the control and control of whiteflies.
    [2]
    The Bacillus subtilis AMEP412 protein according to claim 1, wherein the amino acid sequence of the Bacillus subtilis AMEP412 protein is shown as SEQ ID NO:1.
    [3]
    Use according to claim 1, characterized. that when the AMEP412 protein is fed to adult whiteflies, a concentration of the AMEP412 protein required to achieve a median lethal dose is 15.57 pg/mL, and a concentration of the AMEP412 protein required to achieve a lethality of 90% is 63.27 µg/mL.
    [4]
    Use according to claim 1, characterized in that the AMEP412 protein is ingested by adult whiteflies, enters the body, and concentrates in the gut.
    [5]
    Use according to claim 1, characterized in that the AMEP412 protein has a thermal stability.
    [6]
    Use according to claim 1, characterized in that the AMEP412 protein has a stability against natural degradation.
    [7]
    Use according to claim 1, characterized in that when the AMEP412 protein at a concentration of 100 µg/mL is sprayed in a greenhouse, a whitefly lethality after spraying for two days is 56%.
    类似技术:
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    同族专利:
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    引用文献:
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    AU684068B2|1993-03-25|1997-12-04|Syngenta Participations Ag|Novel pesticidal proteins and strains|
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    MX2016017087A|2016-12-19|2018-06-18|Biokrone S A De C V|A biofungicide composed by a consortium of three strains of subtitlis lactobacilli for the control of fungi, nematodes, and phytopathogens insects.|
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    CN113788882A|2021-08-12|2021-12-14|黑龙江八一农垦大学|Application of bacillus subtilis AMEP412 protein in inhibiting tumor cell proliferation|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
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