CN114807133B - RNAsqu nanometer molecule capable of being used for gray mold control and crop pathogen inhibitor - Google Patents
RNAsqu nanometer molecule capable of being used for gray mold control and crop pathogen inhibitor Download PDFInfo
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- CN114807133B CN114807133B CN202210374919.1A CN202210374919A CN114807133B CN 114807133 B CN114807133 B CN 114807133B CN 202210374919 A CN202210374919 A CN 202210374919A CN 114807133 B CN114807133 B CN 114807133B
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- gray mold
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- botrytis cinerea
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Abstract
The invention provides RNAsqu nanometer molecules capable of being used for preventing and controlling plant gray mold and a crop gray mold inhibitor, which have an RNA sequence shown in SEQ ID NO. 1. The invention also provides a botrytis inhibitor, which at least comprises RNAsqu nanometer molecules. Experiments prove that the solution added with RNAsqu nanometer molecules can obviously inhibit spore germination, hypha growth and plant infection toxicity of botrytis cinerea, and can be used for preventing and controlling plant botrytis cinerea.
Description
Technical Field
The invention relates to protection and treatment of crop gray mold, in particular to RNAsqu nanometer molecules for inhibiting growth and development of gray mold bacteria and a crop germ inhibitor.
Background
The pathogenic bacteria causing Botrytis cinerea (Botrytis cinerea) belonging to the genus Botrytis (Botrytis) half-known fungus. Gray mold is a common disease on vegetables and fruits, which is serious in damage, mainly damages fruits and stems and leaves, and especially serious in damage to crops in greenhouses. Continuous high temperature, insufficient illumination, untimely ventilation and high humidity all cause serious occurrence of gray mold, cause a large number of fallen leaves and fallen fruits and even be out of harvest, and cause serious loss for farmers. At present, chemical pesticides are mainly used for gray mold control methods, and two methods of physical control and biological control are adopted as auxiliary materials. Due to the characteristics of quick propagation, easy variation and the like of the botrytis cinerea, the botrytis cinerea gradually generates drug resistance to various bactericides along with repeated use of a large amount of bactericides for many years, so that the control effect of the chemical bactericides is greatly reduced. Moreover, as the living standard of people increases year by year, the demand for healthy diet by humans is also increasing. The use of large amounts of chemical fungicides in the production of fruits and vegetables, the pollution of the agricultural chemicals to the environment and water resources during the production and use, and the like are receiving increasing attention. Therefore, alternative control measures are urgently needed to resist gray mold, so that the development of novel pollution-free and environment-friendly pesticides is a primary research target of scientists.
Spraying exogenous dsRNA or sRNA molecules with potential of triggering RNAi (exogenous RNAi) is considered to be a novel and environment-friendly biological pesticide, and has good control effect on plant viruses, fungi and insects. However, due to the characteristics of poor stability of RNA itself environment, easy degradation, low plant absorption efficiency, etc., when exposed dsRNA or siRNA is directly sprayed, there are defects of low RNAi induction efficiency, short acting time, etc., so that the effect of inducing gene silencing (SIGS) by spraying requires a delivery vehicle in application to improve the absorption efficiency of dsRNA/siRNA, otherwise, the effect can be maintained only when dsRNA is continuously supplied. Therefore, the selection of suitable vectors to improve dsRNA/siRNA delivery efficiency and RNAi-inducing efficiency is a critical issue faced by current RNAi biopesticides.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides an RNAi biological pesticide capable of being used for preventing and controlling plant gray mold, and the design thinking is that dsRNA/siRNA with inhibition effect on gray mold bacteria is used as a part of RNA nano particles, and the RNAi triggering efficiency of the dsRNA/siRNA is improved and the preventing and controlling capability on gray mold bacteria is enhanced by means of good stability and high-efficiency cell penetrability of the RNA nano particles.
An object of the present invention is to provide an RNA nanomolecular for crop gray mold control, designated RNAsqu, having an RNA sequence shown in SEQ ID No. 1:
SEQ ID NO.1：
Further, RNAsqu nanometer molecules are obtained by the following method: the coding DNA sequence is constructed into an expression vector, and is synthesized in vitro to self-assemble RNAsqu nano-particles.
The invention also provides a crop germ inhibitor, which comprises RNAsqu nanometer molecules.
The beneficial effects of the invention are as follows: after the self-assembled RNAsqu nano-particles for gray mold control are synthesized into RNAsqu nanometers through in vitro transcription, the delivery efficiency and RNAi induction efficiency of dsRNA/siRNA are higher.
Drawings
Fig. 1: influence of RNAsqu molecules on the germination of Botrytis cinerea spores;
fig. 2: RNAsqu inhibiting effect of molecules on the growth of botrytis cinerea hyphae;
Fig. 3: RNAsqu has effect of preventing and treating botrytis cinerea infection on tomato leaves.
Detailed Description
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A preparation method of a functional RNA nano-molecule RNAsqu and a gray mold inhibitor thereof comprises the following steps:
1. Synthesis of DNA template encoding bacteriostatic RNAsqu: according to the sequence of the antibacterial RNAsqu nanometer molecule provided by the invention, a DNA template sequence coded by the antibacterial RNAsqu nanometer molecule is deduced, a T7 promoter sequence is inserted into the upstream of the template sequence, and the template DNA sequence coded by the antibacterial RNAsqu nanometer molecule is sent to a biological company for chemical synthesis.
In vitro transcriptional synthesis of rnasqu: RNAsqu molecules are obtained by using a T7 RNA in vitro transcription kit according to the operation instructions, and diluted to 100 ng/. Mu.L for gray mold control experiments.
Comparative example 1: and designing an upstream primer and a downstream primer (F1/R1) based on a DCL gene sequence of the botrytis cinerea, and then adding a T7 promoter sequence at two ends of the primer to obtain the T7_F1/T7_R1. Finally, using gray mold cDNA as a template, respectively using T7_F1/R1 and F1/T7_R1 as primers to amplify target fragments, respectively using the obtained fragments for in vitro transcription of the template, mixing the obtained two RNAs, and annealing to obtain the DCL-targeted dsRNA.
Comparative example 2: RNA molecules that were unable to target the Botrytis cinerea gene served as negative controls (NC-RNA).
Verification test:
Inhibition of Botrytis cinerea spore germination by RNAsqu
The botrytis cinerea with good growth vigor is selected, conidium is collected, and the concentration is prepared to be 10 4/mL by using PD culture medium. 10. Mu.L of RNAsqu solution with the concentration of 4. Mu.g/. Mu.L was added to a centrifuge tube containing 500. Mu.L of spore solution, and the mixture was incubated for 6 hours at 26℃in the dark to give a final concentration of RNAsqu of 100 ng/. Mu.L, and the result was shown in FIG. 1, with the same amounts of dsRNA and NC-RNA as controls.
As can be seen from FIG. 1, compared with the comparative example, the use of the antibacterial RNA molecules of the examples of the present invention has extremely high germination inhibition efficiency on Botrytis cinerea spores, and all spores hardly germinate; the dsRNA of the comparative example has a certain germination inhibiting effect on botrytis cinerea spores, but the effect is far lower than RNAsqu nanometers of molecules of the invention; NC-RNA molecules have no inhibitory effect on the germination of botrytis cinerea spores, and almost all spores germinate.
Inhibition effect of RNAsqu on Botrytis cinerea mycelium growth
The bacteriostatic activity of RNAsqu molecules on the tested pathogenic fungi was determined using the growth rate method. After filtering and sterilizing RNAsqu solution, the solution was dissolved in PDA medium cooled to 50℃to prepare RNAsqu molecules of PDA medium with a final concentration of 100 ng/. Mu.L, and poured into a petri dish with a diameter of 9cm to cool and solidify, and the equal amounts of dsRNA and NC-RNA were added as a control.
The colony of pathogenic bacteria spore was inoculated onto PDA medium for 24 hr, transplanted to the center of the dish, and placed in 28 deg.C room for 6d of inversion culture, and the result is shown in FIG. 2.
As can be seen from fig. 2, the growth inhibition efficiency of botrytis cinerea hyphae was extremely remarkable using the RNAsqu nm molecule of the present invention as compared with the comparative example.
Control effect of RNAsqu on Botrytis cinerea on different plant leaves
Tomato with regular and consistent growth is selected, and is cultivated for 3d after being sprayed with RNAsqu solution with the concentration of 100 ng/mu L, and dsRNA and NC-RNA solution with the same concentration are used as control. Cutting tomato leaves with the same leaf positions and consistent sizes, and wrapping the water cotton clusters on the leaf stalks. And (3) taking a fungus cake at the edge of a gray mold fungus flat plate with good growth vigor, and inoculating the fungus cake on the back of the leaf blade. Each leaf is inoculated with 1 truffle. After inoculation, the culture was continued for 3d at 26℃and 85% relative humidity.
As shown in FIG. 3, compared with NC-RNA and dsRNA, the RNAsqu nanometer molecule of the invention has extremely strong control effect on gray mold bacteria on living leaves of tomatoes.
Taken together, the RNAsqu nanometer molecule provided by the invention has higher RNAi triggering induction efficiency and extremely obvious inhibition effect on the infectious virulence of gray mold bacteria.
The above examples are only for illustrating the application effect of the present invention, and are not intended to limit the scope of the present invention. Equivalent transformation or modification made according to the essence of the present invention, or the control of botrytis on other plants by using the present invention should be covered within the protection scope of the present invention.
Sequence listing
<110> University of Zhejiang
<120> RNAsqu nm molecule for gray mold control and crop germ inhibitor
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 662
<212> RNA
<213> Unknown (Unknown)
<400> 1
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cgagaguuac aacaauauag gcuuuugcca uccggcauac uuguucaucu acaccgguag 120
augcuagaga ucuuuugccu gucuuauucu caucgucgug ucccuccggg gacacgacga 180
ugagaauaag acaggcuuuu ggcgccgcau cucgaugguc agaaucaccg guagaugcua 240
gagaucuuuu gccaacagaa gcaacaacau ccaagcucuc cggagcuugg auguuguugc 300
uucuguuggc uuuuggccca aggagaaggu gcugauugac acaaggaggc agugucaauc 360
agcaccuucu ccuugggccu uuugaucucu agcaucuacc ggugauucug accaucgaga 420
ugcggcgccu uuugaucucu agcaucuacc gguguagaug aacaaguaug ccggauggcu 480
uuugaucucu agcaucuacc ggugauuauc ucuagcaucu accggugacu uuugaucucu 540
agcauaagcc uccaaugcua gagaucuuuu gccuugaaca ucagaacgag ggcgacccuc 600
cggggucgcc cucguucuga uguucaaggc uuuugucacc gguagaugcu agagauaauc 660
ac 662
Claims (1)
1. The application of RNAsqu nanometer molecules in preparing botrytis cinerea inhibitor is characterized in that the RNA sequence of RNAsqu nanometer molecules is shown as SEQ ID NO. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210374919.1A CN114807133B (en) | 2022-04-11 | RNAsqu nanometer molecule capable of being used for gray mold control and crop pathogen inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210374919.1A CN114807133B (en) | 2022-04-11 | RNAsqu nanometer molecule capable of being used for gray mold control and crop pathogen inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114807133A CN114807133A (en) | 2022-07-29 |
| CN114807133B true CN114807133B (en) | 2024-07-09 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626071A (en) * | 2021-01-22 | 2021-04-09 | 浙江理工大学 | RNA (ribonucleic acid) bacteriostatic agent miRNA482a and crop pathogen inhibitor |
| CN112646817A (en) * | 2021-01-22 | 2021-04-13 | 浙江理工大学 | RNA (ribonucleic acid) bacteriostatic agent siR2 and crop pathogen inhibitor |
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626071A (en) * | 2021-01-22 | 2021-04-09 | 浙江理工大学 | RNA (ribonucleic acid) bacteriostatic agent miRNA482a and crop pathogen inhibitor |
| CN112646817A (en) * | 2021-01-22 | 2021-04-13 | 浙江理工大学 | RNA (ribonucleic acid) bacteriostatic agent siR2 and crop pathogen inhibitor |
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