CN109957572B - Bemisia tabaci lethal gene and application thereof, RNA (ribonucleic acid) interference agent and preparation method and application of RNA interference agent - Google Patents
Bemisia tabaci lethal gene and application thereof, RNA (ribonucleic acid) interference agent and preparation method and application of RNA interference agent Download PDFInfo
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Abstract
The bemisia tabaci lethal gene is related to the activity of α -glucosidase, and the RNA interference agent designed according to the bemisia tabaci lethal gene can inhibit the activity of α -glucosidase, so that the capability of the bemisia tabaci to hydrolyze glucose is reduced, the bemisia tabaci lethal gene is finally dead, and simultaneously the virus disease transmitted by the bemisia tabaci lethal gene can be effectively controlled.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a bemisia tabaci lethal gene and application thereof, and a synthetic method and application of dsRNA and dsRNA.
Background
At present, the control of bemisia tabaci and viral diseases spread by the bemisia tabaci is mainly controlled by using a large area of pesticide, and the large area of the traditional pesticide causes serious safety problems in agricultural production in China, so that the development and the use of a new technology to get rid of the irresistible dependence of people on the traditional chemical pesticide are a good way for solving the problems in the development trend of the new technology for controlling pests based on RNA interference (RNAi).
RNA interference (RNAi) refers to a highly conserved, double-stranded RNA (dsRNA) -induced, highly efficient and specific degradation of homologous mrnas during evolution. According to the lethal gene of bemisia tabaci and the principle of RNA interference technology, an RNA interference preparation is developed, so that bemisia tabaci can be efficiently and safely prevented and treated, and simultaneously, viral diseases transmitted by the bemisia tabaci can be effectively controlled. The technology does not change the genome of the bemisia tabaci, does not influence the ecosystem, can be designed aiming at the specific gene of the bemisia tabaci, plays a supporting role in reducing the application and increasing the effect of chemical pesticides, and realizes the accurate prevention and control of the bemisia tabaci and the virus diseases transmitted by the bemisia tabaci.
Disclosure of Invention
The Bemisia tabaci lethal gene is related to the activity of α -glucosidase, and the RNA interference agent designed according to the Bemisia tabaci lethal gene can inhibit the activity of α -glucosidase, so that the glucose hydrolysis capability of Bemisia tabaci is reduced, and the Bemisia tabaci death is finally caused.
In order to solve the technical problems, the invention provides a bemisia tabaci lethal gene, wherein the bemisia tabaci lethal gene is Alpha-glucosidase, and the DNA sequence of the Alpha-glucosidase is shown as SEQ ID NO. 1.
The cloning method of the bemisia tabaci lethal gene comprises the following steps of:
(1) extracting total RNA of bemisia tabaci by a TRIzol method, and synthesizing a first cDNA chain;
(2) designing a primer pair according to the first strand of the cDNA, and carrying out PCR amplification to obtain a PCR product;
(3) carrying out agarose gel electrophoresis separation on the PCR product to obtain a target DNA fragment;
(4) connecting the target DNA fragment with a vector, and converting the target DNA fragment into escherichia coli to obtain a transformant;
(5) and (3) coating the transformant in an LB culture medium containing X-gal, IPTG and ampicillin for culture to obtain a clone plasmid.
Further, the primer pair comprises an upstream primer and a downstream primer. The DNA sequence of the upstream primer F1 is shown as SEQ ID NO.2, and specifically comprises the following steps: TGAGCATAATGTTGCCAGG, respectively;
the DNA sequence of the downstream primer R1 is shown as SEQ ID NO.3, and specifically comprises the following steps: GCACAAGCGTCACCATAAG are provided.
Based on a general technical concept, the invention also provides an application of the lethal gene of bemisia tabaci in removal of bemisia tabaci.
Based on a general technical concept, the invention also provides an RNA interference agent, wherein the RNA interference agent is dsRNA synthesized according to the Alpha-glucosidase gene segment, and the DNA sequence of the RNA interference agent is shown as SEQ ID NO. 4.
Based on a general technical concept, the present invention also provides a preparation method of the RNA interference agent, which comprises the following steps:
s1, synthesizing a primer pair according to the DNA sequence of Alpha-glucopsidase, and carrying out PCR amplification by using the Bemisia tabaci cDNA as a template to obtain an amplification product;
s2, carrying out agarose gel electrophoresis separation on the amplification product to obtain a target DNA fragment;
s3, synthesizing dsRNA of the target DNA fragment to obtain the RNA interference agent.
In the above preparation method of the RNA interference agent, preferably, the primer pair in S1 includes an upstream primer and a downstream primer, the DNA sequence of the upstream primer is shown as SEQ ID No.5, and the DNA sequence of the downstream primer is shown as SEQ ID No. 6.
In the above method for preparing an RNA interference agent, preferably, the reaction system for PCR amplification in S1 is: 5 μ L reaction buffer, 4 μ L Mg2+4. mu.L dNTP, 2. mu.L cDNA template; 2 μ L of forward primer F2, 2 μ L of reverse primer R2, 0.25 μ L of LEx tap enzyme, 30.75 μ L of ddH2O；
The reaction conditions for PCR amplification are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30sec, annealing at 55-62 ℃ for 30sec, extension at 72 ℃ for 30sec, three steps of cyclic denaturation, annealing and extension for 38 times, and final extension at 72 ℃ for 10 min.
In the above preparation method of the RNA interference agent, preferably, S3 specifically is: and (3) carrying out dsRNA synthesis on the target DNA fragment by using T7RiboMAX Express RNAi system of a PROMEGA dsRNA synthesis kit to obtain the RNA interference agent.
Based on a general technical concept, the invention also provides an application of the RNA interference agent in removal of bemisia tabaci.
In the above application, preferably, the application method comprises the following steps:
(1) uniformly mixing the nutrient solution and the RNA interference agent and placing the mixture at one end of the glass tube facing to the light source;
(2) after the bemisia tabaci was put in from the other end of the glass tube, the glass tube was covered with a shade cloth, and cultivation was performed.
In the above application, preferably, the culture conditions are a light irradiation ratio of 14: 10 and a humidity of 80%.
Compared with the prior art, the invention has the advantages that:
(1) the invention provides a bemisia tabaci lethal gene, wherein the bemisia tabaci lethal gene is Alpha-glucosidase, the Alpha-glucosidase gene codes α -glucosidase, α -glucosidase is a large class of enzymes in a glycoside hydrolase major family, the main function of the bemisia tabaci lethal gene is to hydrolyze glucoside bonds, glucose is released as a product, and the product is an indispensable class of enzymes in a living body sugar metabolism path.
(2) The invention provides a bemisia tabaci lethal gene which can inhibit virus diseases transmitted by bemisia tabaci, such as tomato chlorosis virus diseases, and α -glucosidase gene expression level has close relation with bemisia tabaci virus acquisition capacity, namely, the higher the virus acquisition capacity is, the higher the α -glucosidase expression level is, when α -glucosidase activity in bemisia tabaci bodies is reduced, the transmission efficiency of the bemisia tabaci to the tomato chlorosis virus is also reduced, so that the occurrence of the virus diseases is reduced.
(3) The invention provides an RNA interference agent, which is dsRNA designed based on Alpha-glucosidase, can induce the efficient and specific degradation of homologous mRNA, thereby inhibiting the activity of α -glucosidase, reducing the glucose hydrolysis capacity of bemisia tabaci, and finally leading to the death of the bemisia tabaci.
Drawings
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
FIG. 1 is a graph showing the relationship between the gene expression level of α -glucosidase and the ability of Bemisia tabaci to acquire viruses in example 4 of the present invention.
Detailed Description
The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention.
Examples
The materials and equipment used in the following examples are commercially available.
Example 1:
a gene sequence of a bemisia tabaci lethal gene fragment Alpha-glucopsidase is shown as SEQ ID NO.1, and specifically comprises the following steps:
example 2:
an RNA interference agent, the preparation method comprises the following steps:
(1) cloning of the lethal gene fragment Alpha-glucosidase of bemisia tabaci:
1.1, taking 50 heads of bemisia tabaci, extracting total RNA by a TRIzol method, and synthesizing a first strand of cDNA.
1.2 primer design with primer premier 5 based on the first strand of cDNA.
The DNA sequence of the upstream primer F1 is shown as SEQ ID NO.2, and specifically comprises the following steps: TGAGCATAATGTTGCCAGG are provided.
The DNA sequence of the downstream primer R1 is shown as SEQ ID NO.3, and specifically comprises the following steps: GCACAAGCGTCACCATAAG are provided.
1.3, using the cDNA of 1.1 as a template and the primer designed by 1.2 as a primer to carry out RT-PCR amplification, wherein the RT-PCR amplification system is as follows:
the reaction procedure is as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, three steps of cyclic denaturation, annealing and extension for 35 times, and final extension at 72 ℃ for 10 min.
1.4, carrying out agarose gel electrophoresis separation on a PCR product obtained by PCR amplification, and purifying and recovering a target DNA fragment with the same size as a target band.
1.5, connecting the recovered target DNA fragment into a pEASY-T3 vector by using T3 ligase to obtain a recombinant.
1.6, transforming the recombinant into Escherichia coli T1 to obtain a transformant.
1.7, the transformant was cultured and spread evenly on LB medium containing X-gal, IPTG and ampicillin, and cultured at 37 ℃ overnight.
1.8, selecting a white single colony in an LB culture medium, and screening positive recombinants.
1.9, amplifying the positive recombinants by using LB culture solution containing ampicillin, and extracting clone plasmids.
1.9, sequencing the cloned plasmid by using a full-automatic sequencer to obtain an Alpha-glucopsidase gene fragment shown in SEQ ID NO. 1.
(2) Synthesis of dsRNA of lethal gene fragment Alpha-glucopsidase of bemisia tabaci:
2.1, designing and synthesizing primers according to the verified Alpha-glucosidase gene fragment sequence.
The DNA sequence of the upstream primer F2 is shown as SEQ ID NO.5, and specifically comprises the following steps:
ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGCTACAGGGTGCCAAATCAT。
the DNA sequence of the downstream primer R2 is shown as SEQ ID NO.6, and specifically comprises the following steps:
ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGAAGGCCTCTGCTGAACAAC。
and 2.2, carrying out PCR amplification by using the first strand of the Bemisia tabaci cDNA as a template and using the primer designed by the 2.1.
The PCR system is as follows:
the PCR conditions were: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30sec, annealing at 55-62 ℃ for 30sec, extension at 72 ℃ for 30sec, cyclic denaturation, annealing, extension for 38 times, and final extension at 72 ℃ for 7 min.
And 2.3, carrying out agarose gel electrophoresis separation on the PCR product obtained in the step 2.2, recovering a target DNA fragment with the size consistent with that of a target band, and carrying out sequencing verification on the sequence.
2.4, synthesizing dsRNA of the target DNA fragment by using T7RiboMAX Express RNAiSystem of a PROMEGA dsRNA synthesis kit to obtain dsRNA containing aleyrodids lethal gene fragment Alpha-glucopdase, wherein the sequence of the dsRNA is shown as SEQ ID NO.4, and the method specifically comprises the following steps:
example 3:
the application of the RNA interference agent of the embodiment 2 in the removal of the bemisia tabaci comprises the following steps:
(1) preparing a feeding nutrient solution: a30 wt% sucrose solution (water as solvent) and 5 wt% yeast extract (water as solvent) were prepared, and the sucrose solution and yeast extract were mixed, and after fully dissolved, filtered through a 0.22 μm bacterial filter, and then mixed with the RNA interference agent of example 2 to obtain a feeding nutrient solution (treatment group). A control group containing no RNA interference agent was also set.
(2) Preparing a whitefly feeding device: preparing a transparent glass tube with the length of 50mm and the inner diameter of 20mm, covering one end of the glass tube with a sealing film (the first layer is stretched to be thin as much as possible), adding the feeding nutrient solution obtained in the step (1) onto the sealing film, covering the liquid drops with the sealing film, and removing air bubbles as much as possible to form a small bag containing the feeding nutrient solution.
(3) The bemisia tabaci is placed in the glass tube, the small bag containing the feeding nutrient solution faces one end of the light source, and the bemisia tabaci is quickly attached to the direction of the small feeding bag for taking food by utilizing the light-driving property of the bemisia tabaci FENG. Then, a black cotton plug was inserted, and a light-shielding sleeve capable of covering the glass tube was wrapped. Placing in an incubator under the conditions of illumination of 14: 10 and humidity of 80%.
The mortality rate of bemisia tabaci within 120h after feeding the RNA interference agent is examined, and the examination result is shown in table 1.
Table 1: results table of the effect on mortality of Bemisia tabaci after feeding dsRNA
| Mortality rate | 24h | 48h | 72h | 96h | 120h |
| Treatment group (%) | 0.5 | 9.3 | 32.2 | 56.7 | 75.3 |
| Control group (%) | 0 | 1.3 | 3.6 | 4.2 | 5.4 |
As can be seen from Table 1, the mortality rate of Bemisia tabaci gradually increases with time after the RNA interference agent of example 2 is fed, and the mortality rate of Bemisia tabaci is highest at 120h and reaches 75.3%.
Example 4:
the application method of the RNA interference agent in the example 2 for reducing the virus spread of Bemisia tabaci is the same as that in the example 3, Bemisia tabaci fed with the RNA interference agent (treatment group) and Bemisia tabaci not fed with the RNA interference agent (control group) are respectively placed in the environment of tomato chlorosis virus, the number of viruses obtained by Bemisia tabaci within 120h after feeding is inspected, and the inspection results are shown in Table 2.
Table 2: result table for influence on acquisition of virus amount of bemisia tabaci after feeding dsRNA
| Acquisition of viral load by Bemisia tabaci | 24h | 48h | 72h | 96h | 120h |
| Treatment group (%) | 15.2 | 6.3 | 0 | 0 | 0 |
| Control group (%) | 39.8 | 63.4 | 62.6 | 68.5 | 69.2 |
From the results of table 2, it can be seen that: after the bemisia tabaci is fed with the RNA interference agent of example 2, viruses obtained by the bemisia tabaci gradually decrease along with the increase of time, and the tomato chlorosis virus completely disappears at 72h, while the tomato chlorosis virus content in the bemisia tabaci gradually increases in the control group without the RNA interference agent of example 2, so that the RNA interference agent of example 2 of the application has the effect of reducing the virus transmission in the bemisia tabaci.
Referring to fig. 1, it is seen from fig. 1 that the gene expression level of α -glucosidase in bemisia tabaci has close relation with the virus acquisition capacity of bemisia tabaci, namely the higher the acquired virus amount, the higher the expression level of α -glucosidase, the activity of α -glucosidase in bemisia tabaci is reduced by feeding RNA interference agent, the transmission efficiency of bemisia tabaci to tomato chlorosis virus can be reduced, and the occurrence of virus diseases is reduced.
Example 5:
the application of the RNA interference agent of example 2 in inhibiting virus transfer is to divide the Bemisia tabaci infected with tomato chlorotic virus into two groups, feed the RNA interference agent to the treatment group according to the method of example 3, feed the nutrient solution without RNA interference agent to the control group, culture the Bemisia tabaci and the tomato of the treatment group and the control group simultaneously, examine the number of viruses of the Bemisia tabaci transmitted to the tomato within 60 days after feeding the RNA interference agent, and see Table 3 for the results of examination.
Table 3: results table of viral load transmitted to tomato by Bemisia tabaci after dsRNA feeding
| Viral load of bemisia tabaci transmitted to tomato | 30d | 40d | 50d | 60d |
| Treatment group (%) | 8.2 | 11.9 | 14.3 | 17.7 |
| Control group (%) | 15.7 | 21.3 | 43.7 | 87.6 |
From the results in table 3, it can be seen that: after the bemisia tabaci is fed with the RNA interference agent of example 2, the number of tomato infected by tomato chlorosis virus can be effectively controlled, while the number of tomato chlorosis virus infection is obviously increased in the control group without the RNA interference agent of example 2, which proves that the RNA interference agent of example 2 of the application can inhibit the occurrence of plant virus diseases.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
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Claims (10)
1. The bemisia tabaci lethal gene is characterized in that the bemisia tabaci lethal gene is Alpha-glucosidase, and the DNA sequence of the Alpha-glucosidase is shown as SEQ ID NO. 1.
2. Use of the bemisia tabaci lethal gene of claim 1 for the removal of bemisia tabaci.
3. An RNA interference agent, wherein the RNA interference agent is dsRNA synthesized by the Alpha-glucosidase gene segment according to claim 1, and the DNA sequence of the RNA interference agent is shown as SEQ ID NO. 4.
4. A method for preparing the RNA interference agent of claim 3, which comprises the following steps:
s1, synthesizing a primer pair according to the DNA sequence of Alpha-glucopsidase, and carrying out PCR amplification by using the Bemisia tabaci cDNA as a template to obtain an amplification product;
s2, carrying out agarose gel electrophoresis separation on the amplification product to obtain a target DNA fragment;
s3, synthesizing dsRNA of the target DNA fragment to obtain the RNA interference agent.
5. The method for preparing the RNA interference agent of claim 4, wherein the primer pair in S1 comprises an upstream primer and a downstream primer, the DNA sequence of the upstream primer is shown as SEQ ID NO.5, and the DNA sequence of the downstream primer is shown as SEQ ID NO. 6.
6. The method for preparing the RNA interference agent of claim 5, wherein the reaction system of the PCR amplification in S1 is: 5 μ L reaction buffer, 4 μ L Mg2+4. mu.L dNTP, 2. mu.L cDNA template; 2. mu.L of forward primer, 2. mu.L of reverse primer, 0.25. mu.L of Ex tap enzyme, 30.75. mu.L of ddH2O；
The reaction conditions for PCR amplification are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30sec, annealing at 55-62 ℃ for 30sec, extension at 72 ℃ for 30sec, three steps of cyclic denaturation, annealing and extension for 38 times, and final extension at 72 ℃ for 10 min.
7. The method for preparing an RNA interference agent according to claim 4, wherein the S3 is specifically: and (3) carrying out dsRNA synthesis on the target DNA fragment by using T7RiboMAX Express RNAi system of a PROMEGA dsRNA synthesis kit to obtain the RNA interference agent.
8. The use of the RNA interference agent of claim 3 for the removal of Bemisia tabaci.
9. The application according to claim 8, characterized in that the application method comprises the following steps:
(1) uniformly mixing the nutrient solution and the RNA interference agent and placing the mixture at one end of the glass tube facing to the light source;
(2) after the bemisia tabaci was put in from the other end of the glass tube, the glass tube was covered with a shade cloth, and cultivation was performed.
10. Use according to claim 9, wherein the culture conditions are: the illumination ratio is 14: 10, and the humidity is 80%.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220340A (en) * | 2011-05-12 | 2011-10-19 | 南京农业大学 | Gene silencing technique based Laodelphax striatellus lethal gene fragment Tubulin and dsRNA thereof |
-
2019
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (3)
| Title |
|---|
| ID：Ssa00229;function：Alpha-glucosidase;《whitefly genome database》;20161214;第1页 最后一段，第2页 gene annotation段，第3页 文本框内序列 * |
| Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs.;Meenal Vyas et al.;《PLoS ONE》;20170103;第12卷(第1期);e0168921（第3页 第3段,第4页 第2段，第5页 第2段,第8页 第2段，图2A，表5） * |
| RNA Interference based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly;Amir Raza et al.;《PLoS ONE》;20160422;第11卷(第4期);e0153883 * |
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