CN116875610A - Laodelphax striatellus octopamine receptor gene fragment LsOA2B2 and application thereof - Google Patents
Laodelphax striatellus octopamine receptor gene fragment LsOA2B2 and application thereof Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
The application discloses application of an octopamine receptor gene fragment LsOA2B2 of Laodelphax striatellus in preventing and treating Laodelphax striatellus, belongs to the field of agricultural science, and utilizes an RNA technology to silence LsOA2B2 genes, has obvious inhibition effect on the spawning quantity and the hatching rate of Laodelphax striatellus, and shows that the genes can be used as effective targets for controlling pests by using an RNA interference technology, thereby providing reliable basis for developing control of pests by using the RNA interference technology in the future.
Description
Technical Field
The application belongs to the technical field of agricultural science, and particularly relates to a fragment LsOA2B2 of an octopamine receptor gene of Laodelphax striatellus and application thereof.
Background
Laodelphax striatellus is one of the most important pests on rice, and particularly serious pests occur in Huang-Huai and Yangtze river delta and surrounding areas in China. Besides the damage caused by piercing and sucking phloem rice juice, the Laodelphax striatellus can also be used as a transmission medium of plant viruses, such as rice stripe viruses, so that serious rice diseases are caused, and the loss caused by the plant virus diseases is far greater than the damage caused by direct piercing and sucking. In production, most of the control of insect-borne plant virus diseases depends on pesticides to control the insect-borne virus and further control the occurrence of the diseases. However, the use of pesticides in large quantities not only causes problems of pesticide residues, pest resistance, rampant re-growth, etc., but also threatens the environment and food safety. Therefore, development of an environmentally friendly control strategy to replace the conventional chemical control is urgently required.
In recent years, RNA interference technology has been developed, and is widely used for research of gene functions and pest control. Laodelphax striatellus is a typical pest with strong fertility and short growth cycle. In theory, if the RNAi technology is used for interfering the expression of important reproduction related genes in the Laodelphax striatellus, the fertility of pests can be reduced, so that the population quantity is reduced, and the purpose of controlling the pests is achieved.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present application has been made in view of the above and/or problems occurring in the prior art.
Therefore, the application aims to overcome the defects in the prior art and provide the Laodelphax striatellus octopamine receptor gene fragment LsOA2B2 with the nucleotide sequence shown as SEQ ID NO. 1.
In order to solve the technical problems, the application provides the following technical scheme: comprising the steps of (a) a step of,
the cloning method of the fragment comprises the steps of,
designing upstream and downstream primers P1 and P2 by taking a first chain of the Laodelphax striatellus cDNA as a template, and amplifying the primers by using an RT-PC R method to obtain a gene fragment LsOA2B2;
the nucleotide sequences of the upstream primer P1 and the downstream primer P2 are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
The application also aims to overcome the defects in the prior art and provide dsRNA of the octopamine receptor gene fragment LsOA2B2 of the Laodelphax striatellus with a nucleotide sequence shown as SEQ ID NO. 4.
In order to solve the technical problems, the application provides the following technical scheme:
the synthesis method of the dsRNA comprises the following steps of,
the octopamine receptor gene fragment LsOA3 plasmid is taken as a template, upstream and downstream primers P3 and P4 are designed for PCR amplification, amplified products are separated by agarose gel electrophoresis, and sequencing verification is carried out to obtain dsRNA with a sequence shown as SEQ ID NO.4, and recovery, annealing and purification are sequentially carried out;
the nucleotide sequences of the upstream primer P3 and the downstream primer P4 are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
It is still another object of the present application to overcome the deficiencies in the prior art and to provide the use of the octopamine receptor gene fragment LsOA2B2 of Laodelphax striatellus and its dsRNA for preventing Laodelphax striatellus.
As a preferred scheme for the application of the dsRNA of the octopamine receptor gene fragment LSOA2B2 of the application, the application comprises the following steps: the dsRNA acts on the Laodelphax striatellus by injection.
As a preferred scheme for the application of the dsRNA of the octopamine receptor gene fragment LSOA2B2 of the application, the application comprises the following steps: the dsRNA is injected into the Laodelphax striatellus, so that the spawning quantity and the hatching rate of the Laodelphax striatellus can be reduced.
As a preferred scheme for the application of the dsRNA of the octopamine receptor gene fragment LSOA2B2 of the application, the application comprises the following steps: the injection concentration of the dsRNA is 5000-7000 ng/. Mu.L.
As a preferred scheme for the application of the dsRNA of the octopamine receptor gene fragment LSOA2B2 of the application, the application comprises the following steps: the injection volume of the dsRNA on the nymphs was 23nl and the injection volume on the adults was 36nl.
The application has the beneficial effects that:
the application utilizes RNA technology to silence LsOA2B2 gene, has obvious inhibition effect on the spawning quantity and the hatching rate of the Laodelphax striatellus, and shows that the gene can be used as an effective target point of controlling pests by RNA interference technology; compared with the previous synthesis method, the synthesized LsOA2B2 gene dsRNA is added with an annealing purification step, so that the synthesized dsRNA has higher purity, higher synthesis concentration and better interference effect; compared with the prior gene interference, the RNA interference of the LsOA2B2 gene has a more remarkable inhibiting effect on the spawning quantity and the hatching rate of the Laodelphax striatellus, and provides a reliable basis for future development of pest control by utilizing an RNA interference technology.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a graph showing the result of PCR amplification of a gene fragment in example 1 of the present application.
FIG. 2 is a graph showing the PCR amplification result of dsRNA in example 2 of the present application.
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
The embodiment provides a cloning method of octopamine receptor gene LsOA2B2 fragment, which specifically comprises the following steps:
1) The first strand of the Laodelphax striatellus cDNA is taken as a template, upstream and downstream primers P1 and P2 are designed according to the LsOA3 gene of the Laodelphax striatellus, the nucleotide sequences of the primers are respectively shown as SEQ ID NO.2 and SEQ ID NO.3, and the primers are amplified by an RT-PCR method;
wherein, the reaction system of RT-PCR (50. Mu.L): 25. Mu.L of 2X Phanta Max Master Mix, 2. Mu.L of cDNA template, 2. Mu.L of 10. Mu.M upstream primer P1, 2. Mu.L of 10. Mu.M downstream primer P2, 19. Mu.L of RNase-free ddH 2 O；
The reaction conditions are as follows: denaturation at 95℃for 3min, 15sec at 95℃for 15sec, 15sec at 57℃for 30sec at 72℃for 1min for 42 cycles, extension at 72℃for 5min.
2) Separating amplified product by agarose gel electrophoresis, recovering target DNA fragment, transferring into competent cells by ligation reaction, adding LB culture solution, shake culturing at 37deg.C for 1 hr, and culturing at 37deg.C overnight;
screening a positive recombinant by selecting white single colonies, amplifying the recombinant by using LB culture solution containing Amp antibiotics, extracting cloning plasmids, and sequencing by a full-automatic sequencer (completed by Shanghai biological engineering technology service Co., ltd.) to obtain the Laodelphax striatellus octopamine receptor gene fragment LsOA2B2 with a nucleotide sequence shown as SEQ ID NO. 1.
Example 2
The embodiment provides a synthesis and recovery method of dsRNA of a fragment of a Laodelphax striatellus octopamine receptor gene LsOA2B2, which specifically comprises the following steps:
1) Designing an upstream primer P3 and a downstream primer P4 by taking a Laodelphax striatellus LsOA2B2 gene plasmid as a template according to the verified Laodelphax striatellus lethal gene fragment sequence, wherein the nucleotide sequences are respectively shown as SEQ ID NO.5 and SEQ ID NO.6, and obtaining the dsRNA of the Laodelphax striatellus octopamine receptor gene LsOA2B2 fragment through PCR amplification;
wherein, the PCR system is: 25 μL 2 x Phanta Max Master Mix, 2. Mu.L cDNA template, 2. Mu.L 10. Mu.M upstream primer P1, 2. Mu.L 10. Mu.M downstream primer P2, 19. Mu.L RNase-free ddH 2 O；
The PCR conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15sec, annealing at 58℃for 15sec, elongation at 72℃for 30sec,42 cycles; thoroughly extend at 72℃for 5min.
2) Separating PCR product by agarose gel electrophoresis and observing under ultraviolet lamp, as shown in figure 2, determining nucleotide sequence as shown in SEQ ID NO. 4;
3) By Vazyme CoGel DNA Extraction Mini Kit the kit is recovered and purified to obtain a DNA template, and the concentration of the DNA is measured by an ultraviolet spectrophotometer (Thermo Nanodrop 1000);
4) By T7 riboMAX TM Express RNAi System (Promega) kit for synthesizing dsRNA and configuring a reaction system as riboMAX TM Express T7 2×Buffer 10μL，DNA Template 1-8μL，RNase-free ddH 2 O0-7 mu L, enzyme mix, T7 Express 2 mu L, 20 mu L total, gently mixing by blowing, incubating for 4h at 37 ℃; incubating for 10min at 70 ℃, cooling for 20min at room temperature, and annealing to form dsRNA;
5) Purifying the dsRNA and removing cDNA and ssRNA in the dsRNA;
diluting RNase A Solution by 1:200, adding 1 mu L of newly diluted RNase A Solution and 1 mu L of RQ1 RNase-Free DNase into the system, and incubating at 37 ℃ for 30min;
adding 0.1 times volume of Sodium Acetate and 1 time volume of isopropanol, mixing, centrifuging at 15000 Xg for 10min after ice for 5min, carefully pouring out supernatant, washing with 750 μl of cold 75% alcohol, repeating for one time, air-drying at room temperature for 2min, and dissolving precipitate thoroughly with 20 μl of RNase-Free water;
the synthesized dsRNA is diluted by 10 times, and then the concentration of the RNA is measured by an ultraviolet spectrophotometer (Thermo Nanodrop 1000), and in order to avoid repeated freeze thawing and influence on the interference effect, the RNA is immediately split into 10 mu L/tube and frozen in a refrigerator at-80 ℃ for later use.
Example 3
In this example, the dsRNA synthesized in example 2 was subjected to experiments for spawning after injection of laodelphax striatellus, specifically:
shearing the needle tip to a proper length by scissors before using the microinjection needle to suck a dsRNA sample, wherein the concentration of the sample is 6000 ng/. Mu.L;
selecting unmatched Laodelphax striatellus female insects in the eclosion period of 48 hours, anaesthetizing the Laodelphax striatellus female insects with carbon dioxide, injecting dsRNA into the joint of the female insect forechest and the middle chest by using a microinjection instrument, injecting 36nl of each Laodelphax striatellus female insect, injecting dsGFP with the same volume in a control group, and treating 60 heads each;
the injected Laodelphax striatellus female insects are inoculated into fresh rice seedlings for breeding for 24 hours, laodelphax striatellus female insects with good survival state are selected, female adults are singly placed into a test tube containing the rice seedlings, a male insect which is eclosion in the same period and is not mated is paired, the male insects are self-paired and lay eggs, and each treatment is repeated for 50 times;
transferring paired male and female worms into a new test tube every 5 days for continuous spawning, repeating until the female worms die, and adding the male adults again if the male adults die in the period; counting the number of nymphs hatched from test tube seedlings after daily replacement, dissecting the test tube under a stereoscopic microscope if no nymphs hatch for 5 consecutive days, and counting the number of eggs which are not hatched;
the egg laying amount of the Laodelphax striatellus refers to the sum of the number of hatched nymphs and the number of non-hatched egg grains, the hatching rate refers to the ratio of the number of hatched nymphs to the total egg laying amount, and the results are shown in tables 1 and 2.
TABLE 1 comparison of egg laying amount of female Laodelphax striatellus under different treatment conditions
As can be seen from table 1, the dsRNA injected with octopamine receptor gene fragment LsOA2B2 has a very remarkable inhibitory effect on the oviposition amount of female plant hoppers.
TABLE 2 comparison of hatching rates of Laodelphax striatellus eggs under different treatment conditions
As can be seen from table 2, the dsRNA injected with octopamine receptor gene fragment LsOA2B2 also has a very significant inhibitory effect on the spawning hatchability of female plant hoppers.
In conclusion, the application utilizes the RNA technology to silence the LsOA2B2 gene, has obvious inhibition effect on the spawning quantity and the hatching rate of the Laodelphax striatellus, and shows that the gene can be used as an effective target point of controlling pests by the RNA interference technology; compared with the previous synthesis method, the synthesized LsOA2B2 gene dsRNA is added with an annealing purification step, so that the synthesized dsRNA has higher purity, higher synthesis concentration and better interference effect; compared with the prior gene interference, the RNA interference of the LsOA2B2 gene has a more remarkable inhibiting effect on the spawning quantity and the hatching rate of the Laodelphax striatellus, and provides a reliable basis for future development of pest control by utilizing an RNA interference technology.
It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.
Claims (10)
1. The fragment LsOA2B2 of the octopamine receptor gene of the Laodelphax striatellus is characterized in that: the nucleotide sequence of the gene fragment is shown as SEQ ID NO. 1.
2. The fragment LsOA2B2 of the octopamine receptor gene of laodelphax striatellus as claimed in claim 1, wherein: the cloning method of the fragment comprises the steps of,
the method comprises the steps of taking a first strand of the cDNA of the Laodelphax striatellus as a template, designing upstream and downstream primers P1 and P2, and amplifying the primers by an RT-PCR method to obtain a gene fragment LsOA2B2;
the nucleotide sequences of the upstream primer P1 and the downstream primer P2 are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
3. The dsRNA of the fragment LsOA2B2 of the octopamine receptor gene of the Laodelphax striatellus is characterized in that: the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 4.
4. The dsRNA of the fragment LsOA2B2 of the octopamine receptor gene of laodelphax striatellus of claim 3, wherein: the dsRNA targets and silences the fragment LsOA2B2 of the octopamine receptor gene of the Laodelphax striatellus.
5. The dsRNA of the fragment LsOA2B2 of the octopamine receptor gene of laodelphax striatellus of claim 3, wherein: the synthesis method of the dsRNA comprises the following steps of,
the octopamine receptor gene fragment LsOA2B2 plasmid is taken as a template, upstream and downstream primers P3 and P4 are designed for PCR amplification, amplified products are separated by agarose gel electrophoresis, and the dsRNA with the sequence shown as SEQ ID NO.4 is obtained by sequencing verification, and is sequentially recovered, annealed and purified;
the nucleotide sequences of the upstream primer P3 and the downstream primer P4 are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
6. Use of the fragment LsOA2B2 of the octopamine receptor gene of laodelphax striatellus according to claim 1 for controlling laodelphax striatellus.
7. Use of the dsRNA of the fragment LsOA2B2 of the octopamine receptor gene of the laodelphax striatellus as claimed in claim 6, characterized in that: the dsRNA acts on the Laodelphax striatellus by injection.
8. Use of the dsRNA of the fragment LsOA2B2 of the octopamine receptor gene of the laodelphax striatellus as claimed in claim 7, characterized in that: the dsRNA is injected into the Laodelphax striatellus, so that the spawning quantity and the hatching rate of the Laodelphax striatellus can be reduced.
9. Use of the dsRNA of the fragment LsOA2B2 of the octopamine receptor gene of the laodelphax striatellus as claimed in claim 8, characterized in that: the injection concentration of the dsRNA is 5000-7000 ng/. Mu.L.
10. Use of the dsRNA of the fragment LsOA2B2 of the octopamine receptor gene of the laodelphax striatellus as claimed in claim 9, characterized in that: the dsRNA injection volume was 36nl.
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