Injection method for realizing RNA interference on Apolygus lucorum and application of same in gene screening
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A kind of chlorotic bug, gene technology, applied in the direction of biochemical equipment and methods, determination/inspection of microorganisms, etc.
Inactive Publication Date: 2014-08-20
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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[0007] Although it has been discussed at present, before the technical proposal, it is not known whether introducing dsRNA into Lygus spp. can trigger the RNAi effect and produce a lethal phenotype
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preparation example Construction
[0062] 5. Preparation of dsRNA template
[0063] Using the plasmid with the target gene fragment as a template, it is amplified with a polymerase. The reaction system is 25 μl: 17 μl redistilled water, 2.5 μl 10× reaction buffer, 2 μl dNTPs (2.5 mM), 1 μl plasmid carrying the target gene fragment, 1 μl forward (F) primer (10 mM), 1 μl reverse (R) Primers (10 mM) and 0.5 μl DNA polymerase. The reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55-60°C for 45 s, extension at 72°C for 1 min, and 35 cycles; final extension at 72°C for 10 min.
[0064] 1 μl of the PCR product was detected by electrophoresis on a 1% agarose gel dissolved in 1×TAE buffer. If the length of the obtained band is consistent with the length of the target fragment, and the result shows a single bright band, the remaining PCR products are extracted and purified with phenol-chloroform.
[0065] The steps of phenol chloroform extraction of PCR product:...
Embodiment 1
[0118] Example 1: Injecting and screening the dsRNA of the β-actin gene fragment on the growth and development of the green Lygus bug
[0119] The above injection method was used to inject 3L of Lygus lygus with dsGFP and dsβ-actin respectively. The injection parameters were as follows: the injection volume was 41.6 nl, the concentration of dsRNA was 10 μg / μl, and the injection site was between the hind thorax and abdomen of the green Lygus. The outermost side of the membrane (position marker: II).
[0120] According to the above parameters, observe and record for 7 days after injection, count the mortality rate, and compare with the control injection of dsGFP. The specific values are shown in Table 4 and figure 2 .
[0121] Table 4. Survival rate within 7 days of injection of dsGFP and injection of dsβ-actin
[0122]
[0123] From Table 4 and figure 2 It can be seen that the survival rates of the treatment group (dsβ-actin) and the control group (dsGFP) are signifi...
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Abstract
The invention relates to an injection method for realizing RNA interference on Apolygus lucorum and application of the same in gene screening, belonging to the field of biotechnology. The injection method for realizing RNA interference on Apolygus lucorum is characterized in that the outmost side of an intersegmental membrane of metastethidium and an abdomen is the position for injection of dsRNA. An RNAi platform used for researching gene functions of Apolygus lucorum is established for the first time; an injection method is creatively used to determine an optimal injection position for RNAi of Apolygus lucorum and an optimal injection volume at a specific dsRNA concentration, and changes of the phenotype of Apolygus lucorum after injection is detected and recorded. The method provides a novel technological means and a novel technological platform for screening of novel pest-resistant genes and novel gene resources for development of anti-Apolygus lucorum transgenic crops, thereby realizing development of an economic, effective, environment-friendly and novel method for controlling of harm of Apolygus lucorum.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to an injection method for performing RNAi on Lygus spp. and the application of the method in screening key genes for the growth and development of Lygus spp. Background technique [0002] With the large-scale planting of Bt cotton, the damage of cotton bollworm has been effectively controlled. However, Lygus aeruginosa has gradually become an important pest that harms my country's agricultural production, especially cotton fields. The occurrence area accounts for about 90% of the planting area, seriously threatening cotton. Production, resulting in a loss of 10% to 20% of cotton yield. In addition, the green Lygus not only harms cotton, but also affects the production of crops such as maize, peach, cherry, apple and grape. At present, the control measures for the green Lygus mainly rely on chemical control. However, long-term large-scale use of pesticides for pest control will not onl...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/124C12Q2600/178
Inventor 王桂荣刘方舟杨婷刘杨
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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