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RNA interference feeding method for apolygus lucorum and application of feeding method in gene screening

A technology of RNA interference and Lygus lygus, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc.

Inactive Publication Date: 2017-02-15
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there were a lot of research reports on the RNAi of Lygus green bug some time ago, 1) it mainly focused on the genes related to the olfactory system, and the research on RNAi-mediated insecticides was in a blank state, and 2) the above studies all used microinjection dsRNA delivery method

Method used

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  • RNA interference feeding method for apolygus lucorum and application of feeding method in gene screening
  • RNA interference feeding method for apolygus lucorum and application of feeding method in gene screening
  • RNA interference feeding method for apolygus lucorum and application of feeding method in gene screening

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Experimental program
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Effect test

preparation example Construction

[0072] 5. Preparation of dsRNA template

[0073]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.

[0074] 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.

[0075] The steps of phenol chloroform extraction of PCR product: ...

Embodiment 1

[0149] Example 1: The effect of feeding and screening the dsRNA of the V-ATPaseE gene fragment on the growth and development of the green Lygus bug

[0150] The above-mentioned feeding method was used to feed 3L Lygus viridans with dsGFP and ds V-ATPaseE respectively. The feeding parameters were as follows: the feeding volume was 200ul, and the final concentration of feeding dsRNA was 0.5μg / μl. see results Figure 6 ,7.

[0151] Depend on Figure 6 and Figure 7 It can be seen that the survival rates of the treatment group (dsV-ATPaseE) and the control group (dsGFP) are significantly different, and as the number of days increases, the difference between the two groups gradually becomes larger. On the 7th day after the feeding experiment, the control group still had a high survival rate. According to the statistical results, there is a significant difference between the two (P<0.05).

[0152] Similarly, using the same method of the present application, the energy metabolis...

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Abstract

The invention relates to an RNA interference feeding method for apolygus lucorum and an application of the feeding method in gene screening, and belongs to the field of biotechnology. The RNA interference feeding method for the apolygus lucorum is characterized by comprising the following steps: mixing dsRNA to artificial feed, covering the feed with a sealing film and simulating natural feeding state of the insects (the apolygus lucorum). Feasibility and approach of establishing an RNAi feeding delivery method for apolygus lucorum gene function researches are established for the first time; and with the application of the method, a new technical means and a new technical platform are provided for screening novel insect-screening genes and a new gene resource is provided for the development of an apolygus lucorum resistant transgenic crop, so that a novel economic, effective and environment-friendly method is developed to control the harm of the apolygus lucorum.

Description

technical field [0001] The invention relates to the technical field of biological applications, in particular to a feeding method for performing RNAi on the green ligus bug and the application of the method in screening key genes for the growth and development of the green ligus bug. Background technique [0002] The expansion of Bt cotton planting scale since 1997 has led to the fact that Lygus, which was previously a secondary pest, has replaced cotton bollworm as the main pest that seriously damages agricultural production in my country, especially becoming an important pest in cotton fields in the Yangtze and Yellow River basins. The occurrence area of ​​Lygus even reached about 90%, which not only caused the cotton bolls to fall but also the leaves to wither, thereby seriously affecting the cotton yield. The resulting economic loss yield loss was about 10-20%, and the severe occurrence area exceeded 30%. In addition, the green ligus not only harms cotton, but also affect...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/124C12Q2600/178
Inventor 王桂荣阿布都如苏力·吐孙刘扬
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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