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Method for effectively controlling agricultural pest mites by RNAi

A cloning method, RT-PCR technology, applied in the field of agricultural biology, can solve the problems of difficult operation of RNAi molecular technology and less research on mites, and achieve the effects of reducing experimental costs, reducing mechanical damage, and simplifying experimental operations

Inactive Publication Date: 2015-03-11
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, RNAi induced by dsRNA has been widely used to study the function of insect genes, but there are very few studies on mites (Khila, 2007; Campbell, 2010), mainly because mites are small (the body is mostly in 1 below mm), it is difficult to operate using RNAi molecular technology

Method used

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  • Method for effectively controlling agricultural pest mites by RNAi
  • Method for effectively controlling agricultural pest mites by RNAi
  • Method for effectively controlling agricultural pest mites by RNAi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] (1) Get 100-150 spider mites, and use the TRIzol method to extract total RNA;

[0057] (2) Synthesizing the first strand of cDNA;

[0058] (3) The sequence of the gene fragment was obtained from the transcriptome of the spider mite, and after homology comparison at http: / / www.ncbi.nlm.nih.gov / , it was predicted to be the AK gene, and P1 and P1 were designed using Primer premier 5.0 software. P2, amplified by RT-PCR method;

[0059] Upstream primer (P1): 5'GCGACTTGTGAACCTG 3' (SEQ ID NO.2),

[0060] Downstream primer (P2): 5'TCTGCTGACGCTGAAA 3' (SEQ ID NO.3);

[0061] The PCR reaction program was: denaturation at 94°C for 5 min, 1 min at 94°C, 1 min at 56°C, 1 min at 72°C, 32 cycles, and extension at 72°C.

[0062] PCR reaction system (25μL):

[0063] 10×Ex PCR Buffer 2.5μL

[0064] 2.5mM dNTP Mixture 2.0μL

[0065] cDNA template 3 μL

[0066] 10 μM upstream primer P1 1.0 μL

[0067] 10 μM downstream primer P2 1.0 μL

[0068] 5U / μL Ex Taq 0.25μL

[0069] ...

Embodiment 2

[0077] (1) Extract the recombinant plasmid PMD18-T-AK cloned in the first aspect of the present invention, and perform double digestion with HindIII and SacII to obtain two cohesive ends.

[0078] (2) The digested product is separated by agarose gel electrophoresis, and the target DNA fragment is recovered Figure X.

[0079] (3) The L4440 plasmid was also double digested with HindIII and SacII to obtain the same sticky end as in (1).

[0080] (4) The digested product is separated by agarose gel electrophoresis, and the target DNA fragment is recovered as vector image X.

[0081] (5) Ligate the target fragment obtained in (2) and the L4440 plasmid vector fragment obtained in (4) with T4 DNA ligase, transform into Escherichia coli HT115, and spread on the double antibody LB plate containing (Amp+Tet), 37°C Cultivate overnight;

[0082] (6) Pick a single colony, inoculate it in 2ml LB medium (Amp+Tet), culture overnight at 37°C, extract the plasmid and carry out enzyme digestio...

Embodiment 3

[0088] (1) Cut the double-sided tape into 2cm length and paste it on one end of the glass slide

[0089] (2) Select a healthy female adult mite, gently pick it up with a small brush, and stick its back on the adhesive tape, and be careful not to stick the feet, pedicles and mouthparts.

[0090] Glue 30 heads per piece, arrange them in three rows, then put the slides in a clean, non-toxic and moist tray, and place them in an incubator at 28±1°C

[0091] (3) After 0.5 hours, pick out inactive and dead individuals under a binocular dissecting microscope and make up 30 for use;

[0092] (4) Use a 2.5 μL pipette gun to add an appropriate amount of dsRNA to the back of each worm under a stereo microscope

[0093] (5) Death standard: lightly touch the mite body with a small brush, and those who do not move their limbs are considered dead

[0094] (6) Feed continuously for 48 hours, count the mortality rate every 24 hours, the results are shown in Table 1 and figure 2 , from Table...

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Abstract

The invention relates to the field of a biotechnology, specifically to a method for controlling leaf mites by silencing an energy metabolism gene. The method provided by the invention comprises the following steps: leaf mite lethal gene AK is selected as an RNAi target gene; and the RNAi target gene is connected to a plasmid vector L4440 and is transformed to escherichia coli HT115 so as to obtain a bacterial strain for expression of AK gene dsRNA. The method is an important supplement for a leaf mite control method by mainly using a pesticide. By the RNAi technology, expression of the energy metabolism gene in leaf mites is interfered. The method provides a technical support for controlling leaf mites.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology; more specifically, the present invention relates to a target gene and dsRNA thereof based on RNAi technology for preventing and controlling agricultural pest mite, and describes a method for synthesizing the target gene into a double-stranded RNA preparation and directly applying it to the control of agricultural pest mite . technical background [0002] In my country, there is currently no effective control method for agricultural pest mites, and spraying insecticides is still the main control method. The indiscriminate application of highly toxic and high-residue pesticides has not only led to the decline in the quality of crop products, the increase in the resistance of pest mites, but also endangered people's lives and health. Therefore, there is an urgent need to develop safe, effective and new strategies to control the harm of pest mite populations. [0003] The phenomenon of RNA...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C12N15/70C12N15/66A01N57/16A01P7/02
Inventor 赵伊英刘峰汪小东
Owner SHIHEZI UNIVERSITY
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