Method, primer and kit for detecting blue light phototaxis of aphids

A technology of phototaxis and aphids, applied in the field of agricultural biology, can solve problems such as high technical requirements, unsatisfactory, difficult to operate, etc., and achieve the effect of avoiding heavy workload, high accuracy, and improving efficiency

Active Publication Date: 2017-08-25
湖北香润生态农业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is an urgent need for a rapid identification of aphid phototaxis technology in production. The traditional visual electrophysiologic

Method used

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  • Method, primer and kit for detecting blue light phototaxis of aphids
  • Method, primer and kit for detecting blue light phototaxis of aphids
  • Method, primer and kit for detecting blue light phototaxis of aphids

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Cloning of opsin gene CRY1 from rape aphids (mainly Myzus persicae).

[0053] 1. Aphid total RNA extraction and cDNA reverse transcription

[0054] Use Trizol reagent to extract the total RNA of rape aphids, and the specific steps are as follows: (1) Take about 50 mg of the frozen sample in an ultra-low temperature refrigerator, pour it into a mortar, and grind it thoroughly. The powder was transferred to a clean centrifuge tube with 1 mL of Trizol reagent, shaken vigorously, then left at room temperature for 10 min, centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was transferred to a new centrifuge tube. (2) Add 0.2mL chloroform, shake for 30s, and place at room temperature for 10min. Centrifuge at 12000rpm at 4°C for 10min. (3) Take the upper aqueous phase to a new centrifuge tube, add 400 μL of isopropanol, mix well, place at room temperature for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, and discard the supernatant. (4) Add 1 mL o...

Embodiment 2

[0089] In order to facilitate detection and application, this embodiment provides a primer for detecting blue light phototaxis of rape aphids, which includes primers shown in Seq ID No.5-6.

[0090] Using the cDNA of the rape aphid provided in Example 1 as a template, and the sequence Seq ID No.5-6 as a primer, the full-length sequence of the rape aphid opsin gene CRY1 can be obtained by one-time PCR amplification, and the PCR reaction system is as follows:

[0091] wxya 2 o

13.6μL 10×Buffer 2.0 μL dNTP (2.5mM) 1.6μL cDNA 1.0 μL Upstream primer Seq ID No.5 (10μM) 0.8μL Downstream Primer Seq ID No.6 (10μM) 0.8μL Taq enzyme 0.2 μL overall system 20.0 μL

[0092] The conditions of the PCR amplification reaction were: pre-denaturation at 94°C for 5 minutes; 30 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 30s; and extension at 72°C for 10 minutes.

[0093] After sequencing and comparative analysis, it was fo...

Embodiment 3

[0096] Bioinformatics Analysis and Discrimination of Opsin CRI of Rapeseed Aphid

[0097] 1. Analysis of the Conserved Domain of CRY1 Opsin in Rapeseed Aphid

[0098] The conserved structure analysis of the opsin sequence of rape aphid was completed by SMART (http: / / smart.embl-heidelberg.de / ). Using Smart analysis, the results showed that CRY1 opsin has DNA photolyase protein family and DNA photolyase FAD binding protein family conserved domains, but no binding site ( figure 2 ).

[0099] 2. Prediction of transmembrane structure and subcellular localization of CRY1 opsin in rape aphid

[0100] Using TMHMM Server v.2.0 to analyze the transmembrane region of the protein sequence (http: / / www.cbs.dtu.dk / services / TMHMM / ) to predict the transmembrane structure of the rape aphid opsin. The analysis showed that no transmembrane structure was found for the CRY1 opsin of the rape aphid, and the subcellular location was in the cytoplasm (such as image 3 As shown, in the figure: A: ...

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Abstract

The invention discloses a method, a primer and a kit for detecting blue light phototaxis of aphids and relates to the field of agriculture biotechnology. The method for detecting the blue light phototaxis of the aphids disclosed by the invention comprises the following steps: comparing amino acid sequences of CRY1 proteins of target aphids with amino acid sequences of CRY1 proteins of pea aphids; and determining the blue light phototaxis of the target aphids according to sequence comparing results. The detection method is short in time and high in accuracy; the defects such as heavy workload, long time and high possibility of influence of environment factors and the like of the conventional method for detecting phototaxis are avoided; the method is taken as significant technical renovation; the efficiency is greatly improved; the key technical support is provided for early warning and green control of the aphids; the method has an extremely important application value in the field of control of the aphids and can be applied to the fields of control of the aphids, prediction of aphid situation or early warning of the aphids.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a method for detecting blue light phototaxis of aphids, primers and a kit. Background technique [0002] Aphids are one of the most serious pests in agricultural and forestry production. The main crops that are harmed are cotton, wheat, vegetables, rapeseed and so on. Its harmfulness can make plants unable to grow and develop normally, such as plant nutrient deficiency, growth deformity, reduced yield, and damaged quality. The main species of aphids on rapeseed are Myzus persicae, radish aphid (Lipaphiserysimi) and cabbage aphid (Brevicoryne brassicae), which suck nutrients with adults or nymphs, which not only harm the growth of rapeseed, but also spread a variety of viral diseases , affecting the yield and quality of rapeseed, causing serious economic losses. Therefore, the intensification of rapeseed aphids will cause losses to the entire rapeseed industry, especial...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12Q1/68C12N15/11
CPCC12Q1/6888G01N33/6818G01N2333/43552
Inventor 李春生王永张兴甫
Owner 湖北香润生态农业科技有限公司
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