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Vegetable leafminer odorant-binding protein and application thereof

An odorant binding protein, technology of Liriomyza sativae, applied in the field of insect biology

Active Publication Date: 2015-08-19
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

However, the high resistance of Liriomyza pests to chemical insecticides directly led to the "poisonous cowpea" incident in Hainan Province, my country in 2009-2010 (http: / / english.peopledaily.com.cn / 90001 / 90776 / 90882) happened

Method used

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  • Vegetable leafminer odorant-binding protein and application thereof
  • Vegetable leafminer odorant-binding protein and application thereof
  • Vegetable leafminer odorant-binding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Sequencing of the full-length gene of Liriomyza sativae odorant binding protein OBP

[0025] Materials and Methods

[0026] (1) Extraction of total RNA from Liriomyza sativae adults

[0027] According to the (EASYspin Plus Tissue / Cell RNA Rapid Extraction Kit) instructions:

[0028] 1) Grinding and homogenizing with liquid nitrogen: Freeze the collected worms and various tissues in liquid nitrogen, grind them thoroughly in a grinder, add 350ul tissue lysate, shake vigorously for 20s, and transfer to a 1.5ml centrifuge tube. The lysate (sheared DNA) was pipetted with a 1ml pipette tip.

[0029] 2) After homogenization, the lysate was centrifuged at 13,000 rpm for 3 minutes, and all the supernatant of the lysate was added to the DNA removal column (the removal column was placed in a collection tube).

[0030] 3) Immediately centrifuge at 13000rpm for 60s, retain the filtrate (RNA is in the filtrate).

[0031] 4) Use a micropipette to more accurately estimate...

Embodiment 2

[0110] Tissue expression of embodiment 2OBP in Liriomyza sativae body

[0111] Materials and Methods

[0112] The male and female adults of Liriomyza sativae and different tissues of the head, thorax, abdomen, and feet, as well as the bodies of different developmental stages were collected, frozen in liquid nitrogen and stored at -80°C for later use.

[0113] Total RNA was extracted from each part of male and female adults of Liriomyza sativae and at different developmental stages, and the first strand of cDNA was synthesized. Same method as above

[0114] Design and synthesize primers for quantitative PCR:

[0115] Liriomyza sativae odorant binding protein gene-specific primers are:

[0116] YG-3S: 5'-CCGACAAGTCAGACGGCAAAAA-3' (SEQ ID No.11)

[0117] YG-3A: 5′-TACGACGGGGCACAAACAAAAG-3′ (SEQ ID No.12) amplified fragment length is 234bp

[0118] The β-actin gene-specific primers used as internal reference are:

[0119] actin-F3: 5'-GCCCAAAGCAAAAGAGGT-3' (SEQ ID No.13)

...

Embodiment 3

[0132] Embodiment 3 prepares OBP recombinant protein

[0133] Materials and Methods

[0134] Design of primers for expression of Liriomyza sativae OBP protein

[0135] OBP-O-S: 5′- GATATC GAAGAGTGGAAACGCAAGGAAT-3' (SEQ ID No. 15)

[0136] OBP-O-A: 5′- CTCGAG TTATTCTTCTTTCTTTTGGGCT-3' (SEQ ID No.16) (the underlined part in italics is the restriction site, respectively EcoRV, Xho I restriction site)

[0137] After PCR amplification, it was gel-cut, recovered and purified, and connected to the carrier overnight to obtain recombinant OBP, which was transformed into Escherichia coli competent cells Trans1-T1, PCR amplification was detected by electrophoresis, and then sequenced. After correct sequencing, it was transformed into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression.

[0138] By constructing prokaryotic expression vector ( Figure 4 , 5), IPTG induced expression, affinity chromatography purification obtained OBP recombinant protein with higher ...

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Abstract

The invention relates to a separated vegetable leafminer odorant-binding protein, its encoding protein and an application thereof. The protein can bind to odors matters of 4-ethyl benzaldehyde, azulene, 3,4-dimethyl acetophenone and p-ethylacetophenone, and combination of the protein and the odors matters can be used for preventing and treating vegetable leafminer.

Description

technical field [0001] The invention relates to the field of insect biotechnology, in particular to a novel odorant binding protein of Liriomyza sativae and its application. Background technique [0002] Liriomyza sativae Blanchard is a polyphagous pest that damages a variety of host plants. At present, the use of chemical pesticides is still an important means of control. However, the high resistance of Liriomyza pests to chemical insecticides directly led to the "poisonous cowpea" incident in Hainan Province, my country in 2009-2010 (http: / / english.peopledaily.com.cn / 90001 / 90776 / 90882) happened. How to continuously and effectively control the damage of Liriomyza sativae is a severe problem currently facing. [0003] With the development of molecular biology, it is a potential effective method to control the chemosensitivity of target pests to host plants. The host selection of Liriomyza sativae is mainly determined by the adult's avoidance response to the host volatiles...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/11A01N47/44A01P17/00
Inventor 王海鸿吉青战张林雅雷仲仁
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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