A screening method for plant-derived attractants based on tea geometrid odor-binding protein

A technology of odor binding protein and screening method, applied in the field of screening of plant-derived attractants, can solve problems such as difficult to effectively design and develop, complex volatile components of tea tree leaves, etc.

Inactive Publication Date: 2016-04-13
HANGZHOU ACAD OF AGRI SCI +1
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Problems solved by technology

Among them, using its olfactory behavior to design its attractant has become an important means of comprehensive management of tea geometrids, and the main representative technology is to identify the volatiles of tea geometrids on tea tree leaves by means of chemical instrument analysis. The composition of the compound is very complex, so it is difficult to effectively design and develop an olfactory attractant formula suitable for the tea geometrid

Method used

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  • A screening method for plant-derived attractants based on tea geometrid odor-binding protein
  • A screening method for plant-derived attractants based on tea geometrid odor-binding protein
  • A screening method for plant-derived attractants based on tea geometrid odor-binding protein

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Embodiment 1

[0024] 1) Specific steps for collecting total RNA from the antennae of the tea geometrid;

[0025] Materials: Tea geometrid larvae were captured in Hangzhou tea gardens, and artificially reared to adults in the laboratory for experimental use. Larva rearing conditions are: temperature 26±1°C, L:D=12:12, RH 70%-80%. Total RNA was extracted from the antennae of adult tea geometrids by TRIzol method, and the first strand of cDNA was obtained by reverse transcription using Super-Script?ⅡReverse Transcriptase System (Takara).

[0026] 2) The specific steps of obtaining the full length of the tea looper odor binding protein by RT-PCR;

[0027] ① Design of primers for tea geometrid EoblGOBP2. According to the gene sequence of the tea geometrid common odor binding protein GOBP2 (EoblGOBP2) in Genbank (Genbank accession number is: ACN29681.1 (12), primers were designed using PrimerPremier5.0 software. For the convenience of later expression, the target gene was cloned into the expres...

Embodiment 2

[0052] Tests demonstrating the effectiveness of the botanical attractant obtained by the present invention.

[0053] 1) Insects to be tested. The mature larvae of Tea Geometrid were collected from the artificially reared population in this laboratory. The feeding conditions are: temperature (26±1)°C, relative humidity (75±5)%, photoperiod L:D=12:12 (light 12h, dark 12h). After pupation and eclosion, the sexually mature adults were collected for experiments, and they were starved for 3 hours before the experiments.

[0054] 2) Experimental instruments and reagents. The experimental instrument used the antenna potentiometer (Netherlands Syntech Company); various standard volatiles of tea tree leaves were purchased from Bailingwei, and each compound was dissolved in liquid paraffin, and made into a 0.1 (v / v) solution for EAG determination. Add 10uL of the sample solution to be tested dropwise on the filter paper strip (2cm×0.5cm) in advance, let the solvent volatilize for a wh...

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Abstract

The invention discloses a tea geometrid odorant binding protein-based plant attractant screening method and belongs to the technical field of bioengineering. The tea geometrid odorant binding protein-based plant attractant screening method comprises the following processes of collecting tea geometrid antenna total RNA, acquiring total length of a tea geometrid odorant binding protein by RT-PCR, constructing a prokaryotic expression vector of the tea geometrid odorant binding protein, inducing the expression of the recombinant tea geometrid odorant binding protein by IPTG, carrying out the purification of the recombinant tea geometrid odorant binding protein by nickel sepharose gel affinity column, acquiring a binding reaction spectrum of the recombinant tea geometrid odorant binding protein and tea leaf smell volatiles by a competitive fluorescent combination method, and determining that the tea leaf smell volatile reducing relative fluorescence intensity of 1-NPN to less than 50% and having the binding constant of 13-45 micromoles per liter is the plant attractant of tea geometrid. The tea geometrid odorant binding protein-based plant attractant screening method provides a novel means for screening and designing a formula of a plant tea geometrid smell information attractant.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for screening plant-derived attractants based on tea geometrid odor-binding protein. Background technique [0002] tea inchworm Ectropisobliqua Prout, belonging to Lepidoptera and Geometridae, is a major tea tree pest widely distributed in tea-producing provinces in the Yangtze River Basin. Because it likes to eat tea tree leaves, has many generations, and reproduces quickly, all leaves will be eaten when it is serious. The light caused the tea industry to reduce production or even fail. For a long time, the control of tea inchworms has mainly relied on chemical pesticides, which can easily cause the problem of pesticide residues in commercial tea and seriously threaten the normal development of my country's tea trade industry. In view of this, it is necessary to develop new types of pollution-free tea in addition to chemical pesticides. Biological co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C12N15/12C12N15/63C07K14/435
Inventor 余继忠李红亮赵磊黄海涛崔宏春
Owner HANGZHOU ACAD OF AGRI SCI
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