Protein associated with sesquiterpene synthesis and encoding gene and application thereof

A technology for encoding genes and proteins, applied in the field of proteins and their encoding genes and applications, can solve the problems of wheat fps gene isolation and cloning, expression characteristics, enzyme activity characteristics expression and anti-aphid function research, etc.

Active Publication Date: 2013-10-23
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on the isolation and cloning, expression characteristics, enzyme activity characteristics, expression in plants (Arabidopsis thaliana) and aphid resistance function of wheat fps gene

Method used

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  • Protein associated with sesquiterpene synthesis and encoding gene and application thereof
  • Protein associated with sesquiterpene synthesis and encoding gene and application thereof
  • Protein associated with sesquiterpene synthesis and encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the acquisition of common wheat Tafps-B1 cDNA

[0051] 1. Obtaining of common wheat Tafps-B1 cDNA

[0052] 1. Extraction of common wheat total RNA and synthesis of cDNA

[0053] The total RNA of common wheat was extracted according to the instruction manual in the RNA simple total RNA extraction kit of Tiangen Biochemical Technology Co., Ltd. Agarose gel electrophoresis detection and concentration determination by NanoDrop.

[0054] The first strand of cDNA was synthesized using the TransScript First-Strand cDNA Synthesis SuperMix kit from Beijing Quanshijin Biotechnology Co., Ltd. The method refers to the instruction manual in the kit. The specific operation is as follows:

[0055] First-strand cDNA synthesis

[0056] 1) join

[0057]

[0058] 2) Mix gently and incubate at 42°C for 30min.

[0059] 3) Inactivate TransScript RT Enzyme by heating at 85°C for 5 minutes. The first strand of cDNA synthesized by the reaction was used as a template for...

Embodiment 2

[0073] Embodiment 2, the functional identification of Tafps-B1

[0074] 1. Application of Tafps-B1 in the synthesis of sesquiterpene compound farnesol

[0075] 1. Carrier Construction

[0076]Using T-Tafps-B1 sequencing vector as template, high-fidelity enzyme TranStart FastPfu DNA Polymerase and primer efpps1F (5′-ATGGCGGCGGCGGCGGTGGCGTTG-3′) / efps1R (5′- CTATTTCTGCCTCTT-3', the part of the primer in bold italics is the 3'UTR sequence), and a 1074bp target fragment with an A-terminal (with the nucleotide sequence 1 in the sequence listing) was obtained. The above-mentioned target fragment with the A-terminus was ligated with the vector pEASY-E1 according to the pEASY-E1 Expression Kit manual of Quanshi Jin Biological Co., Ltd., and the obtained ligation product was transformed into common Escherichia coli and spread on LB containing Amp (50mg / ml) Plate overnight.

[0077] Single clones were picked and detected by T7F (5′-TAATACGACTCACTATA-3′) and the downstream primer efps...

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Abstract

The invention discloses a protein associated with sesquiterpene synthesis and an encoding gene and an application thereof. The protein is a protein represented by the following 1) or 2): 1) a protein composed of an amino acid sequence represented by a sequence 2 in a sequence table; 2) a protein derived from 1), wherein the amino acid sequence represented by the sequence 2 in the sequence table is subjected to the replacement and / or deletion and / or addition of one or several amino acid residues and the protein is associated with sesquiterpene synthesis and / or aphid repelling. The experiment proves that a nucleotide sequence of Tafps-B1c deoxyribonucleic acid (DNA) of common wheat and a Tafps-B1 encoding protein sequence are obtained; the Tafps-B1 protein expressed in escherichia coli has the farnesyl pyrophosphate (FPP) synthesis activity; the wheatTafps-B1gene transformed arabidopsis is obtained by using an agrobacterium-mediated transformation method, and results show that Tafps-B1 transformed plants can obviously repel aphid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein related to the synthesis of sesquiterpene, its coding gene and its application. Background technique [0002] Peach aphid is a wide-feeding pest with about 350 kinds of hosts. It mainly harms tobacco, peach, plum, plum, pear and other fruit trees and vegetables such as cabbage, radish, pepper and spinach, causing great economic losses. In recent years, due to factors such as global warming and changes in farming systems, the reproductive ability and adaptability of aphids have been significantly enhanced, and their harm has become increasingly serious. At present, aphid control is mainly based on spraying pesticides, but a large amount of pesticides are not only harmful to humans and animals, but also cause serious environmental pollution. Breeding aphid-resistant wheat and vegetable varieties is the most effective way to prevent aphid damage. However, due to the lack of e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N5/10C12N1/21C12P5/00C12P7/04A01N31/02A01N27/00A01P17/00C12N15/82A01H5/00C12R1/19
Inventor 夏兰琴张彦马有志
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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