Plant aphid resistance realization method based on 35S promoter

A promoter and plant technology, applied in the field of bioengineering, can solve problems such as pesticides easily polluting the environment, and achieve the effect of being conducive to popularization and low cost

Inactive Publication Date: 2018-03-06
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pesticides play an important role in resisting pests, however, pests are gradually develo...

Method used

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  • Plant aphid resistance realization method based on 35S promoter
  • Plant aphid resistance realization method based on 35S promoter
  • Plant aphid resistance realization method based on 35S promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Cloning of EβF Gene of Peppermint

[0021] 1. Extraction of total RNA from the peppermint genome: Take the peppermint leaf tissue, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and follow the instructions in the TIANGEN kit. Instructions for extracting total RNA. The quality of total RNA was identified by agarose gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0022] 2. Cloning of the peppermint EβF gene: using the extracted total RNA as a template, cDNA was synthesized under the action of PowerScript reverse transcriptase; gene-specific primers were designed according to the sequence of the EβF gene, and amplified from the total cDNA by PCR EβF gene, and sequenced.

[0023] Through the above steps, the full-length 1650bp of the peppermint EβF gene was obtained, its nucleotide sequence is shown in SEQ ID NO.1, and its protein coding sequence was deduced as shown ...

Embodiment 2

[0030] Construction of a plant overexpression vector containing the EβF gene: The partial sequence of the EβF gene was constructed on the transformed plant overexpression vector (pCAMBIA1305.1). In order to facilitate the construction of the expression vector, the forward primer introduced the NcoI enzyme cutting site point, the restriction site of BstEII was introduced in the reverse primer, and the primers are shown in Table 3;

[0031] Table 3 PCR primers constructed by pCAMBIA1305.1-EβF vector

[0032] Primer name

[0033] The plant overexpression vector pCAMBIA1305.1 after described transformation, all introduced the restriction site of XhoI in EPSPS gene forward primer and reverse primer in commercially available pCAMBIA1305 carrier, constructs the plant overexpression vector after the construction pCAMBIA1305.1.

Embodiment 3

[0035] Genetic transformation of wheat with EβF overexpression vector mediated by Agrobacterium tumefaciens to obtain transgenic wheat plants

[0036] 1. The acquisition of the Agrobacterium tumefaciens engineering bacterium of the plant overexpression vector containing the EβF gene: the plant overexpression vector containing EβF in Example 2 is transferred into Agrobacterium tumefaciens (such as EHA105, which is publicly available in the market) The biological material for sale can be purchased from CAMBIA Company in Australia, the strain number is Gambar 1), and PCR verification is carried out. The results showed that the plant overexpression vector containing EβF had been successfully constructed into the strain of Agrobacterium tumefaciens.

[0037] 2. Transformation of Fielder spring wheat with EβF gene mediated by Agrobacterium tumefaciens

[0038] 2.1. Pre-cultivation of immature embryos: immature seeds 13-14 days after flowering and pollination (immature embryo size 1...

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Abstract

The invention discloses a plant aphid resistance realization method based on a 35S promoter. The method comprises the following steps: cloning to obtain EbetaF synthetase genes EbetaFS from peppermintMenthaXpiperita, constructing an expression vector, transferring the vector into agrobacterium, infecting wheat embryos with the agrobacterium, catalyzing FPP to synthesize trans-beta-farnesene in wheat, synthesizing aphid alarm toxin trans-beta-farnesene in the wheat, and transporting to transgenic wheat to release the trans-beta-farnesene, thereby achieving the effect of dispelling aphids.

Description

technical field [0001] The invention relates to a technology in the field of bioengineering, in particular to a method for realizing plant aphid resistance based on a 35S promoter. Background technique [0002] Aphids are one of the main pests in agricultural production. The temperature rises during the wheat growth period in the main wheat planting areas of my country, and "warm winter" occurs, which leads to rampant aphid damage. Economic losses caused by aphids and the viruses they spread in wheat production every year Very serious. Since the main varieties of wheat in my country are not very resistant to aphids at present, how to effectively control aphids has become one of the major problems in wheat production. Pesticides play an important role in resisting pests, but pests are gradually developing resistance to pesticides. In addition, pesticides are easy to pollute the environment. Genetic modification has made a major contribution to helping crops resist pests with...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/82A01H5/00A01H4/00A01H6/46
CPCC12N9/88A01H4/00C12N15/8286C12Y402/03047
Inventor 唐克轩马嘉伟付雪晴张婷婷赵静雅谢利辉黎凌孙小芬
Owner SHANGHAI JIAO TONG UNIV
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