Application of arabidopsis transcription factor gene PIF3 in insect stress resistance of plants

A dicotyledonous plant and transgenic cell line technology, applied in the field of genetic engineering, can solve problems affecting plant resistance to biological stress, achieve good potential application value, and improve the effect of plant insect resistance

Active Publication Date: 2019-11-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on PIFs focuses on the regulation of plant light signaling pathways, and there is no report on the impact of plant resistance to biotic stress.

Method used

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  • Application of arabidopsis transcription factor gene PIF3 in insect stress resistance of plants
  • Application of arabidopsis transcription factor gene PIF3 in insect stress resistance of plants
  • Application of arabidopsis transcription factor gene PIF3 in insect stress resistance of plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The cloning of embodiment 1, PIF3 gene and the construction of expressing PIF3 gene vector

[0060] 1. Cloning of PIF3 gene

[0061] The nucleotide sequence of the cDNA of the PIF3 gene is sequence 1, and the amino acid sequence of the encoded PIF3 protein is sequence 2.

[0062] The CDS (Sequence coding for aminoacids in protein) protein coding region DNA sequence 1 of the PIF3 gene was artificially synthesized, and the upstream primer F and downstream primer R were designed according to the sequence analysis of the coding region.

[0063] Upstream primer F: 5'-CAAGGGTACCATGCCTCTGTTTGAGCTTTT-3' (SEQ ID NO: 3)

[0064] Downstream primer R: 5'-CAAGCTCGAGGACGATCCACAAAACTGATC-3' (SEQ ID NO: 4)

[0065] Using the DNA molecule shown in Sequence 1 as a template, PCR amplification was performed with upstream primer F and downstream primer R to obtain a PCR amplification product of 1575 bp.

[0066] Second, the construction of the vector expressing PIF3

[0067] The vector ...

Embodiment 2

[0069] Embodiment 2, the application of PIF3 gene in regulating plant insect resistance

[0070] 1. Construction of transgenic PIF3 plants expressing PIF3 gene

[0071] The PIF3-expressing vector 35S:PIF3-YF prepared in Example 1 was transformed into the Agrobacterium EHA105 strain to obtain a recombinant Agrobacterium expressing PIF3. Then, the recombinant bacteria were transformed into wild-type Arabidopsis col-0 plants to obtain PIF3-transformed Arabidopsis seedlings.

[0072] The above transformation method is specifically: inoculate the recombinant Agrobacterium expressing PIF3 in 5 mL of LB liquid medium containing antibiotics (rifampicin 50 μg / m L, spectinomycin 50 μg / m L), and cultivate overnight at 28° C. at 220 rpm; 5 mL of LB liquid medium containing recombinant Agrobacterium expressing PIF3 was transferred to 250 mL of liquid medium, and cultured at 28°C and 220 rpm for 16 hours; the bacterial liquid was collected in a 250 mL centrifuge bucket, and centrifuged at ...

Embodiment 3

[0090] Embodiment 3, PIF3 gene regulates plant insect resistance

[0091]1. Construction of pifQ mutants (pif1, pif3, pif4, pif5)

[0092] The purchased pif1, pif3, pif4, pif5 mutant Arabidopsis (these mutants are only pif1, pif3, pif4 or pif5 gene inactivation compared with the wild type.) sow, and select the Arabidopsis that has just flowered, bolted and grown well. Mustard plants, pif1 and pif5 were crossed, and pif3 and pif4 were crossed. Pick the unflowered buds of the female plant and carefully remove the stamens, petals and sepals completely with pointed tweezers, leaving the stigma. Get the stamens of the male parent flowers that have just bloomed and smear the stigma of the female parent repeatedly, and get final product when you see that the small hole above the stigma is filled with anthers. After 2-3 days, it is observed that the stigma elongates and becomes thicker, which means that the hybridization is successful. If the change is not too large or slowly wither...

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Abstract

The invention discloses an application of an arabidopsis transcription factor gene PIF3 in insect stress resistance of plants. The invention provides an application of any one of the following substances 1) to 3) in regulating and controlling insect resistance of plants: 1) protein PIF3; 2) a DNA molecule for encoding the protein PIF3; and 3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the DNA molecule for encoding the protein PIF3. Experiments prove that after the PIF3 gene disclosed by the invention is over-expressed, arabidopsisshows an anti-bemisia tabaci phenotype. In order to conveniently identify and screen transgenic arabidopsis plants, the used vector can be processed, for example, a selective marker or an antibioticmarker with resistance is added to plants. The PIF3 gene provided by the invention provides a gene resource for culturing new varieties of insect-resistant plants, and has a relatively good potentialapplication value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the application of Arabidopsis thaliana transcription factor gene PIF3 in plant resistance to insect stress. Background technique [0002] Agricultural insect pests are one of the main agricultural disasters in my country. It has the characteristics of many types, great impact, and frequent outbreaks. The scope and severity of its occurrence and severity often cause heavy losses to my country's national economy, especially agricultural production. , also by carrying pathogens or parasites, etc., causing disease epidemics and outbreaks, the double harm of diseases and insect pests has seriously affected the national economy and people's livelihood. Common agricultural pests in my country include whitefly, rice planthopper, corn borer, cotton bollworm, cotton aphid, wheat aphid, wheat red spider, locust, etc. In recent years, the frequent outbreaks of various crop dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29A01H5/00A01H6/20
CPCC07K14/415C12N15/8286
Inventor 叶健龚雨晴赵平芝
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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