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Cloning and application of tobacco neonicotinoid synthesis regulation gene NtERF91

A technology for regulating genes and neonicotinoids, applied in the field of genetic engineering

Pending Publication Date: 2020-01-10
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, as an alkaloid that accumulates less in tobacco alkaloids, the transcriptional regulator genes that regulate its metabolism and accumulation have not been reported yet.

Method used

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  • Cloning and application of tobacco neonicotinoid synthesis regulation gene NtERF91
  • Cloning and application of tobacco neonicotinoid synthesis regulation gene NtERF91
  • Cloning and application of tobacco neonicotinoid synthesis regulation gene NtERF91

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Experimental program
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Embodiment 1

[0060] Embodiment 1—the isolation and cloning of NtERF91 gene, comprises the following steps:

[0061] 1. Determine the NtERF91 gene sequence:

[0062] By analyzing the transcriptome data of tobacco root tissues treated with jasmonin, one ERF family gene positively regulated by jasmonin was obtained, and sequence alignment revealed that the gene was NtERF91. Design gene cloning primers according to this gene sequence and carry out PCR reaction, obtain target product; Forward primer NtERF91-BamH I:

[0063] GGATCC ATGTTAACTAGTGTCAATACCACGT;

[0064] Reverse primer NtERF91-Xho I: CTCGAG TCAATTTTCAGCCAATTTTCTCTTC; in order to construct the overexpression vector, the primer restriction sites (underlined) BamH I and Xho I in the above primers;

[0065] 2. Use the Trizol kit (Invitrogen) to extract RNA from the roots of tobacco variety Coker176, and operate according to the instructions provided by the kit.

[0066] 3. Using the first-strand cDNA obtained by reverse transcript...

Embodiment 2

[0071] Overexpression vector construction

[0072] 1. The cloned NtERF91 was connected to the TOPO vector

[0073] (1) Using the cDNA of the root of the tobacco variety Coker176 as a template, using NtERF91 gene-specific primers to amplify, obtain a gene fragment with a size of about 0.5 kb, purify and recover;

[0074] (2) TOPO clone the recovered NtERF91 gene fragment, connect to -BluntⅡ-TOPO (3.5kb) vector was used to transform Escherichia coli DH5α competent cells, the plasmid was extracted for PCR detection, and the plasmid with amplified product size of about 0.5kb was selected to extract DNA, and the constructed vector was named pTOPO-NtERF91;

[0075] (3) Since the forward and reverse primers of the gene have recognition sites of BamH I and Xho I respectively, these two enzymes were selected for double-enzyme digestion detection on the plasmid DNA sample, and two fragments were produced as a result of the enzyme digestion, with a size of 3.5 kb and 0.5kb, indicating...

Embodiment 3

[0084] genetic transformation of tobacco

[0085] (1) Transformation of expression vector into Agrobacterium

[0086] Take out the Agrobacterium competent cells from the -80°C refrigerator, place them on ice to dissolve, and add 4 μL of the recombinant expression vector pK2GW7-NtERF91; quick-freeze in liquid nitrogen for 1 minute, transfer to a 37°C water bath for 5 minutes, and then ice-bath for 2 minutes, add to the mixture Add 1mL LB liquid medium, culture at 28°C and 220rpm for 3-4 hours; spread the culture on LB solid medium containing spectinomycin 100mg / L and rifampicin 25mg / L, and incubate at 28°C for 2-3 hours Days, Agrobacterium clones containing the target vector can be seen;

[0087] (2) Tobacco Transformation

[0088] a. Pick the Agrobacterium clone containing the target vector, streak it on an LB plate containing spectinomycin and rifampicin, and culture it at 28°C for 2-3 days; scrape the streaked plaque and inoculate it on a plate containing spectinomycin and...

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Abstract

The invention discloses a tobacco neonicotinoid synthesis regulation gene NtERF91 and a cloning method and application thereof. The nucleotide sequence of the nicotine synthesis regulation gene NtERF91 is shown as SEQ ID: No.1, and the coded amino acid sequence is shown as SEQ ID: No.2. The invention further discloses a cloning method of the neonicotinoid synthesis regulation gene NtERF91. The cloning method comprises the following specific steps: A, determining the sequence of the NtERF91 gene; B, extracting tobacco RNA, and performing reverse transcription to obtain a first chain cDNA; C, designing and synthesizing a specific primer according to the NtERF91 gene sequence, and performing PCR amplification by taking the cDNA as a template; D, recovering and purifying a PCR product; and E,constructing an overexpression vector containing the NtERF91 gene. The overexpression vector is overexpressed in tobacco through agrobacterium-mediated transformation, and a transgenic plant is prepared. The neonicotinoid content of the obtained transgenic plant is more than 4 times that of a control group. The result shows that the tobacco NtERF91 gene has a great application prospect in the aspect of cultivating tobacco with high neonicotinoid content.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a cloning method and application of a tobacco annicotine synthesis regulating gene NtERF91. Background technique [0002] Anonicotinoid is a kind of alkaloid in Solanaceae plants (such as tobacco, tomato). In tobacco, the proportion of anonicotinoids in total tobacco alkaloids is low. Tobacco alkaloids account for 2%-4% of the total dry weight of tobacco. Among the four alkaloids in tobacco, nicotine accounts for about 95% of the total alkaloids, and neonicotinoid, nornicotine, and pseudobasine only account for the remaining 5%. In nature, these tobacco alkaloids are biologically active and act as natural toxins in the tobacco defense system against predation by insects or herbivores. [0003] Neonicotinoids are often used in animal models and cell lines to study whether they can treat nicotine addiction, Alzheimer's disease, thyroiditis, multiple scler...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8243
Inventor 隋学艺高玉龙王丙武宋中邦李文正李梅云赵璐袁诚张洪博师君丽李永平孔光辉曾建敏邹聪明刘勇黄昌军吴兴富
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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