Application of arabidopsis thaliana Sec14p-like gene in plant cell lipid droplet fluorescent marker

A plant cell, Arabidopsis technology, applied in applications, plant peptides, plant products, etc., can solve the problems of high probe price, cytotoxicity, high consumption, etc., and achieve the effect of wide application prospects.

Active Publication Date: 2018-09-14
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from different aspects, these probes still have some disadvantages. For example, some probes have a certain excitation wavelength and emission wavelength, which will limit their use when the research sample will emit autofluorescence under similar excitation wavelengths; Or the prepared solution has certain cytotoxicity, so it cannot be observed by staining for a long time
Some probes are expensive and cost a lot, just a few milligrams cost thousands of dollars
These issues hinder the development and conduct of lipid droplet research

Method used

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  • Application of arabidopsis thaliana Sec14p-like gene in plant cell lipid droplet fluorescent marker
  • Application of arabidopsis thaliana Sec14p-like gene in plant cell lipid droplet fluorescent marker
  • Application of arabidopsis thaliana Sec14p-like gene in plant cell lipid droplet fluorescent marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cloning and vector construction of Sec14p-like gene

[0039] (1) PCR amplification of Sec14p-like gene

[0040] The vector was constructed in GATEWAY mode, and primers were designed according to the published Arabidopsis thaliana gene AT1G14820 sequence. The size of the amplified product was 817bp, of which the gene coding region was 756bp, and the stop codon at the end of the coding region for the expression of the fusion protein had been removed.

[0041] Sec14p-like-F: ggggacaagtttgtacaaaaaagcaggcttcATGGAGGAAAGCCAAG

[0042] Sec14p-like-R:ggggaccactttgtacaagaaagctgggtcAACATTATTGTTTGTTAGAG

[0043] Arabidopsis RNA was extracted, reversed into cDNA, and PCR amplified using the above primers. The base sequences of the primer pair are respectively shown in SEQ ID NO.3 and SEQ ID NO.4.

[0044] (2) Recovery of target fragments and construction of intermediate vectors

[0045] The target DNA fragment was recovered by agarose gel electrophoresis ( figure 1 ...

Embodiment 2

[0051] Example 2: Recombinant vectors pEarleygate101-Sec14p-like-YFP and pEarleygate103- Tobacco transient transformation and lipid droplet labeling analysis of Sec14p-like-GFP

[0052] (1) Transform the recombinant vector into Agrobacterium

[0053] The recombinant vector pEarleygate101-Sec14p-like-YFP was transformed into Agrobacterium EHA105 by freeze-thaw method, and the single clone of Agrobacterium on the plate was picked and inoculated into 1ml liquid resistant LB medium (streptomycin+rifampicin+ kanamycin) and cultured in a shaker at 28°C for 24 hours.

[0054] (2) Transiently transformed tobacco

[0055] The bacterial solution was transferred to 10ml liquid medium (containing 10mM MES and 40uM AS), and cultured in a shaker at 28°C for 16 hours. Centrifuge the bacterial liquid to collect the bacterial cells, and use 10mM MgCl 2 Resuspend bacteria in solution to OD 600 =1, finally add AS to a final concentration of 200uM and let stand for 3 hours. Get the three...

Embodiment 3

[0060] Example 3: The recombinant vector pEarleygate101-Sec14p-like-YFP stably transforms Arabidopsis thaliana and lipid droplet markers note analysis

[0061] (1) Arabidopsis planting and transformation

[0062] Sow Arabidopsis Col-0 seeds in the culture soil, transplant the seedlings into large pots after seven days, and manage and cultivate them normally in the greenhouse with a temperature of 20-22°C, a relative humidity of 50-70%, and a light intensity of 100-120 μmol / (m 2 .s), 16h light and 8h dark.

[0063] Take the Agrobacterium EHA105 transformed with the recombinant vector pEarleygate101-Sec14p-like-YFP in Example 2, inoculate it into liquid LB medium, and culture it overnight at 28° C. and 200 rpm. The bacteria liquid was collected by centrifugation at 4000rpm for 10min. Resuspend Agrobacterium with transformation medium to OD 600 Approximately equal to 1.0. Pour the bacterial solution into a petri dish to prepare for flower dipping infection. Immerse all A...

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Abstract

The invention provides application of an arabidopsis thaliana Sec14p-like gene in a plant cell lipid droplet fluorescent marker. According to the application, the arabidopsis thaliana Sec14p-like geneis used as specificity position lipid droplets of the plant cell lipid droplet marker in plant cells. The invention further provides application of the gene in fluorescent detection of the lipid droplets in the plant cells. The application comprises the following specific steps: connecting encoding protein of the gene with fluorescent protein to obtain fused protein; transforming the fused protein into a target plant to mark the plant lipid droplets. Compared with other lipid droplet fluorescent markers with similar functions, the Sec14p-like gene provided by the invention can be connected with different fluorescent protein genes according to specific requirements to be transformed into the plant, and the fused protein with different fluorescent protein is expressed, so that interferenceof plant spontaneous fluorescence is effectively avoided; the Sec14p-like gene can be stably transformed into the plant and the plant lipid droplets are stably marked for a long period; self growth and development of the plant are not influenced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the application of Arabidopsis thaliana Sec14p-like gene in the fluorescent labeling of plant cell lipid droplets, more specifically, to the application of Arabidopsis thaliana Sec14p-like gene and its encoded protein in plant cells Applications in Fluorescent Labeling of Lipid Droplets. Background technique [0002] Cells are the main organizational units of life systems, composed of various organelles, such as: cell wall, cell membrane, nucleus, chloroplast, Golgi apparatus, mitochondria, endoplasmic reticulum, ribosomes, vacuoles, etc. Organelles play an important role in enzyme, cell and many tissue activities, such as cell proliferation, apoptosis, drug resistance, ion transport, muscle contraction and so on. Monitoring the morphology of organelles is of great significance for the study of the behavior of living cells. Lipid droplets are the main storage place fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/70C12N15/82A01H5/00A01H6/20A01H6/82G01N21/64
CPCC07K14/415C12N15/70C12N15/8242G01N21/6486
Inventor 左开井王俊吕萌荔
Owner SHANGHAI JIAO TONG UNIV
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