Method based on spinach green RNA visualization and application thereof

A spinach, gene gun technology, applied in the field of biology, can solve problems such as unsuccessful

Active Publication Date: 2021-01-29
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although fluorescence technology based on spinach green RNA aptamer has...

Method used

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  • Method based on spinach green RNA visualization and application thereof
  • Method based on spinach green RNA visualization and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Construction of lysine transfer RNA-spinach green RNA-lysine transfer RNA, referred to as KSK

[0022] Clone lysine transfer RNA and spinach green RNA, directly synthesize the KSK fragment containing Age I at the 5' end and Sma I restriction site sequence at the 3' end, and construct lysine transfer RNA-spinach green RNA-lysine transfer RNA. Lysine transfer RNA nucleotide sequence as shown in SEQ ID No.1, spinach green RNA nucleotide sequence as shown in SEQ ID No.2, lysine transfer RNA-spinach green RNA-lysine transfer RNA sequence As shown in SEQ ID No.3.

Embodiment 2

[0023] Embodiment 2: KSK transfers into vector

[0024] KSK was subcloned into pEAQ-HT vector to obtain pEAQ-HT / KSK, the nucleotide sequence of which is shown in SEQ ID No.4.

[0025] Using the PVX / KK vector containing the KK sequence as a template, use the following primers to obtain the PCR-amplified KK product, recover the PCR product and digest it with Nru I and Xho I, and connect it to the linearized DNA after digestion with the same two enzymes pEAQ-HT empty vector was obtained.

[0026] (pEAQ-HT / K-K For 5'-ATA TCGCGA CAATCACAGTGTTGGCTTGCAAAC-3' underlined is the NruI restriction site; pEAQ-HT / K-K Rev5'-ATA CTCGAG TTGACCCTATGGGCTGTGTTG-3' is underlined for XhoI digestion recognition site) The pEAQ-HT / KSK vector is directly synthesized KSK fragment containing Age I at the 5' end and Sma I restriction site sequence at the 3' end. The linearized pEAQ-HT empty vector (pEAQ-HT plasmid recovered after double digestion with Age I and Sma I) was obtained.

Embodiment 3

[0027] Embodiment 3: Onion epidermis observes sufficiently strong green fluorescence

[0028] Use the particle gun method to attack pEAQ-HT / KSK into onion epidermal cells without chloroplasts, place the onion epidermis on a hypertonic medium, and the hypertonic medium includes: 1 / 2 MS medium with 0.8% plant gel, pH =5.8, 46.67g / L contains sorbitol, 46.67g / L mannitol. After 12 hours of bombardment, the onion epidermis was soaked in 100 µM DFHBI solution for 30 min, and then strong enough green fluorescence was observed under confocal laser microscopy.

[0029]The specific method of the gene gun method is as follows: the pEAQ-HT / KSK plasmid was extracted using the QIAprep Spin Miniprep Kit kit from Qiagen Company, concentrated and then adjusted to a final concentration of 1 µg / µl. Prepare the gold powder that wraps the pEAQ-HT / KSK plasmid by weighing 1.5 mg of gold powder with a diameter of 1 µm, washing once with 70% ethanol, washing twice with 100% ethanol, and centrifuging q...

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Abstract

The invention discloses a method based on spinach green RNA visualization and application thereof, and belongs to the technical field of biology. The invention comprises the application of plant tRNAin protecting and/or stabilizing the interaction between a spinach green RNA aptamer and a fluorescein molecule DFHBI and realizing the visual application of the spinach green RNA in the plant body. The method comprises the following steps: constructing lysine transport RNA-spinach green RNA-lysine transport RNA, subcloning a product into a pEAQHT vector to obtain pEAQHT/KSK, attacking the pEAQHT/KSK into chloroplast-free onion epidermis cells by using a gene gun method, putting onion epidermis on a hypertonic culture medium, infiltrating the onion epidermis into a DFHBI solution, and observing strong enough green fluorescence under a laser confocal microscope. According to the invention, the visualization of RNA in living cells of plants is realized by utilizing methods such as molecularsignal marking, RNA binding marker protein, the RNA-based aptamer and the like.

Description

technical field [0001] The invention belongs to the technical field of biology, and in particular relates to a spinach green RNA visualization method and its application. Background technique [0002] RNA primarily acts as a messenger to transfer genetic information from DNA to proteins. However, RNA has a much wider range of functions. Accumulating evidence confirms that RNA can regulate gene expression at transcriptional, post-transcriptional and translational levels. In plants, cellular mRNAs, small interfering RNAs (siRNAs), microRNAs, and pathogenic viral and viroid RNAs can move between cells via plasmodesmata and spread to distant tissues via phloem highways. Some of these mobile RNAs act as intracellular and intercellular as well as systemic signals to control plant defense, growth and development. For example, the mobile gibberellin-insensitive gene ( GAI ) mRNA can regulate leaf morphology in Arabidopsis, tomato and squash. CmNACP The systematic movement affe...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/11G01N21/64
CPCC12N15/8207C12N15/11G01N21/6402G01N21/6458
Inventor 洪益国俞志明
Owner HANGZHOU NORMAL UNIVERSITY
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