Method for detecting ath-miRNA170-3p targeted MSH2 by using tobacco dual luciferase report system

A dual-luciferase and reporting system technology, applied in fluorescence/phosphorescence, measuring devices, and material analysis through optical means, can solve problems such as time-consuming, heavy workload, and high false positive rate

Pending Publication Date: 2020-11-17
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

Although these assays work well, validating the function of miRNAs involves analyzing molecular and morphological phenotypes in stable transgenic/mutant lines, so they have the disadvantage of being very time-consuming and labor-intensive; in some cases , the disadvantage of post-translational regulation target site technology is that the false positive rate is too high; considering that many plant miRNAs regulate protein expression levels significantly greater than mRNAs, ...

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  • Method for detecting ath-miRNA170-3p targeted MSH2 by using tobacco dual luciferase report system
  • Method for detecting ath-miRNA170-3p targeted MSH2 by using tobacco dual luciferase report system
  • Method for detecting ath-miRNA170-3p targeted MSH2 by using tobacco dual luciferase report system

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Embodiment Construction

[0046] 1 Materials and methods

[0047] 1.1 Main Instruments

[0048]

[0049] 1.2 Main reagents and kits

[0050]

[0051] 1.3 Main Consumables

[0052]

[0053] 2. Construction of Arabidopsis ath-miRNA170-3p overexpression GUS vector

[0054] 2.1 Extraction of Arabidopsis genomic DNA

[0055] Genomic DNA of Arabidopsis thaliana was extracted using Plant Genomic DNA Kit (Plant Genomic DNA Kit, CW0553S), and the specific steps were as follows:

[0056] (1) Take about 100 mg of fresh leaves of Arabidopsis thaliana, add liquid nitrogen and grind them thoroughly.

[0057] (2) Collect the ground powder into a centrifuge tube (prepared by yourself), add 700 μl 65°C preheated Buffer GP1, quickly invert and mix well, then place the centrifuge tube in a 65°C water bath for 20 minutes, and centrifuge upside down during the water bath Mix the sample several times.

[0058] (3) Add 4 μl of RNase A solution (CW0601S) with a concentration of 100 mg / ml (CW0601S), shake and mi...

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Abstract

The invention relates to the technical field of biology, in particular to a verification method for detecting ath-miRNA170-3p targeted MSH2 by using a tobacco dual luciferase report system. The tobacco dual luciferase reporting system comprises fluorescein luciferase (F-Luc) and renilla luciferase (R-LuC). The R-Luc is used as an internal reference, and restriction enzyme cutting sites AvrII and AgeI are embedded into a 3 'UTR region of the FLuc. The method disclosed by the invention is beneficial to clarification of an arabidopsis thaliana MSH2 gene expression mechanism regulated and controlled by the athmiRNA170-3p and screening of the biomarker, and has important significance on research on response to adversity stress of the arabidopsis thaliana MSH2 gene regulated and controlled by the ath-miRNA170-3p.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a verification method for detecting ath-miRNA170-3p targeting MSH2 by using a tobacco dual-luciferase reporter system. Background technique [0002] Small RNAs (microRNAs, miRNAs) are about 22 nucleotides (nt), highly conserved endogenous non-coding single-stranded small RNAs. Its expression has obvious tissue and time specificity, and can be complementary or partially complementary to the specific sequence of the 3' untranslated region (3'UTR) of the target gene mRNA, inducing the shearing of the target gene mRNA or inhibiting its translation, thus in The post-transcriptional level regulates the growth, development and response of organisms to stress (Crosby et al., 2009; Noman et al., 2017; Sarver et al., 2009; Orangi et al., 2019). Mature miRNAs can simultaneously block the translation of multiple target genes, and interfering with miRNAs can change the expression levels of multi...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 赵强王鹤潼张延召刘宛谢甫绨贾春云巩宗强台培东
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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