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

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...

AI Technical Summary

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, in vitro slicing technology only detects mRNA cleavage at target sites, the disadvantage of this technology is false positive rate. The negative rate is too high; the disadvantage of high-throughput sequencing methods is that they can only resolve specific mRNA sequences (Liu and Axtell, 2015)

There is no report on the use of a dual luciferase reporter system infecting tobacco leaves with Agrobacterium to verify whether Arabidopsis ath-miRNA170-3p targets the Arabidopsis MSH2 gene

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