Method for detecting peanut metallothionein mRNA expression level by real-time quantitive PCR

Abstract

The invention discloses a method for a detecting peanut metallothionein mRNA expression level by real-time quantitive PCR, comprising the following steps: 1) connecting metallothionein gene obtained from peanut tissues with vectors, and constructing a recombinant plasmid as a standard substance for real-time quantitive PCR; 2) extracting the recombinant plasmid containing target genes, calculating a molecule copy number, diluting the recombined plasmid into different times to serve as a standard substance template for real-time quantitive PCR reaction; and 3) extracting the total RNA of required tissues, performing reverse transcription on the total RNA to be changed into cDNA serving as a real-time quantitive PCR template, carrying out real-time quantitive PCR to obtain the copy number of an initial template, and detecting and analyzing the mRNA expression level thereof.

Description

technical field [0001] The invention relates to a method for detecting the expression level of peanut metallothionein mRNA, in particular to a method for detecting the expression level of peanut metallothionein mRNA by real-time quantitative PCR. Background technique [0002] Metallothioneins (MTs) are a class of Cys-rich complexes that form non-toxic or low-toxic complexes through the chelation of -SH on Cys residues with heavy metal ions such as cadmium, and can also be induced by stress conditions such as heavy metals. Free radicals and ROS undergo oxidation-reduction reactions, thereby reducing oxidative damage. The expression behavior of plant metallothionein genes is diverse and complex, and its expression characteristics are different due to different species, different MT types, different tissues and organs, and different external conditions, and are closely related to the growth and development stages of plants. Real-time quantitative PCR technology refers to the a...

AI Technical Summary

Problems solved by technology

[0006] In summary, the disadvantages of the prior art are: high cost, low efficiency, time-consuming and similar problems

At present, the most conventional methods include Northern hybridization, ribonuclease protection assay, etc., but these methods have some disadvantages: Northern blot requires many reagents, high-quality RNA, cannot detect low-abundance mRNA, low sensitivity, and can measure the same It is clumsy when the samples have different mRNA abundances; the detection sensitivity of ribonuclease protection assay is higher than that of Northern hybridization, and it can detect mRNAs with different abundances in the same sample at the same time, but it needs to use antisense nucleic acid probes that are exactly complementary to the target mRNA, so Problems arise if the experiment produces RNA-RNA hybrids that are prone to cleavage by RNases

Images