Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites

Abstract

The invention provides an extration method of plant RNA by utilizing guanidinium isothiocyanate-chloroform extractive and Tris purification. The invention improves traditional guanidinium isothiocyanate method, adopts the two steps of chloroform extration after cracking and phenol-adding for purification, and respectively extract high-quality total RNA from the leaf blade and seed case of red-skinned pears, the leaf blade, fruit flesh and seed case tissues of grapes, the fruit of strawberries and the callus of arnebia euchroma. The extraction buffer liquid in the method contains guanidinium isothiocyanate, sodium dodecyl sarcosinate, sodium citrate, soluble polyvinylpyrrolidone and beta-mercaptoethanol, which can not only crack plant cells efficiently and release RNA, but also effectivelyinhibit the activity of RNA enzyme, and can also prevent the oxidation of phenolic compounds and suppress the interference of secondary metabolites. In the second step, Tris purification is utilized for crude extraction of RNA solution, which can eliminate the polysaccharides and protein which can coprecipitate with RNA. The invention is simple in operating steps, economic and practical, good in stability, and suitable for extracting high-quality total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites.

Description

technical field [0001] The invention relates to the technical field of plant molecular biology, in particular to a method for extracting and purifying total RNA from plant tissues rich in polysaccharides, polyphenols and secondary metabolites. Background technique [0002] The extraction of RNA is one of the basic contents of molecular biology. High-quality RNA is a necessary prerequisite for gene cloning, gene expression, and gene function analysis. For molecular biology research such as RT-PCR, Northern, real-time PCR, and cDNA library construction, total RNA with high purity and integrity must first be obtained. The complex and diverse components of plant tissues, especially higher plant tissues, make the extraction of RNA from plant tissues more difficult than other biological materials. For various plants of the genus Pyrus, Citrus, and Chinese herbal medicine, it is difficult to isolate and purify RNA in tissues, which seriously hinders the progress of their molecular...

AI Technical Summary

Problems solved by technology

[0005] Many plants, especially their fruits, are rich in secondary metabolites such as aromatic phenolic compounds, polysaccharide compounds, and flavonoids, making the extraction of nucleic acids very difficult

Especially for the extraction of total RNA, conventional methods are difficult to work

Although some studies have reported that acetone can remove some flavonoid pigments, and ethylene glycol butyl ether or high-concentration LiCl long-term precipitation can remove some polysaccharides, but these methods are not very effective in dealing with RNA extraction and purification obstacles.

Due to the many processing steps, the entire extraction process takes a long time, which will greatly increase the possibility of RNA being degraded

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