Method and application of complete acquisition of ribosome nascent peptide chain complex in plant tissue

Abstract

The invention discloses a method of completely acquiring ribosomal nascent peptide chain compound from plant tissues and application of the method. The method includes the steps of washing fresh plant tissues with water, quickly freezing the fresh plant tissues to -80 DEG C with liquid nitrogen, and extracting RNC (ribosome nascent-chain complex); or storing the plant tissues, quickly frozen by the liquid nitrogen, at the temperature not higher than -80 DEG C, and after stable storage and transport at such temperature, and extracting the RNC. The step of extracting the RNC includes adding the liquid nitrogen to grind the plant tissues to obtain powder; adding pre-cooled cell lysis buffer into the powder in the liquid nitrogen environment, and performing grinding and mixing; placing mixture on ice, and allowing lytic reaction after the ice thaws; performing centrifuging, and extracting the RNC form supernate. The method has the advantages that the RNC in the plant tissue sample can be conveniently and effectively acquired, the requirement of the RNC sample for long-term storage and transport is met, and analysis on the genetic functions is facilitated.

Description

technical field [0001] The invention belongs to the technical field of life sciences, and in particular relates to a method and application for completely obtaining ribosome nascent peptide chain complexes in plant tissues. Background technique [0002] At present, most of the investigations on the gene expression level of plants stay at the transcription level, that is, the detection of gene expression is to detect the total RNA directly obtained by crushing the cells, and then extracting the total RNA. The amount of mRNA in the total RNA is used as the expression level of the gene. However, gene expression cannot be equal to protein expression, which has always been an unresolved problem in the plant world. [0003] In recent years, it has been found that translation regulation is an important regulatory level in life. When the mRNA is translated, the ribosome binds to the mRNA chain and moves, and gradually synthesizes the protein polypeptide chain (nascent-chain) accord...

AI Technical Summary

Problems solved by technology

However, for plant tissues, since the cell walls containing cellulose and polysaccharides can block the entry of various foreign substances, and the cell walls are tightly connected with few gaps, it is difficult for translation elongation inhibitors to penetrate into the inner cells of the tissue in a short time. Immobilize ribosomes; for animal cells, detergents such as Triton X-100 can be used to quickly and gently break the cell membrane to release the cell contents, while plant tissues have hard cell walls, and the cell walls between cells are thick and tightly connected, only Triton X Detergents such as -100 do not break down the strong plant cell walls, making extraction of RNC from plants difficult

Therefore, the RNC extraction process of plant tissue samples cannot follow the strategy of extracting RNC from animal cells. How to quickly combine translation elongation inhibitors with ribosomes and obtain intact RNC from plant tissue samples is a huge challenge for laboratories and commercial services.

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