Preparation method and application of paddy rice green protoplast

Abstract

According to the invention, the culture time of paddy rice is shortened by adjusting the growing conditions of paddy rice seedlings; a paddy rice green protoplast is prepared by an enzyme method; the preparation method is optimized, so vacuum pumping and the usage of a highly toxic reagent are avoided; and thus the preparation method of the paddy rice green protoplast is simpler and safer. The protoplast prepared by the method of the invention has a high cell survival rate, and a high transformation rate, is applicable to various analysis such as subcellular localization of target gene codingprotein, protein interaction, western blotting and the like, and is especially applicable to instantaneous expression research of light / chloroplast related gene.

Description

technical field [0001] The invention relates to a method for preparing protoplasts, in particular to a method for preparing rice green protoplasts. The invention also relates to the application of the prepared rice green protoplast in the study of transient expression of light / chloroplast related genes. Background technique [0002] Rice is one of the important food crops and model organisms. With the deciphering of the genome sequence, it is of great significance to identify the function of its genes. The method of transient gene expression in plants is widely used in gene function identification and high-throughput analysis of gene function due to its rapidity and high efficiency. Using the protoplast transient gene expression system, the function of rice genes can be quickly identified, and experimental systems can be established for cytology, physiology and genetics. Therefore, the efficient and rapid preparation of rice protoplasts is the basis and key to carry out the...

AI Technical Summary

Problems solved by technology

[0004] However, the cells of embryogenic cells, suspension cell lines and etiolated seedlings are all in an abnormal state, without forming normal chloroplasts, and are not a cell system in a normal physiological state. Therefore not suitable for many studies, especially those related to chloroplasts and photosynthesis

Based on this, the researchers explored using rice green seedlings to prepare green protoplasts, Bart R, Chern M, Park C-J, Bartley L, Ronald P: A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2006, 2(1):13 and Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL: A highly efficient transient protoplast system for analyzing defense gene expression and protein-protein interactions in rice. Mol Plant Pathol 2006, 7(5):417-427 Two articles using 2-week-old and 2-month-old rice plants respectively, and using vacuum in the method , adding antibiotics, using highly toxic mercaptoethanol and other steps to isolate green protoplasts, but the transformation efficiency of the isolated green protoplasts is very low, not as high as that of protoplasts from etiolated seedlings

Moreover, only the application of plant resistance gene expression analysis and siRNA-mediated gene silencing research

[0005]The rice green protoplasts prepared by the prior art generally have a low transfection rate, and it is difficult to meet the needs of transient gene expression

Such as Bart R, Chern M, Park C-J, Bartley L, Ronald P: A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2006, 2(1):13 and Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL: A highly efficient transient protoplast system for analyzing defense gene expression and protein-protein interactions in rice. Mol Plant Pathol 2006, 7(5): 417-427 pointed out that the transformation efficiency of the isolated rice green protoplast is lower than that of the etiolated seedling protoplast, and the highest transformation efficiency of the reported rice yellow seedling protoplast can only reach 70%. %

At the same time, the rice green protoplasts prepared by the existing method have high transformation efficiency for small plasmids, but the transformation efficiency for plasmids larger than 12 kb is very low, only reaching 25% to 30%, which also limits the transformation efficiency of large fragment genes. Research

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