Method for efficiently converting gene module and line for rice leaf sheath protoplast

Abstract

The invention discloses a method for efficiently converting a gene module and line for a rice leaf sheath protoplast, and relates to a conversion method of the rice leaf sheath protoplast. The methodcomprises the steps of adding an MMg solution to the rice leaf sheath protoplast to obtain a rice leaf sheath protoplast suspension; adding plasmid and a polyethylene glycol-calcium solution of whichthe percentage by mass is 40% to the rice leaf sheath protoplast suspension, performing uniform mixing, and performing standing; and adding a W5 solution, performing uniform mixing, performing centrifugation, removing supernatant, performing suspension once again and precipitation on the W5 solution, performing centrifugation, removing supernatant, adding carbenicillin, performing uniform mixing,and performing culturing to complete transformation of the protoplast. According to the conversion method of the rice leaf sheath protoplast, preparation, plasmid concentration, conversion time and the like of the PEG solution are adjusted, so that the conversion efficiency is improved to 80%, intelligent reformation devices can exert functions in 80% of plant cells, available data can be providedfor research on functions of the devices at the initial stage, and convenience is provided for research on functions of the intelligent reformation devices for crops at the later stage.

Description

technical field [0001] The invention relates to the field of plant cell biology technology, in particular to a method for efficiently transforming gene modules and circuits by rice leaf sheath protoplasts. Background technique [0002] Protoplast refers to the naked cell obtained by removing the cell wall from the plasmolysis of plant cells under enzymatic or mechanical action. Due to the loss of the barrier effect of the cell wall, it is easier to absorb exogenous genetic materials such as DNA, virus particles, organelles, and chromosomes. Under suitable conditions, protoplasts have the ability to regenerate into complete plants, and are ideal materials for transforming recipients, which not only greatly reduces the experimental period, but also helps to obtain real-time detection data for in vivo experiments. PEG-mediated transient transformation of plant protoplasts has played a huge role in modern cell biology and molecular biology, and has been widely used in the resea...

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Problems solved by technology

Compared with the dicot Arabidopsis, the preparation of protoplasts in monocot rice is more cumbersome due to its special structure. Although there have been a few reports on the application of rice protoplast transient expression system, the free transformation efficiency of protoplasts is limited. The application of this technology has a very low yield, and there is a lack of methods for efficient transformation of genes, gene modules, gene circuits and other vectors, so it has not been widely used in rice gene function research

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