Method for efficiently converting gene module and line for rice leaf sheath protoplast
A protoplast and leaf sheath technology, applied in biochemical equipment and methods, genetic engineering, introduction of foreign genetic material using vectors, etc., can solve the problems of limitation, cumbersome preparation of protoplasts, low yield, etc., and achieve the effect of a reliable technology platform
A protoplast and leaf sheath technology, applied in biochemical equipment and methods, genetic engineering, introduction of foreign genetic material using vectors, etc., can solve the problems of limitation, cumbersome preparation of protoplasts, low yield, etc., and achieve the effect of a reliable technology platform
CN110656126AInactive Publication Date: 2020-01-07THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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Embodiment 1
[0034] The preparation and transformation method of rice leaf sheath protoplast of embodiment 1
[0035] 1. Solution configuration
[0036] 1.1 Configuration of enzymatic hydrolysis solution
[0037] The enzymatic solution is composed of cellulase RS, isolated enzyme R10, mannitol, CaCl 2 , BSA, β-mercaptoethanol, carbenicillin and ddH 2 O composition; wherein, the content of the cellulase RS in the enzymolysis solution is 15g / L; the content of the isolated enzyme R10 in the enzymolysis solution is 7.5g / L; the mannitol in the enzymolysis solution The content of the liquid is 0.6mol / L, the content of the MES in the enzymolysis solution is 0.01mol / L, and the CaCl 2 The content in the enzymolysis solution is 1mmol / L, the content of the bovine serum albumin (BSA) in the enzymolysis solution is 1g / L, and the volume of the β-mercaptoethanol in the enzymolysis solution The percentage content is 0.04%, and the content of the carbenicillin in the enzymolysis solution is 50 mg / L.
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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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Patent Timeline
07 Jan 2020
Publication
CN110656126A
- IPC
- C12N15/82; C12N5/04
- CPC
- C12N5/04; C12N15/8206
- Inventors
- 谷晓峰; 刘青