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Method of transient expression of exogenous genes by endosperm protoplasts

A protoplast and transient expression technology, applied in the field of cell engineering, can solve problems such as reducing the yield and integrity of protoplasts, affecting the amount of protoplast preparation, activity and transformation efficiency of exogenous genes, and insufficient understanding of accumulation process and regulation. Achieve the effect of large quantity, high conversion efficiency and high activity

Inactive Publication Date: 2020-05-29
SICHUAN AGRI UNIV
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Problems solved by technology

[0004] Due to the important role of endosperm in grass seed development and crop production, the functional analysis of genes related to endosperm development and storage material synthesis and accumulation has always been a research hotspot, but the current understanding of the process and regulation of endosperm development and storage material accumulation is still unclear Far from enough
There are no reports on the transient transformation system of endosperm protoplasts in gramineous crops, partly because the endosperm contains highly differentiated cells that affect the protoplast preparation, activity, and transformation efficiency of exogenous genes.
Starch granules in highly differentiated endosperm cells reduce protoplast yield and integrity, study shows

Method used

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  • Method of transient expression of exogenous genes by endosperm protoplasts
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  • Method of transient expression of exogenous genes by endosperm protoplasts

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Embodiment 1

[0035] 1. Material collection and processing:

[0036] Field planting management of corn materials, bagging pollination, marking the date of pollination, taking corn ears 6-10 days after pollination ( figure 1 )conduct experiment. In corn 1-4 days after pollination, the endosperm cells are not fully developed and are too small to be picked; in corn endosperm 10 days after pollination, there are too many amyloplasts, and the storage materials begin to accumulate, and the protoplasts are easily broken when preparing protoplasts and the transformation efficiency is low. Low. Corn endosperm ( figure 2 ) experiment, and the experimental results showed that the maize endosperm prepared 8 days after pollination had the highest yield, the best vigor and the highest transformation efficiency. Take corn ears 8 days after pollination, bring them back to the laboratory for processing, select corn kernels with good growth and consistency in the middle, peel off the outer seed coat with...

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Abstract

The invention discloses a method of transient expression of exogenous genes by endosperm protoplasts. The method of transient expression of the exogenous genes by the endosperm protoplasts comprises the following steps: (1) cutting grains of graminaceous crops in a prefilling stage, and conducting enzymolysis for 4-8 h; (2) then adding an enzymolysis stop solution, conducting filtering, collectingfilter liquor, and conducting centrifugation to obtain protoplasts; (3) resuspending the protoplasts, conducting centrifugation after ice-bath is conducted for 20-40 min, removing supernate, then conducting resuspending and purification continuously; (4) mixing the protoplasts purified in the step (3) and plasmids, then adding a PEG-Ca2<+> solution, and allowing standing and transformation for 15-30 min; and (5) then adding a W5 solution continuously, conducting centrifugation after even mixing is conducted, removing supernate, then conducting resuspending, conducting centrifugation after culture is conducted at 20-30 DEG C for 6-18 h, removing supernate, and then conducting resuspending again. The endosperm protoplasts produced through the method of transient expression of the exogenousgenes by the endosperm protoplasts are large in number and high in activity, and the protoplast transformation efficiency is high so that strong technical support can be provided for subsequent relative studies of endosperm character-related gene function analysis, endosperm cell signaling, endosperm bioreactors and the like.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a method for transiently expressing exogenous genes in gramineous endosperm protoplasts. Background technique [0002] A protoplast is a plant or fungal cell from which the cell wall has been removed, including intact organelles such as the cell membrane, cytoplasm, and nucleus. With complete organelles and no cell wall shielding, protoplasts are more likely to absorb and integrate exogenous nucleic acids and proteins, making protoplasts a fast and convenient system for gene function analysis. The protoplast-based transient expression system has been widely used in the research of plant molecular biology and cell biology. played a huge role. The isolation technology of protoplasts has been widely reported in various tissues of various plants, but the transient expression system of protoplasts is currently reported mainly in the young plants of model plants ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/04
CPCC12N5/04C12N15/8206
Inventor 黄玉碧胡育峰高蕾李炀平宋大林黄焕焕张军杰刘汉梅余国武刘应红
Owner SICHUAN AGRI UNIV
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