Method for performing transient expression by introducing foreign gene into poplar bioplast

A protoplast, exogenous gene technology, applied in the field of genetic engineering, to achieve the effect of easy operation and realization

Inactive Publication Date: 2012-03-14
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This system has been applied in plants such as Arabidopsis thaliana, maize (Sheen 2001), tobacco (Locatelli et a

Method used

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  • Method for performing transient expression by introducing foreign gene into poplar bioplast
  • Method for performing transient expression by introducing foreign gene into poplar bioplast
  • Method for performing transient expression by introducing foreign gene into poplar bioplast

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: The influence of different enzyme liquid combinations on poplar protoplast separation

[0031] In the preparation of the enzyme solution, according to the enzymes commonly used in the separation of plant protoplasts, select cellulase R-10 (Cellulase R10, 5000U / g), RS (Cellulase RS, 16000U / g), Sigma cellulase (840U / ml) , pectinase Y23 (Pectolyase Y23, 1000U / g), Sigma pectinase (Pectinase (sigma), 3800U / ml), pectinase R-10 (Macerozyse R10, 3000U / g), respectively dissolved in different combinations In the enzyme solution, the separation experiment of poplar mesophyll protoplasts was carried out, and the combination and concentration of enzymes are shown in Table 1.

[0032] Table 1 Enzyme solution composition and concentration

[0033]

[0034]The experimental results showed that the yield and quality of mesophyll protoplasts isolated under various enzyme solution composition conditions were different. In the same type of enzyme solution combination, as ...

Embodiment 2

[0039] Embodiment 2: Effect of PEG concentration on conversion efficiency

[0040] PEG acts as a medium for transformation to aggregate protoplasts and plasmids. In this experiment, PEG4000 was used to transform poplar mesophyll protoplasts. The initial concentration gradient of the configured PEG solution is 20% g / ml, 30% g / ml and 40% g / ml, and about 6×10 4 1 protoplast, 10 μg of plasmid DNA and different concentrations of PEG4000, the PEG4000 added is 1 / 2 of the total transformation system volume, that is, the concentration of PEG during transformation is 10%, 15%, and 20%, respectively. Incubate at 25°C for 15 minutes. Before transformation, the PEG solution was sterilized by filtration through a 0.45 μm mixed cellulose microporous membrane. Experiments were repeated three times.

[0041] Since the volume of PEG added during conversion is 1 / 2 of the total volume of conversion, the final concentrations of PEG during conversion are 10%, 15% and 20%, respectively. After b...

Embodiment 3

[0044] Plasmid DNA is used as a vector to bring the target gene into protoplast cells and express it in a short time. Its content has a great influence on the conversion efficiency. According to the above-mentioned transformation method, plasmids of different masses were added to the transformation system, and their effects on the transformation efficiency were detected. Set the plasmid gradient as: 5 μg, 10 μg and 15 μg. Plasmids of different qualities were added to the transformation system, about 6×10 4 protoplasts and optimized PEG4000. Wherein, PEG4000 added is 1 / 2 of the volume of the total transformation system, and incubated at 25° C. for 15 minutes. Experiments were repeated three times.

[0045] according to Figure 5 As shown, when the number of protoplast cells is constant, the transformation efficiency will increase with the increase of the plasmid concentration within a certain range. The experiment stabilized the number of protoplasts at 6×10 4 , when add...

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Abstract

The invention discloses a method for performing transient expression by introducing a foreign gene into a poplar bioplast. The method comprises the following steps of: introducing a transient expression vector containing a foreign gene into a poplar bioplast by using PEG (Polyethylene Glycol)-Ca<2+>; culturing; and expressing the foreign gene in the bioplast. A poplar bioplast transient expression system constructed with the method is easy to operate and realize, a place with broad prospects is opened up for the research of functional genes of a poplar, and the transformation efficiency of the method can be up to 70 percent.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for transferring exogenous genes into poplar protoplasts through chemical induction and performing transient expression. Background technique [0002] Poplar has been used as a model species in forests due to its rich species, wide distribution, strong adaptability, rapid growth, small genome, and easy genetic transformation (Bradshaw et al., 2000; Brunner et al., 2004) , Carry out extensive research in the fields of physiology and biochemistry, genetics, molecular biology and cell biology. In 2006, the U.S. Department of Energy completed the Poplar Whole Genome Sequencing Project (Tuskan et al.), which laid a solid foundation for the study of functional genomes of poplar and even forest trees. influences. [0003] So far, a variety of molecular and genome-based research technologies have been successfully applied to poplar, such as genome seque...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
Inventor 黄敏仁谭碧玥陈英胥猛徐立安潘惠新王光萍王明庥
Owner NANJING FORESTRY UNIV
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