Electrotransformation method of isolated chloroplast of cucumis stativum

A chloroplast and electrotransformation technology, applied in the field of plant genetic engineering, can solve the problems of low success rate, long cycle, difficult transformation operation, etc., and achieve the effects of high cost, long cycle and easy to master.

Inactive Publication Date: 2014-08-06
NANKAI UNIV
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  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of difficult operation, long period and low success rate of chloroplast transformation at the existing tissue and cell level, and use cucumber (Cucumis sativum) as ma

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  • Electrotransformation method of isolated chloroplast of cucumis stativum
  • Electrotransformation method of isolated chloroplast of cucumis stativum
  • Electrotransformation method of isolated chloroplast of cucumis stativum

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Embodiment 1

[0038] The specific steps of the electrotransformation, incubation and post-transformation effect identification method of cucumber isolated chloroplasts of the present invention include:

[0039] 1) Electrotransformation and incubation of isolated cucumber chloroplasts:

[0040] Cucumber chloroplasts ( figure 1 ), centrifuge the freshly extracted cucumber chloroplasts (1000g / 8-10min), resuspend the chloroplast precipitate with 0.33mol / L (or 0.33mol / L, the same below) sorbitol solution, and then centrifuge again, repeat such washing for 3-10min 4 times. Finally, resuspend the chloroplasts with 0.33mol / L sorbitol solution, and control the concentration of the chloroplast suspension to 5~6×10 6 / mL, ice bath.

[0041] Take 150ul of chloroplast solution to a 1.5ml centrifuge tube, add about 1ug of transformation plasmid (two kinds of chloroplast expression vectors with exogenous IL6 gene and GFP gene respectively), mix well and transfer to a pre-cooled electroporation cup to ...

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Abstract

The invention relates to an electrotransformation method of isolated chloroplast of cucumis stativum. The method comprises the following steps: by taking chloroplast separated from sterile cotyledon of cucumis stativum as a raw material, establishing an electrotransformation system of isolated chloroplast of cucumis stativum in proper volume and under proportion of chloroplast concentration and expression vectors; after transformation, carrying out short-time incubation, centrifugalization and DNase I digestion of chloroplast to carry out PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription-Polymerase Chain Reaction) identification by taking extracted transformed chloroplast DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid). The result shows that the chloroplast expression vector not only can be efficiently transformed to chloroplast, but also expression of an exogenous gene can be detected in the transcriptional level. The method provided by the invention can be used for overcoming the problems that the chloroplast of a higher plant cell is long in transformation period, difficult in technology and high in cost and the like, and provides an effective novel path for establishment of the higher plant chloroplast transformation vector, identification of the functions of key elements and basic theory and application and research of regulation of chloroplast gene expression and cell engineering and the like.

Description

【Technical field】: [0001] The invention belongs to the technical field of plant genetic engineering, and relates to methods such as electrotransformation, incubation and detection of isolated chloroplasts of higher plants represented by cucumbers. 【Background technique】: [0002] Chloroplast is a plant-specific organelle inherited from the maternal line. It not only has a relatively independent environment in the cell, but also has a high copy number. Chloroplast genetic transformation can realize the fixed-point integration of foreign genes, gene expression in a prokaryotic manner, and facilitate simultaneous transformation of multiple genes. It is an important way to overcome scientific bottlenecks such as gene escape, ecological environmental pollution, and food safety panic caused by nuclear transgenes. [0003] There are multiple copies in higher plant cells. Usually, a gene gun can be used to inject the exogenous expression vector into some chloroplasts in the cell. If...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N5/10C12Q1/68
Inventor 白艳玲刘盛海路瑶王宏刚徐海津王勇张秀明乔明强
Owner NANKAI UNIV
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