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A method for constructing a genetic transformation system using non-isolated grape fruits

A non-in vitro, fruit technology, applied in the field of genetic engineering, can solve the problems of inability to form a genetic transformation method, strong randomness, and high failure rate, and achieve the effects of complete observation of subcellular localization, efficient transient transformation, and shortening of the experimental period.

Active Publication Date: 2021-11-02
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the EL31-EL35 period of grape fruit development, various physiological and biochemical activities are gradually vigorous, which is the best period for studying transgenic fruits. But it is compact, the cell gap is extremely small, and the microenvironment of the cell fluid is very complicated. It is difficult to inject the bacterial fluid directly by inserting the syringe into the fruit epidermis, because the dense structure inside the fruit and the liquid microenvironment will generate huge resistance, which makes it difficult for researchers to inject bacteria. Pushing the bacterial liquid through injection normally, forcibly increasing the force of the injection will cause the bacterial liquid to spray out from the needle pricking part of the fruit, resulting in a very low final conversion rate
For example, the inventor once directly injected the Agrobacterium liquid into the non-isolated grape fruit at the EL31-EL35 stage for instantaneous transformation without any auxiliary means, but the results of such experiments were highly random and difficult to repeat, and The failure rate of transformation is high, and it is impossible to form a stable and reproducible genetic transformation method suitable for future industrialized grape fruit transgenic research

Method used

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  • A method for constructing a genetic transformation system using non-isolated grape fruits
  • A method for constructing a genetic transformation system using non-isolated grape fruits
  • A method for constructing a genetic transformation system using non-isolated grape fruits

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1 Comparison of effects of non-isolated fruit injection of grapes

[0045] Preparation of infection solution: pick out the monoclonal plaques of FaVPT1 OE Agrobacterium and Agrobacterium aeruginosa (as a control), and put them in 5 mL of LB liquid medium containing corresponding resistance and rifampicin, respectively, and shake the bacteria solution at 28°C. The activated bacterial liquid was transferred to 50 mL of LB liquid medium (added with kanamycin and rifampicin) for mass expansion, and the bacteria were shaken overnight at 28° C. and 220 rpm. On the second day, the cells were enriched by centrifugation at 5000g for 5 min, and the cells were enriched with infection buffer (MES-KOH (pH 5.7) 10mM, MgCl 2 10mM) to resuspend the bacterial solution, adjust the OD after washing twice to make the bacterial suspension OD 600 The value is 0.8-1.0, add AS to the final concentration of 200 μM, and activate the cells at 28°C for 2-3h.

[0046] Selection of grap...

Embodiment 2

[0055] The effect of embodiment 2 on fruit development

[0056] According to the method of the present invention in Example 1, a 1mL syringe (Jiangsu Zhiyu Medical Equipment Co., Ltd. disposable sterile syringe with needle, injection needle specification: 0.45×16RWLB, namely diameter 0.45mm, length 16mm) was used in the EL34 period. Punch 30 holes on the surface of the grape fruit (injection methods such as figure 2 ). Fruit before injection image 3 A. The puncture was healed when observed on the third day after injection, and the fruit could develop normally ( image 3 B), about 65% of the FaVPT1 OE fruits were obviously colored on the 7th day, while only a few CK fruits began to change color, and the coloring was not obvious ( image 3 C), indicating that the mechanical damage to the fruit by this method will not inhibit the fruit development, and the effect of phenotypic function identification can be achieved. The experiment was repeated 3 times. Compared with CK f...

Embodiment 3

[0057] Example 3 Gene expression analysis

[0058] According to the method of the present invention in Example 1, a 1mL syringe (Jiangsu Zhiyu Medical Equipment Co., Ltd. disposable sterile syringe with needle, injection needle specification: 0.45×16RWLB, namely diameter 0.45mm, length 16mm) was used in the EL34 period. Punch 30 holes on the surface of the grape fruit (injection methods such as figure 2 ). The fruits on the 7th day after injection of FaVPT1 OE and CK were collected, peeled and cut off the pulp at the injection site, and stored in liquid nitrogen for future use. Use Huayueyang GK RNA extraction kit (Cat. No. 0416-50) to extract RNA from samples (follow the instructions in the instructions), reverse-transcribe them into cDNA, and use fluorescence quantitative PCR technology to detect FaVPT1 (which can promote fruit ripening and coloration). ) expression. The experiment was repeated 3 times.

[0059] The results showed that the expression level of FaVPT1 i...

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Abstract

The invention relates to a method for constructing a genetic transformation system using non-isolated grape fruit, which can be specifically used for research work such as the transient transformation of grape fruit with Agrobacterium, the study of gene expression patterns and subcellular localization, and the like. The method of the present invention not only performs long-term operations such as in vitro culture and callus induction, but also solves the technical problem that it is impossible to inject liquid into the grape fruit, and further realizes efficient instantaneous transformation and clear subcellular localization observation; The time for gene function identification is shortened, the transformation efficiency can be greatly improved, and the stability and repeatability of the results are very good.

Description

technical field [0001] The invention relates to a method for constructing a genetic transformation system by utilizing non-isolated grape fruits, and belongs to the field of genetic engineering. Background technique [0002] my country is the world's largest producer of grapes. The area under viticulture has reached 11.985 million mu. The output of fresh table grapes has ranked first in the world, and the area under viticulture has jumped to the second place in the world. Due to its huge industrial and economic value, the improvement of the quality of grapes (mainly refers to the fruit) has always been widely concerned. Grape fruit quality, including edible quality centered on sugar content and appearance quality centered on color and lustre, are closely related to grape genetics and cultivation conditions, so functional genomics has become an essential link between genes and traits. Methods. With the completion of the grape genome sequencing, in the past ten years, resear...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/84C12N15/29A01H5/08A01H6/88
CPCC07K14/415C12N15/8205C12N15/8212
Inventor 黄芸白倩沈元月郑珍珍
Owner BEIJING UNIV OF AGRI
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