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Method for transferring gene by injecting plant ovaries

An ovary and plant technology, applied in the field of high-efficiency gene transformation, can solve the problems of transformation principle, controllable incidence depth and transformation efficiency difference, etc., to achieve the effect of improving transformation efficiency, solving difficult problems, and saving financial expenses

Inactive Publication Date: 2010-04-21
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The three gun types are different in terms of conversion principle, controllability, depth of incidence and conversion efficiency.
In the present invention, we found that using 1-2% DMSO to dissolve expression vector plasmid DNA to carry out genetic transformation of plants by ovary injection method can effectively improve the transformation efficiency, which has not been reported in the current literature

Method used

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  • Method for transferring gene by injecting plant ovaries
  • Method for transferring gene by injecting plant ovaries
  • Method for transferring gene by injecting plant ovaries

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Amplification of AI fragment of muskmelon fruit acid invertase gene

[0030] Melon inbred line 01-3 (Cucumis melo L.) was sown in the solar greenhouse of the Horticultural Station of Shandong Agricultural University, and the total RNA was extracted from the fruit 5 days after flowering. RNA PCR Kit, pMD18-T vector and T 4 DNA ligase was purchased from Dalian Bao Biological Co., Ltd.; DNA Extraction Kit was purchased from MBI; Escherichia coli DH5α was provided by the Genetic Engineering Laboratory of the College of Life Sciences, Shandong Agricultural University.

[0031]RNA was extracted by guanidinium isothiocyanate (GTC)-acidic phenol-chloroform extraction method. Gene-specific primers were designed according to the sequence published in Genebank (AF490425). According to the instructions of the RNA PCR Kit, reverse transcription: Take 1 μL (about 1 μg) of total RNA of melon fruit in 8.5 μL of RNase-free sterile double-distilled water, bathe in 70°C water for 10...

Embodiment 2

[0046] In this example, the transformation method is the same as in Example 1, except that the plasmid carrier DNA is dissolved in dimethyl sulfoxide aqueous solution and sterile double distilled water (control), the DNA concentration is 3 μg / μl, and the dimethyl sulfoxide The concentration of sulfone was 1% by weight. The identification results showed that the conversion rate of DMSO as a plasmid solvent was 1.93%, which was about 3 times that of using sterile double distilled water as a plasmid solute.

Embodiment 3

[0048] In this example, the transformation method is the same as in Example 1, except that the plasmid carrier DNA is dissolved in dimethyl sulfoxide aqueous solution and sterile double distilled water (control), the DNA concentration is 50 μg / μl, and the dimethyl sulfoxide The concentration of sulfone was 1% by weight. The identification results showed that the conversion rate of DMSO as a plasmid solvent was 2.15%, which was about 3.3 times that of using sterile double distilled water as a plasmid solute.

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Abstract

The invention relates to a novel plant genetic transformation method. An ovary injection method is utilized to efficiently and directly introduce vector plasmid DNA dissolved in dimethyl sulfoxide solution into plants so as to obtain transgenic plants.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a high-efficiency gene transformation method. technical background [0002] Biotechnology, also known as bioengineering, is based on life sciences, using the characteristics and functions of organisms to design and construct new species, new varieties, and new strains with expected traits, and to combine them with engineering principles , to process and produce, and to provide the society with a comprehensive technical system of goods and services. The introduction of exogenous genes into plant cells through genetic engineering technology is an important way of modern genetic breeding, and the transformation and integration of exogenous genes into the genome of recipient plant cells is an important part of plant genetic engineering. However, the bottleneck of current research lies in the efficiency of genetic transformation, so the research on efficient genet...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N15/82
Inventor 于喜艳田红梅闻小霞孔庆国
Owner SHANDONG AGRICULTURAL UNIVERSITY
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