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A kind of method for preparing transgenic gapdh gene cotton

A cotton, coding gene technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of undiscovered genes and complex mechanisms, and achieve the effect of improving the ability of salt and alkali tolerance

Active Publication Date: 2016-03-30
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous development of biotechnology and the elucidation of complex mechanisms of stress resistance in physiology, genetics, and molecular biology, some major achievements have been made in the study of plant salt tolerance, and many genes related to salt tolerance have been discovered, but due to their Mechanistic complexity and many undiscovered genes related to salt resistance

Method used

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  • A kind of method for preparing transgenic gapdh gene cotton
  • A kind of method for preparing transgenic gapdh gene cotton
  • A kind of method for preparing transgenic gapdh gene cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the construction of GAPDH overexpression vector

[0037] 1. Design PCR primers

[0038] The primer sequences are as follows:

[0039] GAPDH-F: 5'-ATGGCTACATCAATGGCGAAGAC-3';

[0040] GAPDH-R: 5'-TTAGGCGGCCCACCTCTGGGCG-3'.

[0041] 2. PCR amplification

[0042] (1) Extract the RNA of Dunaliella salina and reverse transcribe it into cDNA.

[0043] (2) Using cDNA as a template, PCR amplification of GAPDH gene was carried out with primer pair GAPDH-F / GAPDH-R.

[0044] The PCR amplification system is:

[0045]

[0046] The PCR reaction procedure is as follows:

[0047] 94°C for 3min; 94°C for 30s, 58°C for 30s, 72°C for 90s, 35 cycles; 72°C for 10min; 4°C+∞.

[0048] The nucleotide sequence of the GAPDH gene is shown in SEQ ID No.1.

[0049] The amino acid sequence of GAPDH protein is shown in SEQ ID No.2.

[0050] 3. Agarose gel electrophoresis to detect and recover PCR amplified fragments.

[0051] The results of agarose gel electrophoresis figu...

Embodiment 2

[0063] Embodiment 2, biolistic live transformation cotton

[0064] 1. Extract pBI121-GAPDH plasmid.

[0065] 2. The transformation recipient material is: non-insect-resistant cotton material Shanghai 749513. The transformation time is set from mid-July to mid-August during the full flowering period of cotton, and Shanghai 749513 is self-crossed.

[0066] 3. Collect the flowers of each recipient material on the afternoon of the first day of transformation, and then place the flowers on the test bench at room temperature (the flowers of each material should be marked), and the flowers whose stamens have been removed must cover the stigma with a wax tube .

[0067] 4. Start collecting pollen at around 9:00-10:00 the next morning. Put the collected pollen in a clean petri dish and mark it.

[0068] 5. Take 10 μl of gold powder and plasmid suspension and add it to the hole of the gene gun GDS-80, and gently tap the vicinity of the sample hole of the gun twice with your hand to m...

Embodiment 3

[0075] Example 3, Molecular detection of transgenic cotton T0 generation seeds

[0076] 1. Extract the DNA of the germination material of the transgenic non-insect-resistant cotton material Shanghai 749513T0 generation plant.

[0077] 2. Using the DNA of each plant as a template, two pairs of primers of the GAPDH gene are used to amplify and detect. The detection primers are listed in Table 1.

[0078] Table 1 detection primers

[0079]

[0080] GAPDH-F1 and GAPDH-R1 are a pair of primers, and GAPDH-F2 and GAPDH-R2 are a pair of primers.

[0081] Three, 1% agarose gel electrophoresis detection.

[0082] The result is as Figure 4 with Figure 5 shown.

[0083] Figure 4 It is the result of molecular detection of GAPDH gene (using GAPDH-F1 and GAPDH-R1 as primers).

[0084] Figure 4 In, M: MarkerDL2000; 1-6 are Shanghai 749513T0 generation plants.

[0085] Figure 4 It shows that with GAPDH-F1 and GAPDH-R1 as primer amplification, the plants numbered 1, 2, 3, 4, ...

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Abstract

The invention discloses a method for preparing GAPDH (reduced glyceradehyde-phosphate dehydrogenase) transgenic cotton. GAPDH proteins are as shown in a formula (1) or (2), wherein the formula (1) represents proteins as shown in SEQ ID No.2; (2) the formula (2) represents proteins which are obtained by substitution and / or deletion and / or addition of one or a few of amino acid residues of an amino acid sequence as shown in the SEQ ID No.2. According to the invention, the GAPDH transgenic cotton is obtained for the first time, a foundation is established for cultivation of saline-alkaline tolerance cotton variety and improvement of saline-alkaline tolerance capacity of the cotton, and economic and social benefits are achieved.

Description

technical field [0001] The invention relates to a method for preparing transgenic GAPDH cotton. Background technique [0002] Soil salinization is a global ecological problem and one of the main factors of land desertification and cultivated land degradation in the world today. Land salinization has a huge impact on agricultural production and ecological environment. How to develop and utilize saline-alkali land has become the focus of increasing attention. The high concentration of salt in the soil will cause ion imbalance, oxidative damage, water shortage and nutrient deficiency in the plant, and lead to the destruction of biological macromolecules, growth retardation, and even plant death, which seriously affects the growth and development of plants, resulting in crop yield reduction or failure , restricting agricultural production and development. The impact of specific salt stress on plants is manifested in: the impact on seed germination, the impact on plant photosyn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12N15/82A01H5/00
Inventor 叶武威阴祖军孔静静王德龙王俊娟樊伟莉王帅
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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