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Cotton dehydrin similar gene and application thereof

A dehydrin, cotton technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as yield reduction

Inactive Publication Date: 2013-02-27
BIOCENTURY TRANSGENE CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most crops are sensitive to adverse environmental conditions and often experience reduced yields due to environmental stress

Method used

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  • Cotton dehydrin similar gene and application thereof
  • Cotton dehydrin similar gene and application thereof
  • Cotton dehydrin similar gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cloning of cotton dehydrin-like gene GhDh1

[0031] 1. Processing of cotton material

[0032] The F1 generation seeds of Ezamian No. 11 germinated for 15 days, and the whole plant material was taken for 8 hours of drought induction, and frozen in a -70 degree refrigerator.

[0033] 2. Extraction of cotton total RNA

[0034] A. After grinding 0.1 g of cotton leaves with liquid nitrogen, add 0.5 ml of plant RNA extract (purchased from invitrogen), and shake until thoroughly mixed.

[0035] B. Leave at room temperature for 5 minutes.

[0036] C. Centrifuge at 12,000 rpm at 4°C for 1 minute, and transfer the supernatant to a new RNase-free centrifuge tube.

[0037] D. Add 0.1ml 5M NaCl and mix gently.

[0038] E. Add 0.3ml chloroform, mix up and down.

[0039] F. Centrifuge at 12,000 rpm at 4°C for 10 minutes, and transfer the upper aqueous phase to a new RNase-free centrifuge tube.

[0040] G. Add isopropanol equal to the volume of the obtained water, mix w...

Embodiment 2

[0142] The construction of embodiment 2GhDh1 gene plant expression vector

[0143] see Figure 4 1. The GhDh1 gene cDNA obtained by PCR amplification was ligated to pGEM-TEasy with T4 DNA ligase to obtain plasmid pGEM-GhDh1, and pGEM-GhDh1 and pBI121 were simultaneously double-digested with BamH1 and Sacl to obtain the GhDh1 fragment and the linear pBI121 vector respectively. T4 DNA ligase ligated the GhDh1 fragment and the linear pBI121 vector to obtain the GhDh1 gene plant expression vector pBI121-GhDh1.

Embodiment 3

[0144] Embodiment 3 Utilizes the transformation method mediated by Agrobacterium to obtain transgenic GhDh1 tobacco

[0145] 1. Preparation of Agrobacterium tumefaciens LBA4404 Competent Cells

[0146] 1) Pick a fresh LBA4404 single colony and inoculate it in LB liquid medium containing an appropriate amount of antibiotics, and culture it at 28°C until the logarithmic growth phase;

[0147] 2) Centrifuge at 8000rpm at 4°C for 5 minutes, and collect the bacteria in a small centrifuge tube;

[0148] 3) resuspend and wash the cells with 600 μl ice-cold 500 mM CaCl2;

[0149] 4) Centrifuge at 8000 rpm for 5 minutes at 4°C, add 100 μl of ice-cold 500 mM CaCl2 to the cell pellet, mix well and set aside (the effect is best after 24-48 hours).

[0150] 2. Transformation of Agrobacterium tumefaciens LBA4404

[0151] 1) Add 1 μl of plant expression vector plasmid DNA to Agrobacterium competent cells, mix gently, freeze in liquid nitrogen for 5 minutes, and incubate at 37°C for 5 minu...

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Abstract

Dehydrin-like gene of cotton GhDh1, the nucleotide sequence of which is shown as SEQ ID NO: 1, is provided. Expression vector comprising gene GhDh1 is constructed and used to transform tobacco through Agrobacterium tumefaciens, and then the tobacco transformed with gene GhDh1 is obtained. The result of simulated arid test proves that the tobacco transformed with gene GhDh1 gets enhanced drought tolerance compared with the control tobacco, and the expression of gene GhDh1 in tobacco can improve the tobacco seed's tolerance of drought stress during seed germination.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a cotton dehydrin-like gene, a plant expression vector constructed by the gene and its application in the development of drought-resistant, cold-resistant and salt-alkali-resistant transgenic plants. Background technique [0002] In nature, plants are exposed to a variety of environmental conditions, water deficit, cold and freezing, heat, salt and oxygen deficiency are the main environmental factors affecting plant growth. Most crops are sensitive to adverse environmental conditions and often experience reduced yields due to environmental stress. During the occurrence of stress, a series of physiological and biochemical changes will occur in plants, such as sugar, lipid, protein and nucleic acid, as well as photosynthesis, nitrogen fixation and respiration, etc. to adapt to the stress. Different stages of plant growth, including seed germination, seed maturation, and se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N5/10C07K14/415A01H5/00
CPCC07K14/415C12N15/8273
Inventor 孙超陈文华王君丹崔洪志
Owner BIOCENTURY TRANSGENE CHINA
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