Glycine max(L.)Merr Williamus 82 medium-small G protein gene GmRAB7 and application thereof

A technology of G protein and soybean, which is applied in the field of biogenetic engineering to achieve the effect of improving the salt tolerance and salt tolerance of crops

Inactive Publication Date: 2020-04-10
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report about the soybean small G protein gene GmRAB7 and its role in improving plant salt stress tolerance

Method used

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  • Glycine max(L.)Merr Williamus 82 medium-small G protein gene GmRAB7 and application thereof
  • Glycine max(L.)Merr Williamus 82 medium-small G protein gene GmRAB7 and application thereof
  • Glycine max(L.)Merr Williamus 82 medium-small G protein gene GmRAB7 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Cloning of GmRAB7

[0024] 1.1 Extraction of Williams 82 total RNA

[0025] (1) Put the Williams 82 plant material into a mortar, and use liquid nitrogen to grind it into powder (directly applied to the following experiments or frozen in a -80°C ultra-low temperature refrigerator for later use);

[0026] (2) After the liquid nitrogen volatilizes, immediately transfer 100-200mg of plant powder into a 1.5ml centrifuge tube, then quickly add 1ml of Trizol extract, vortex and shake to fully dissolve the sample into the extract, and place at room temperature for 5min;

[0027] (3) 4°C, 12,000rpm, centrifuge for 10min, transfer 0.9ml of supernatant to a new 1.5ml centrifuge tube, add 0.2ml of chloroform, vigorously shake and mix for 15sec, and leave at room temperature for 2-5min;

[0028] (4) 4°C, 12,000rpm, centrifuge for 10min, transfer 0.4ml of supernatant to a new 1.5ml centrifuge tube, add 0.4ml of isopropanol, invert 15 times to mix the solution, and place a...

Embodiment 2

[0130] Example 2 Functional verification of the Williams 82 small G protein gene GmRAB7 expressed in Arabidopsis

[0131] 2.1 Transformation of Arabidopsis by flower infection

[0132] (1) When Arabidopsis thaliana (Col-0 wild type) grows to 1 cm of bolting, the tip is reduced to induce the formation of lateral inflorescences;

[0133] (2) One day before transformation, add 1 ml of activated Agrobacterium GV3101 containing the expression vector plasmid to 40 ml YEP medium containing corresponding antibiotics and 50 μg / ml rifampicin, and shake at 28°C to culture to OD 600 about 1.0-1.2;

[0134] (3) At room temperature, centrifuge at 4,200 rpm for 10 min, collect the cells, and resuspend the cells with a dip solution (5% sucrose, 0.02% Silwet L-77) to make the OD 600 about 0.8;

[0135] (4) drop Agrobacterium on the inflorescence with a pipette to carry out dip infection, and after all inflorescences are infected, put Arabidopsis thaliana into a vacuum desiccator and vacuumi...

Embodiment 3

[0147] Example 3 Detection of salt tolerance of transgenic soybeans

[0148] (1) The preparation method of transgenic soybean is the same as that in 2.1-2.3.

[0149] (2) Surface disinfection of soybean seeds (same as 2.2).

[0150] (3) The soybean GmRAB7 overexpressing plants and wild-type soybean seeds were soaked in water for 4 hours, placed in a white porcelain plate and covered with gauze, and germinated for 2 days, during which the gauze was kept moist. After 2 days, put the bean sprouts into the seed germination bags, and put about 8 plants in each bag. When placing beans, pay attention to the different positions of seeds between bags to avoid position effects. The seed germination bag was placed in a light incubator, and cultivated under 25-degree long-day light. Water was added to the seed germination bag regularly every day, adding 20ml each time, once a day. The seeds were cultured in seed germination bags until the first pair of true leaves grew, and 20 ml of 15...

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Abstract

The invention discloses a Glycine max(L.)Merr Williamus 82 medium-small G protein gene, the gene is named as Glycine max(L.)Merr Williamus 82NAC membrane-bound transcription factor gene GmRAB7, and anucleotide sequence of cDNA of the gene is shown as SEQ ID No.1. The invention also discloses an application of the gene in improving the salt tolerance of plants. The experiments prove that the salttolerance of the GmRAB7 transgenic plant is greatly improved compared with that of a non-transgenic plant, the gene is expected to play an important role in cultivating salt-tolerant plant varieties,and a new theoretical basis and a new practical basis are provided for improving the salt tolerance of crops.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, in particular to a small G protein gene in soybean (Glycine max (L.) Merr) Williams 82 (Williams 82) - GmRAB7 and its application. Background technique [0002] Plants are exposed to the natural environment and are often affected by various environmental stresses, such as drought, extreme temperatures, nutrient deficiencies, and pests and diseases. These environmental stresses not only restrict the growth and development of plants, but also lead to the reduction of food crops. [0003] The growth and development of higher plants and their response to environmental changes are achieved by regulating the expression of target genes. RAB protein is a member of the small G protein family, widely distributed in eukaryotes, mainly located in the inner membrane of the cytoplasm, involved in vesicle transport, cytoskeleton assembly, cell signal transduction, etc. In the transducti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H6/54A01H6/20
CPCC07K14/415C12N15/8273
Inventor 向凤宁李朔刘振华
Owner SHANDONG UNIV
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