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Soybean drought-resistant zinc finger protein gene STF-2

A technology of STF-2 and zinc finger protein is applied in the field of genetic engineering to achieve the effect of improving comprehensive stress tolerance and drought resistance of plants

Inactive Publication Date: 2012-10-03
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still relatively few studies on soybean zinc finger proteins at home and abroad. Only SCOF-1 has been analyzed clearly. The zinc finger protein encoded by it is a typical double zinc finger structure C2H2 type zinc finger protein can significantly improve the tolerance of transgenic Arabidopsis to low temperature (Kim et al., 2001)

Method used

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  • Soybean drought-resistant zinc finger protein gene STF-2
  • Soybean drought-resistant zinc finger protein gene STF-2
  • Soybean drought-resistant zinc finger protein gene STF-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Select the soybean variety "Jinong 18". After the seedlings grow for a week, culture them at 4°C for 24 hours, take the leaves, grind them with liquid nitrogen, add them to the 1.5ml EP tube containing the lysate, shake and mix well, and use the kit RNAiso Plus Extract total RNA, use formaldehyde denaturing gel electrophoresis to identify the quality of RNA, and then use a spectrophotometer to measure the concentration of RNA; perform reverse transcription according to the instructions of the reverse transcription kit from the US company FERMENTAS, and synthesize the first strand of cDNA; plant C2H2 type zinc The conserved sequence of the finger protein was used as a probe to search the soybean genome database. After alignment and sequence splicing, a 1115bp soybean C2H2-type zinc finger protein sequence was obtained. According to this sequence, primers on both sides were designed: 5' GCTCATTCACTCATA 3', 5' TGATGAAAACAATTGTTA 3', using RT-PCR method for cDNA cloning, PCR...

Embodiment 2

[0022] Soybean C2H2 zinc finger protein gene STF-2 cDNA sequence, design primers to amplify the complete coding reading frame, add XbaI restriction site upstream, add SacI restriction site downstream, upstream primer: GGG TCTAGA ATGGCTTTGGAGGCTTTGA, downstream primer: TTTGAG AAACAAACGCGGCCTCT, obtained in Example 1 The cDNA was used as a template for PCR amplification, and the STF-2 The cDNA was cloned into the PMD-18T vector, and further cloned into the binary expression vector pBI121; the GFP The gene is linked to the above vector and constructed into pBI121 -STF-2-GFP Vector, pBI121 -STF-2-GFP Transformed into onion epidermal cells by Agrobacterium infection for transient expression, and found that pBI121 was rubbed by laser confocal fiberscope -STF-2-GFP The gene chimeric product is localized in the nucleus, indicating that soybean STF-2 Genes are transcription factors localized in the nucleus, see figure 1 , where a is pBI121- GFP Transient expression in onion...

Embodiment 3

[0024] The expression vector pBI121- STF-2-GFP Transform into Agrobacterium, and use the method of Agrobacterium infection to further transfer into tobacco, and perform PCR, RT-PCR, Southern hybridization detection on the obtained transgenic plants, and then analyze the stress tolerance of the T2 generation of transgenic plants and control plants ; Acquisition of drought-tolerant transgenic plants: the transgenic STF-2 Genetic tobacco and control were placed in an incubator at 25°C for 15 days without watering and the relative humidity was 40%, and then returned to normal watering at 25°C for 12 hours.

[0025] The transgenic tobacco and the control were placed in a 25°C incubator for continuous cultivation without watering for 15 days. The relative humidity of the incubator was controlled at 40%. The growth conditions were observed and recorded every day. It was found that after 10 days of stress, the leaves of the control plants appeared withered and curled. phenomenon, af...

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Abstract

The invention discloses a soybean zinc finger protein gene STF-2 which is a C2H2 type zinc finger protein gene related to drought and newly discovered in soybeans. Experiments show that: the STF-2 encoded protein can be located in the nucleus; after over-expression, the gene can obviously improve the drought resistance of the transgenic plants, and has a function of improving the comprehensive stress resistance of the plants; the gene disclosed by the invention comes from soybeans and has an optimized codon suitable for the dicotyledons such as soybeans and the like, and the genetic engineering receptor thereof is mainly suitable for the dicotyledons such as soybeans, tobaccos, cotton and the like; and moreover, the gene is also suitable for the monocotyledons such as rice, wheat, corn and the like.

Description

[0001] technical field [0002] The invention belongs to the field of genetic engineering, in particular to a transcription factor for plant drought stress response, specifically soybean zinc finger protein gene STF-2 and its application. technical background [0003] Zinc finger protein is a kind of transcription factor, which is widely involved in the expression regulation and growth process of organisms. The most studied is the C2H2 zinc finger protein, especially for Arabidopsis ( Arabidopsis thaliana ), Petunia ( Petunia hybrida Vilm) and rice ( Oryza sativa ) and other plants have been studied more. The study found a variety of plant C related to plant stress tolerance 2 h 2 type zinc finger proteins, for example, a STZ gene, which can encode a double-zinc-finger C 2 h 2 type zinc finger protein, trans STZ Genetic tobacco has significantly improved tolerance to cold and high-salt conditions (Sakamoto et al., 2000); the zinc finger protein gene obtained ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
Inventor 王丕武宋冰付永平洪洋晏佳琳李翠崔程程邢飞王贺张卓李勃王鹏马建杜鹃王丹
Owner JILIN AGRICULTURAL UNIV
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