KR20040050633A - Transcription factor related to freezing tolerance isolated from potaot - Google Patents

Abstract

PURPOSE: A gene related to freezing tolerance of potato and freezing-tolerant plants using the same are provided, thereby operating transcription factor through the signal transfer process when the plant is placed at low temperature to amplify the expression of other genes, so that the plant can grow at low temperature. CONSTITUTION: The gene StEREBP related to freezing tolerance of potato is isolated from potato and has the nucleotide sequence set forth in SEQ ID NO: 1, wherein the gene StEREBP is isolated by the steps of: freezing potato treated at 4 deg. C for 8 hours and untreated potato and pulverizing them; extracting RNA from the pulverized potatoes; isolating poly A+RNA from the extracted total RNA; subjecting the poly A+RNA to RNase H-reverse transcriptase to synthesize first strand cDNA; removing RNA using RNase and synthesizing second strand cDNA using DNA polymerase I; inserting the synthesized cDNA into Uni-zap XR vector; in vitro packaging the cDNA inserted vector; infecting E. coli with the cDNA inserted vector to determine Phage titer and amplify the genes; and sequencing the genes.

Description

Potato dynamics-related genes and dynamic crops using the same {TRANSCRIPTION FACTOR RELATED TO FREEZING TOLERANCE ISOLATED FROM POTAOT}

The present invention relates to genes related to potato resistance, and more particularly, to isolate genes that are specifically induced during poor environmental stress in potatoes, to analyze their functions, and to use them for development of poor environmentally adaptive crops.

In general, when plants are in an environment unsuitable for plant growth such as drought, salt, low temperature, and high temperature, they have a characteristic of adapting to a poor environment and surviving by changing the metabolic process and intracellular substance biosynthesis.

In particular, the EREBP gene is a transcription factor and has similar characteristics to the domain of C repeat binding factor (CBF) isolated from Arabidopsis.

General plants undergo transcriptional signaling during poor environmental conditions, and transcription factors amplify the expression of several genes. Therefore, many attempts have been made to use transcription factors for the development of disaster-tolerant crops.

However, it is very doubtful how much environmental resistance is actually increased when a gene with amplified gene expression during environmental stress is put into a plant.

The present invention increases the adaptability to low temperatures by increasing metabolic regulators or osmotic regulators in plants when the plants are in poor environmental conditions such as low temperature, East Sea, etc. Its purpose is to use it for the development of same-sex plants.

Figure 1 is a nucleotide sequence of the promoter gene StEREBP gene (800bp, 206aa) isolated from the potato of the present invention.

Figure 2 is a analysis of Northern expression by cold treatment of the StEREBP gene of the present invention,

2a is a state of expression by temperature.

2b is a state of expression over time.

Figure 3 shows the transformation vector and expression of the StEREBP gene of the present invention,

2a is a schematic view of the RD29A promoter.

2B is a schematic of 35S promoter.

Figure 4 is a clone of Northern expression analysis of tobacco transformants and potato transformants of the StEREBP gene of the present invention.

The above object can be achieved through the StEREBP gene, which is characterized by having a nucleotide sequence of the sequence list attached to the present invention.

Hereinafter, specific embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In this example, clones related to dynamic resistance were isolated from reverse northern analysis in cDNA library prepared by low temperature treatment on potatoes. The nucleotide sequence of the isolated clone was analyzed to confirm its homology with the EREBP clone, and the expression characteristics of this gene were compared by Northern analysis. Cloning the StEREBP gene into a plant transformation vector, the vector was constructed to be controlled by an environmental stress-specific expression promoter or a 35S promoter that is constantly expressed. Was done as follows.

<Step 1> Gene isolation and sequencing for selection of environmentally specific clones from cDNA library

RNA was recovered from pot and untreated ground part of potato treated at 4 ℃ for 8 hours, ground with liquid nitrogen, RNA was extracted with BRL's Trizol reagent, and cDNA gene bank was prepared by Stratagene's Lambda ZAP II. cDNA synthesis kit and Gigapack II gold packaging extract were prepared according to the method suggested by the manufacturer. Total RNA isolated from trizol was isolated from poly A + RNA by Qiagen's oligotex, poly A + RNA was used as a template, RNase H- reverse transcriptase was used to synthesize first strand cDNA, and RNA was removed using RNase H. Second strand cDNA was synthesized using DNA polymerase I. EcoRI adapters were ligation into the synthesized cDNA and treated with XhoI to create EcoRI and XhoI sites and ligation to EcoRI and XhoI treated Uni-zap XR vectors. The ligation solution was packaged using an in vitro packaging kit (Stratagene), infected with E. coli , measured for phage titers, amplified, and used as a preservation stock. The number of plaques was about 700,000.

The nucleotide sequence of the selected gene was determined using ABI's big dye terminator, and the homology analysis was confirmed to belong to EREBP (ethylene responsive element binding protein, transcription factor) using US NCBI database.

<Step 2> Northern Expression of StEREBP Gene

After 8 hours treatment at 4 ℃ and 250mM NaCl in the pollen, after the time indicated for each sample in Figure 2, the leaves were collected and extracted RNA with BRL company's Trizol reagent and in agarose containing formaldehyde A total of 10 μg of RNA was electrophoresed, transferred to the membrane by capillary transfer, and Northern hybridization was performed by labeling EcoRI and XhoI cut fragments of StEREBP cDNA clone with Takara's labeling method. As a result, the StEREBP gene was confirmed that the expression is amplified at low temperature treatment through FIG. 2A, and as shown in FIG. 2B, the StEREBP gene was increased from 4 hours and the expression of StEREBP was amplified at 24 hours treatment.

<Step 3> Tobacco and potato transformation of StEREBP gene

In order to introduce the isolated StEREBP gene into tobacco, the chimeric gene produced by connecting the structural gene of the StEREBP gene to the rd29A promoter isolated from Arabidopsis was cloned into pBI121, a plant transformation vector, and infected with Agrobacterium (LB4404). (Figure 3)

<Step 4> Potato Transformation of StEREBP Gene and Northern Expression Analysis of Transformant

Agrobacterium infected with the StEREBP gene was incubated overnight in YEP (bacto-peptone, l0g; bacto-yeast extract.l0g; NaC1.5g; phytagar, l5g; DW, 1L) in liquid medium, and then diluted 1/40 times in sterile water. . Meanwhile, Agrobacterium was diluted by cutting the leaves of the plant (stock name: Xanthi) plants grown for 3-4 weeks in MSO medium (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g; DW, 1 L) to 5-l0 mm2. Incubate in 50 ml of culture medium in 7 ml of MSO medium (MS salt 4.3 g, sucrose 30 g, MES 0.5 g / L pH5.6-5.8) and incubate for 3-4 days at 28 ° C dark conditions. After washing 3 ~ 4 times with MS selection medium containing antibiotics and hormones (MSO medium, 500µg / ml carbenicillin, l00µg / ml kanamycin, NAA (1mg / ml) l00µl, BA (1mg / ml) 1, phyto agar 8g / L) was incubated for 2-3 weeks. When the shoot was re-differentiated from the callus of MS selection medium, the shoot was transferred to MSO solid medium to regenerate roots. Regenerated clones were transfected to confirm gene expression by PCR, followed by Northern method, and then purified to grow plants for harvesting in greenhouses.

For potato transformation, the vector used for tobacco transformation was used as it was. After infection with Agrobacterium (LBA 4404), the infected Agrobacterium was treated with YEP (bacto-peptone, l0g; bacto-yeast extract.10g; NaC1.5g; phytagar, 15g; DW). 1L) overnight in liquid medium, and then diluted 1/40 times in sterile water.

Meanwhile, the internodes of the stock potatoes (cultiform name: Sumi superior) plants grown in storage medium PM (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g DW, 1 L) for 3-4 weeks were diluted to a size of 3-5 mm and diluted. After soaking in Agrobacterium culture medium for about 5 minutes, it was transferred to a plate made of Whatman No.1 filter paper sterilized on the surface of callus organic solid medium PM and co-cultured in the dark for 48 hours. After 48 hours transfer to PC medium containing (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g; NAA, 0.1 mg; BA, 0.5 mg; carbenicillin, 500 mg; kanamycin, 50 mg; DW, 1 L) After 4 weeks of incubation, it was again taken with kanamycin containing organic medium PS (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g; carbenicillin, 500 mg; BA, 5 mg; GA₃, 0.3 mg; DW, 1 L). Transfer was incubated for 4-8 weeks, the shoots were transferred to the PM medium and incubated.

StEREBP gene transgenic plant, control group transformed with only pBI121 vector, except StEREBP gene, ground surface from untransformed plant, ground in liquid nitrogen, extracted with Trizol reagent of RNA BRL, and extracted from agarose containing fomaldehyde. A total of 30 ㎍ of RNA was electrophoresed and transferred to the membrane by capillary transfer, and the SATAEREBP gene was labeled by Takara's labeling method to perform northrn hybridization.

As a result, as shown in the expression analysis of FIG. 4, it was confirmed that the StEREBP gene was not expressed in the transgenic tobacco and pBI121 vector transformed plants, but expressed only in the transgenic potatoes.

<Step 5> Stability Analysis of StEREBP Gene Transgenic Tobacco and Potato

Transformed plants confirmed the expression of the StEREBP gene, StEREBP gene was excluded and the transformants of the control transformed only pBI121 vector was purified and cultivated in a greenhouse. Germinated plants capable of germinating and growing in medium containing Kanamycin were used for bioassay analysis to select transgenes during T1 generation.

In addition, the pBI121 vector transgenic tobacco with only 35S promoter and selection marker was transferred to MS medium grown in kanamycin medium among T1 strains of transformants. there was.

As described above, in the StEREBP gene of the present invention, when the plant is placed at a low temperature, a transcription factor is activated through a signal transduction process by an external environment, which amplifies the expression of various genes, thereby enabling growth at low temperatures. Therefore, by introducing and expressing such low temperature-induced transcription factors in plants, it can be usefully used for the development of cold-to-freeze-resistant crops.

Claims (2)

Potato immunity related gene isolated from potato and characterized by having the nucleotide sequence of SEQ ID NO: 1. Dynamic crops using the gene of claim 1.