Short-day sensitive cell death SSD1 gene and coded protein and application thereof

A cell death and gene encoding technology, applied to the short-day light-sensitive cell death SSD1 gene and its encoded protein and application fields, can solve problems such as complex biological cells

Inactive Publication Date: 2013-02-06
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Therefore, the mechanisms leading to c

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  • Short-day sensitive cell death SSD1 gene and coded protein and application thereof
  • Short-day sensitive cell death SSD1 gene and coded protein and application thereof
  • Short-day sensitive cell death SSD1 gene and coded protein and application thereof

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Experimental program
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Embodiment 1

[0020] Embodiment 1: Arabidopsis EMS mutagenesis and screening

[0021] 1. Chemical mutagenesis treatment

[0022] Arabidopsis wild-type (Col-0) seeds were placed in distilled water and stirred for 30 minutes. After standing at 4°C for 12 hours, the seeds were transferred to 100 mM phosphate buffer (pH 6.5), and 0.2% ( v / v) of ethyl methanesulfonate (EMS), sealed and placed on a shaker in a water bath (25°C) for 12 hours, then rinsed the seeds with distilled water.

[0023] 2. Mutation plant culture

[0024] The rinsed seeds were vernalized at 4°C for 3 days, and sown in artificial soil (Northeast black soil: vermiculite = 1:1, v / v). Grow in the light culture room, the culture temperature is 22±2℃; the light intensity is 80-120μmolm -2 the s -1 ; relative humidity 65%; photoperiod 16 hours light, 8 hours dark. Harvesting mature seeds is M 1 Generation, M 1 M 2 Substitute, dry for later use.

[0025] 3. Screening of Mutants

[0026] Will M 2 After surface disinfectio...

Embodiment 2

[0028] Embodiment 2: Map-based cloning method to isolate SSD1 gene

[0029] 1. Construction of map-based cloning populations

[0030] Crossing the ssd1 mutant (Col-0 background) with another ecotype Landsberg erecta (Ler) gave F 1 Generation heterozygote; F 1 F 2 generation; seeding F 2 Generation seeds were screened for individuals with cell necrosis under short-day conditions, and a row of ssd1 mutants were planted in the same seedling tray as a control. A total of 2500 strains of F 2 Genetic populations are used for map-based cloning of genes.

[0031] 2. Selection and design of molecular markers

[0032] According to the Arabidopsis genome-wide DNA polymorphism database published on TAIR (www.arabidopsis.org), molecular markers were selected: NT7123, EAT, F21M12, NGA63, F16J7-TRB, JV18 / 19, F21J9-83201. According to literature reports, the molecular marker SGCSNP9772 was designed, see Table 1. In addition, based on the alignment of the Ler-sequenced expressed sequen...

Embodiment 3

[0049] Embodiment 3: Genetic complementation experiment of SSD1 gene

[0050] 1. Construction of Complementary Vectors

[0051] The high-fidelity enzyme TransStart FastPfu DNA Polymerase (TransGen) was used to amplify a fragment containing the SSD1 gene from Arabidopsis wild-type Col-0, with a full length of 3175bp. CCCGGG ATGGCGTTGCTGAAGTCTTT-3') and RP(5'-cga GAGCTCAC TTATTGTTAATGGGTTGG-3'). The amplification reaction system is 50μl, of which 5×TransStart FastPfu Buffer: 10μl; 2.5mM dNTP: 5μl; 10mM forward primer FP: 2μl; 10mM reverse primer RP: 2μl; template Col-0 DNA: 0.5-2ng; TransStart FastPfu DNA Polymerase: 1μl; add ddH 2 O to make up to 50 μl. The amplification program was: 95°C pre-denaturation for 2 minutes; 95°C for 20s, 55°C for 20s, 72°C for 2min, 30 cycles; 72°C for 5 minutes.

[0052] Carrier plasmid pROK2 and purified PCR product were digested with SmaI and SacI, and T4 DNA Ligase (TransGen) was used to connect the digested and purified AtSSD1 gene and ...

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Abstract

The invention discloses a short-day sensitive cell death SSD1 gene and a coded protein and application thereof. The SSD1 gene has a sequence as shown in SEQ ID NO:1. According to the invention, by using a model plant arabidopsis thaliana as a test material, through EMS (ethyl methanesulfonate) mutagenesis, an ssd1 mutant is obtained through screening. The SSD1 gene is separated by adopting a map-based cloning method, a gene mutational site is found, and the cell death is caused by deletion of the gene under the condition of short day. The SSD1 gene is necessary for the growth of arabidopsis thaliana under the condition of short day, and has a wide homology in plants. The finding of the biological function of the gene and the relevant research on a homologous gene are beneficial to deepening of the research on the controlling of the plant cell death by photoperiod, have important scientific significance in researching a molecular mechanism of the plant cell death, and also have important application value in improvement of crop varieties in a short-day region.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a short-day sunshine-sensitive cell death SSD1 gene, its encoded protein and its application. Background technique [0002] Cell death is a common life phenomenon in organisms. When cells are severely damaged and involve the nucleus, irreversible changes such as metabolic cessation, structural destruction, and loss of function appear, which is cell death. Generally speaking, cell death can be divided into two types: physiological cell death and nonphysiological cell death. Physiological death is the cell death controlled by the death program in the cell. In the process of growth, development and morphogenesis, organisms use physiological death to control the number of cells, or as a defense mechanism to eliminate infected, mutagenized or infected cells. damaged cells. Non-physiological death is caused by unexpected external factors during the growth and development of ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 任春梅彭文韩成云谢道昕朱旗彭志红陈峰
Owner HUNAN AGRICULTURAL UNIV
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