Short-day sensitive cell death SSD1 gene and coded protein and application thereof
A technology of cell death and genetics, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the complex problems of biological cells
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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 80-120μmol m -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 disinfecti...
Embodiment 2
[0028] Embodiment 2: Map-based cloning method to isolate SSCD1 gene
[0029] 1. Construction of map-based cloning populations
[0030] Crossing the sscd1 mutant (Col-0 background) with another ecotype Landsberg erecta (Ler) yielded F l Generation heterozygote; F l F 2 generation; seeding F 2 Generation seeds were screened for individuals with cell necrosis under short-day conditions, and a row of sscd1 mutants was 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 s...
Embodiment 3
[0048] Embodiment 3: Genetic complementation experiment of SSCD1 gene
[0049] 1. Construction of Complementary Vectors
[0050] The high-fidelity enzyme TransStart FastPfu DNA Polymerase (TransGen) was used to amplify a fragment containing the SSCD1 gene from Arabidopsis wild-type Col-0, with a full length of 3175bp, and the primer FP (5'- gta CCCGGG ATGGCGTTGCTGAAGTCTTT-3’) and RP (5’-cga GAGCTC ACTTATTGTTAATGGGTTGG-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 is: 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.
[0051] The carrier plasmid pROK2 and the purified PCR product were digested with SmaI and SacI, and T4 DNA Ligase (TransGen) was used to connect the d...
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