Corn raffinose synthesis key enzyme gene and application thereof
A key enzyme gene, raffinose technology, applied in application, genetic engineering, glycosyltransferase, etc., to achieve the effect of improving drought resistance
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Embodiment 1
[0015] Example 1. The present invention cloned the coding region of the maize ZmRAFS gene by PCR method, and constructed the gene plant expression vector.
[0016] Dehydration treatment was performed on hydroponic three-leaf stage corn seedlings. RNA was extracted from leaves dehydrated for 2 hours and reverse transcribed into cDNA.
[0017] Using cDNA as a template, the coding region of ZmRAFS was amplified by PCR with the upstream primer 5'-CGCGGATCCTGGCTCCCAACCTCAGCAAGAAG-3' and the downstream primer 5'-TGCTCTAGAGGTAGACGTACTGGACGCGACACAG-3'. The amplification program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 1 min 20 s, 35 cycles; 72°C final extension for 10 min. The amplified product was gel-cut and recovered, and the fragment was digested with BamHI and XbaI, and then combined with the pC sGFPBT vector (for stable transformation of Arabidopsis, see figure 1 )connect. The ZmRAFS ORF fragment on the vect...
Embodiment 2
[0019] Embodiment 2, the molecular identification of transgenic strain
[0020] The mRNA expression level of transformed plants was identified by RT-PCR. The expression level of ZMRAFS protein in transformed plants was identified by Western blot.
[0021] The RT-PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 28 cycles; final extension at 72°C for 8 min.
[0022] For Western blot steps, refer to the following prior art.
[0023] Gu,L.,et al.,ZmGOLS2,a target of transcription factor ZmDREB2A,oferssimilar protection against biotic stress as ZmDREB2A.Plant Mol Biol,2016.90(1-2):p.157-70.
[0024] The ZmRAFS primary antibody was prepared from rabbits immunized in the research group, and the dilution factor was 1:5000; the secondary antibody (goat anti-rabbit) was purchased from Kangwei Company, and the dilution factor was 1:10000.
Embodiment 3
[0025] Example 3. Drought resistance identification of maize mutant lines and Arabidopsis transgenic lines.
[0026] Drought resistance test of maize under drought stress: the zmrafs-1 mutant and co-segregated NS plants were planted in the greenhouse, and one NS and one zmrafs-1 material were planted in the same pot. The soil is mixed with the surface soil of the field and the substrate at a ratio of 1:1, and part of the soil is dried and weighed to calculate the soil moisture content. The drought treatment was started 55 days after sowing, the control group was watered normally, the water supply was stopped in the treatment group, and the soil water loss rate was recorded every day. At the same time, the phenotype changes of the leaves of the plants were observed and recorded by taking pictures.
[0027] Drought resistance test of transgenic Arabidopsis seedlings: Weigh the same weight of nutrient soil in a four-hole small pot, then absorb water from the bottom to make the so...
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