Plant expression vector based on Arabidopsis thaliana (At)-pri-miR828 gene and construction and application thereof
A technology of pot2-at-pri-mir828 and plant expression vector, which is applied in the field of bioengineering and can solve problems such as poor results
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Embodiment 1
[0040] Example 1: Construction of plant expression vector pC2300-pOT2-At-pri-miR828.
[0041] Plant material: Columbia ecotype (Columbia-0) wild type Arabidopsis ( Arabidopsis thaliana ) Seed and wild type tomato ( Solanum lycopersicum L. Ailsa Craig) seeds.
[0042] Strains and plasmids: Escherichia coli ( E.coli ) Strain DH5α, Agrobacterium strain GV3101, modified cloning vector pOT2 with 35s promoter, and modified with Pac I single restriction site expression vector pC2300.
[0043] Enzymes and chemical reagents: LA Taq DNA polymerase, common Taq DNA polymerase, T 4 DNA ligase, purification and recovery kit, plasmid extraction kit, reverse transcription kit, fluorescence quantification kit, Marker DL2000 and Marker DL2000 were all purchased from Takara, China; Tans2K TM Plus II DNA Marker was purchased from Beijing Quanshijin Company; DNA restriction endonuclease Hin d III, Eco R I, Pac I was purchased from NEB, UK; TRizol was purchased from Invitrogen, USA. The PC...
Embodiment 2
[0056] Example 2: Genetic transformation of tomato by plant expression vector pC2300-pOT2-At-pri-miR828.
[0057] 1. Agrobacterium-mediated transformation of tomato with leaf disc method.
[0058] Sterile seedling culture: first soak tomato seeds with 75% ethanol for 5 minutes, pour out the ethanol and rinse with sterile water; then use saturated Na 3 PO 4 ·12H 2 Soak in O solution for 20 minutes, and rinse once with sterile water; finally, soak in 50% NaClO solution for 10 minutes, and rinse with sterile water 4-6 times. Evenly inoculate the sterilized seeds in MSR3 medium (4.4g·L -1 MS+30g·L -1 Sucrose+1mL·L -1 R3 vitamins+10g·L -1 Agar powder, pH 5.9), placed in an incubator at 26°C (16h light) / 18°C (8h dark) for about 8-10 days.
[0059] Pre-culture: When the aseptic seedling grows until the cotyledons are fully expanded but the true leaves do not grow out, cut the cotyledons and cut off the two tips of the cotyledons, and place them in MS+2-4D liquid culture medium (4.4g·L -1 ...
Embodiment 3
[0068] Example 3: Real-time fluorescent quantitative PCR analysis of transgenic tomato plants.
[0069] The tender leaves of one transgenic plant were selected from the two miR828 overexpression transgenic tomato lines that were positive by PCR, and the total RNA was extracted with TRizol and treated with DNaseⅠ. RNA concentration and quality were identified with a nucleic acid protein detector (Nanodrop ND-1000, thermo) and electrophoresis. Take tomato Sly - U6 As an internal reference gene, the expression of miR828 gene was detected by real-time fluorescence quantitative method; Sly-CAC It is an internal reference gene to detect miR828 target genes Sly - myb-like1 (SGN320618) Expression level.
[0070] miR828 The sequences of the three primers are shown in Table 1, namely miR828 stem-loop RT primer, miR828 forward primer and miR828 universal reverse primer. The synthesis of the first strand of cDNA is based on PrimeScript TM 1st Strand cDNA Synthesis Kit (TaKaRa, China) m...
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