Genetic engineering application of soybean ribosomal protein coding gene GmRPL12
A ribosomal protein and gene-encoding technology, which can be used in genetic engineering, application, plant genetic improvement, etc., and can solve problems such as different and unpredictable functions
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Embodiment 1
[0023] 1) Cloning of soybean ribosomal protein encoding gene GmRPL12
[0024] Take soybean variety Kefeng No. 1 as the material, take its roots, grind them with a mortar, add to a 1.5mL EP tube containing lysis solution, and after sufficient shaking, move it to a 1.5mL EP tube to extract total RNA (Total RNA). RNA Kit (Tiangen, Beijing, China). Identify total RNA quality with formaldehyde denaturing gel electrophoresis, and spectrophotometer measures RNA content. The total RNA obtained is a template, according to the reverse transcription kit (TaKaRa Primer) provided by Japanese TaKaRa company Script TM RT reagent kit, Japan) for reverse transcription, and after obtaining the first strand of cDNA, PCR amplification is performed. The PCR program is as follows: The PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, and annealing at 60°C for 15 seconds , 72 ℃ extension for 45 seconds, a total of 35 cycles, the last 72 ℃ incub...
Embodiment 2
[0035] 1) Construction of plant expression vector
[0036] Design primers containing BamHI and KpnⅠ double restriction sites and only the full length of the candidate gene, and use the CDS plasmid containing the target gene as a template for PCR amplification (see SEQ ID NO. 9 and SEQ ID NO. 10 for primer sequences) . The PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 45 seconds, a total of 35 cycles, and a final incubation at 72°C for 5 minutes, followed by constant temperature at 4°C. The CDS sequence of the soybean GmRPL12 gene with a length of 576 bp in the complete coding region is shown in SEQ ID NO.1. The amplified product and pMDC83 empty vector were double digested with BamHI and KpnⅠ, and the digested vector was purified. The PCR product was combined with the digested pMDC83 linear vector according to Novozan Entry One Step Cloning Kit (Vazyme, C114-01...
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