Lycium chinense Miller GGPS1 gene for improving stress resistance of plant and recombinant vector comprising the gene
A technology of recombinant vectors and genes, applied in the direction of plant gene improvement, introduction of foreign genetic material using vectors, plant products, etc.
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Embodiment 1
[0049] Cloning of geranylgeranyl pyrophosphate synthase gene LmGGPS1 from Lycium barbarum
[0050] GGPS1 3' end cloning:
[0051] Using Trizol reagent, extract total RNA from 100 mg of fresh Lycium barbarum leaves, and design upstream primers according to the Unigene sequence of the transcriptome. The upstream primer of the geranyl geranyl pyrophosphate synthase gene LmGGPS1 in Lycium barbarum is LmGGPS1-F1:5'TAGGTGGCGGGAACGAAGAT- 3', using the 3' FULLRACE Core Set Ver.2.0 (TaKaRa, Japan) kit to amplify the complete gene sequence. Specific steps: ①Using totalRNA as a template, use 3'RACE Adapter primers for reverse transcription reaction to synthesize 1st Strand cDNA. The reaction system is as follows:
[0052] RNA 2μL. 3' RACE Adapter 1μL 5×M-MLV Buffer 2μL dNTP Mixture 1μL RNase Inhibitor 0.25 μL Reverse Transcriptase M- MLV 0.25 μL RNase Free ddH 2 o 3.5 μL
[0053] Reaction conditions: 42°C, 60min; 70°C, 1...
Embodiment 2
[0064] Construction process of cloning vector pMD19-T-LmGGPS1
[0065] The LmGGPS1 gene shown in the sequence listing is connected to the pMD19-T carrier, and the reaction system is as follows:
[0066] LmGGPS1 full-length recovery product 4μL pMD19-T vector 1μL Solution I 5μL
[0067] LmGGPS1 PCR fragment is the reclaimed in embodiment 1 GGPS1 full length product.
[0068] Reaction conditions: 16°C, 30min. The ligation product was transformed into competent E-Coli.TOP10, spread on LB agar plate medium containing 40ml 25mg / ml X-Gal, 16ml 50mg / ml IPTG, 100mg / L Amp and cultured to form a single colony. Select white colonies, colony PCR method confirms the length of the insert in the T vector, which is consistent with expectations, and sends the vector to Huada Gene Company for sequencing. We obtained the 1074bp base sequence of the gene, blasted it at NCBI, and compared it with solanum The family homology is high, indicating that the gene was s...
Embodiment 3
[0070] Construction process of Escherichia coli expression vector pET28a-LmGGPS1
[0071] The Escherichia coli containing the pMD19-T-LmGGPS1 plasmid obtained in the expanded culture experiment example 2 was extracted, the plasmid was double-digested with BamHI and SalI, and the pET28a empty vector was double-digested with BamHI and SalI, purified and ligated , to obtain the Escherichia coli expression vector pET28a-LmGGPS1, and connect the digested products of the two.
[0072] The connection system is as follows:
[0073] pET28a empty vector fragment 2μL pMD19-T-LmGGPS1 restriction fragment 5μL 5×T4 DNA ligase buffer 2μL T4 DNA ligase 1μL
[0074] The ligation product was transformed into competent Escherichia coli BL21. Spread on LB plates containing 100mg / L kana resistance and culture at 37°C. After 12 hours, a single colony was picked for colony PCR verification, and the colony PCR verification positive bacteria were shaken to extra...
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