Panax notoginseng transcription factor gene PnMYB1 and application thereof
A technology of transcription factor and Panax notoginseng, which is applied in the fields of molecular biology and genetic engineering, can solve the problems of few reports and unclear functions of 4R-MYB, and achieve the effect of increasing expression and increasing yield.
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Embodiment 1
[0023] Example 1: PnMYB1 Cloning and sequence analysis of full-length genes
[0024] The total RNA of Panax notoginseng cells treated with methyl jasmonate for 3-6 h was extracted by the improved guanidine isothiocyanate method ( figure 1 ), and refer to NucleoTeap mRNA (MACHERGY-NAGEL) kit instructions for the isolation of mRNA ( figure 2 ); Take 1 μg mRNA to construct a cDNA library according to the Matchmaker™ Gold Yeast One-Hybrid Library Screening System, and use the Trimmer-2 cDNA normalization kit to normalize the library.
[0025] Design the bait sequence with added restriction sites Xho I and Hind III --- three repeated JERE sequences, and synthesize the sequences into double strands by annealing. Digest the pAbAi vector and the JERE bait sequence with XhoⅠ and HindⅢ, recover the target fragment from the gel, connect the recovered JERE bait fragment to the linear pAbAi vector fragment overnight at 4°C, and then transform Escherichia coli competently, pick a single...
Embodiment 2
[0028] Embodiment 2: plant expression vector construction
[0029] according to PnMYB1 Primers were designed for the 5' and 3' end sequences of the gene ORF box and restriction sites were added. The upstream primer: 5'-GGATCCATGGGGAGGAGCCCTTGCTGTGC-3'; the downstream primer: 5'-GAATTCTCAAGACAGCCAATCTCCTCCGGAC-3'. for amplification PnMYB1 Gene ORF. PCR reaction conditions: 94°C, 5 min; 94°C, 30 s; 63.5°C, 30s; 72°C, 50s, 32 cycles; 72°C, 10 min. The PCR product was separated with 1% agarose gel, recovered from the gel and connected to the pGEM-T vector, transformed into competent Escherichia coli, picked a single clone and shaken, and sent to sequence after PCR detection of the bacterial solution.
[0030] The Escherichia coli plasmid pGEM-T- PnMYB1 As well as the plasmid of the plant expression vector pCAMBIA2300S, 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. Plasmid pGEM-T- PnMYB1 Carry out double enzy...
Embodiment 3
[0033] Example 3: Genetic transformation of Panax notoginseng mediated by Agrobacterium
[0034] The preserved recombinant plasmid pCAMBIA2300S- PnMYB1 Agrobacterium EHA105 was inoculated at 1% inoculum in 5 mL of LB liquid medium containing 50 mg / L Kana and 25 mg / L rifampicin, and cultured at 28°C until turbid. Take 1 mL of turbid bacterial liquid and spread it on LB solid medium containing 50 mg / L Kana and 25 mg / L rifampicin, and incubate at 28°C for 48 hours. A proper amount of Agrobacterium was scraped from the LB solid medium and cultured in MGL liquid medium containing 40 mg / L acetosyringone at 200 rpm at 28°C until the OD600 was 0.6. Stop the shaking and use for dipping.
[0035] The notoginseng callus in good growth state was inoculated on MS solid medium containing 40 mg / L acetosyringone, and pre-cultured for 3 days. The notoginseng callus pre-cultured for 3 days was completely immersed in the above-mentioned Agrobacterium solution with an OD600 of 0.6 for shaking c...
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