Salvia miltiorrhiza erf-ⅶ transcription factor smerf73 and its application
A technology of tanshinone and dihydrotanshinone, which is applied in the fields of application, genetic engineering, and plant gene improvement, and can solve problems such as interactions that are rarely reported
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Embodiment 1
[0065] Example 1. Discovery of ERF-VII group transcription factor SmERF73
[0066] 1. The discovery of ERF-VII transcription factor SmERF73
[0067] Using biological / abiotic elicitor induction, gene cloning and other experiments and bioinformatics analysis methods, YE / Ag + An ERF subfamily gene was obtained by screening the transcriptome after induction of hairy roots of Salvia miltiorrhiza, which was named SmERF73, as shown in SEQ ID No. 1, and the encoded protein was shown in SEQ ID No. 2.
[0068] 2. Construction of a plasmid containing the SmERF73 gene
[0069] Taking the cDNA of Salvia miltiorrhiza as the template, the primer pair consisting of primer Isotig10181-F and primer Isotig10181-R was used for PCR amplification. , Beijing Quanshijin Biotechnology Co., Ltd.) to obtain a Blunt Zero plasmid containing the SmERF73 gene (which has been verified by sequencing).
[0070] Isotig10181-F: 5'-GTACAGATTTGGAAGACATTTCACC-3';
[0071] Isotig10181-R: 5'-CAACACAAACTACCAACACTC...
Embodiment 2
[0072] Example 2. Transcriptional characteristic analysis of SmERF73
[0073] 1. Subcellular localization
[0074] 1. Construction of E3025-SmERF73 plasmid
[0075] The plasmid containing the SmERF73 gene prepared in Example 1 was used as a template, and the primer pair consisting of primer E3025-SmERF73-F and primer E3025-SmERF73-R was used for PCR amplification, and the amplified product was digested with Nco I and Kpn I double enzymes Then, it was ligated with the E3025-GFP vector that was also double-enzyme digested with Nco I and Kpn I to obtain the E3025-SmERF73 plasmid (which has been verified by sequencing).
[0076] E3025-SmERF73-F: 5'-CCCATGGACATGTGTGGCGGTGC-3';
[0077] E3025-SmERF73-R: 5'-GGGGTACCAGTGTTCACCGGAAACT-3'.
[0078] 2. Preparation of DNA gold powder suspension
[0079] (1) Weigh 20 mg of PVP (Code No. 0507-500G, Amresco, USA), add 1 mL of 100% absolute ethanol, put it into a screw tube, and dilute it with ethanol to 0.01-0.1 mg / mL, that is, take 25 μ...
Embodiment 3
[0114] Example 3. Transgenic analysis of SmERF73
[0115] 1. Construction of the interference vector
[0116] 1. Using the Blunt Zero plasmid containing the SmERF73 gene prepared in Example 1 as a template, a primer pair consisting of primer pK7GWIWG2D-SmERF73-F and primer pK7GWIWG2D-SmERF73-R was used to carry out PCR amplification, and the amplified product was purified and then subjected to BP reaction .
[0117] pK7GWIWG2D-SmERF73-F: 5'-caccGCCCACGCTTACGACAGAGAG-3';
[0118] pK7GWIWG2D-SmERF73-R: 5'-GTGTTCACCGGAAACTTCATCG-3'.
[0119] BP reaction system: 1 μL water, 1 μL Salt, 1 μL PCR purified product, 0.5 μL pENTR / SD / D-TOPOVector (Invitrogen company). The above reagents are from pENTR Directional Cloning Kits (Code No. K2525-20), Invitrogen, USA.
[0120] BP reaction conditions: ligation at 22°C for 1.5h.
[0121] 2. Take the BP reaction product of step 1 and carry out the LR reaction.
[0122] LR reaction system: 2 μL TE Buffer (pH8.0), 1 μL BP reaction product,...
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