Primers, kits and applications for controlling Arabidopsis leaf senescence
A technology of leaf senescence and Arabidopsis thaliana, applied in the field of plant genetic engineering, can solve problems such as difficult to be used and poor agronomic traits
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Embodiment 1
[0049] Cloning of TaNACS gene and construction of overexpression vector
[0050] Using Chinese spring wheat cDNA as template, PCR amplification was carried out with SEQ ID NO.1 as upstream primer and SEQ ID NO.2 as downstream primer. The template concentration was 50ng / ml, and the amplification procedure was as follows:
[0051] 95°C for 3min; 33 cycles of 95°C for 30s, 59°C for 30s, 72°C for 1min; 72°C for 10min.
[0052] The PCR amplified product was connected to the pCambia3301 overexpression vector (ubi-driven overexpression) by infusion recombinase, finally forming the recombinant vector pCambia3301-TaNACS.
[0053] The connection method is:
[0054] (1) Digest the pCambia3301 vector with EcoRI and BamHI, and recover the fragments for later use;
[0055] (2) Establish the ligation system: 1.5 μL of the digested pCambia3301 vector; 2.5 μL of PCR fragments; 1 μL of infusion enzyme:
[0056] (3) Establish reaction conditions: 50°C for 15 minutes, place on ice for 5 minute...
Embodiment 2
[0059] 1. Obtain TaNACS gene induction expression vector
[0060] Using the pCambia3301-TaNACS vector as a template, using SEQ ID NO.3 as an upstream primer, and SEQ ID NO.4 as a downstream primer for PCR amplification, the amplification procedure:
[0061] 95°C for 3min; 95°C for 30s, 60°C for 30s, 72°C for 1min, 35 cycles; 72°C for 10min.
[0062] After the amplified fragment was purified and recovered according to the kit instructions, it was connected to the intermediate vector pDonor207 by BP enzyme. After sequencing, the positive clone was selected to extract the plasmid, named TaNACS-in-pDonor207, and the vector was recombined with the final vector pER8 by LR enzyme. , forming an inducible expression vector named TaNACS-pER8; transforming the inducible expression vector into Escherichia coli DH5a for positive identification and sequencing;
[0063] 2. Obtain a 35S-driven TaNACS gene constitutive overexpression vector
[0064] Using the aNACS-in-pDonor207 vector obtain...
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