A kind of tobacco kup7 protein and its coding gene and application
A technology encoding genes and proteins, applied in the field of genetic engineering, can solve the problems of lack of absorption capacity, large potassium consumption, unknown tobacco KUP function, etc., and achieve the effect of promoting potassium ion absorption and transport.
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Embodiment 1
[0039] The acquisition of embodiment 1 KUP7 gene
[0040] Get 0.5g of fresh tobacco leaves, use the Trizol method to extract the total RNA of tobacco cells, then use the cDNA synthesis kit of TaKaRa company to synthesize cDNA, and further use the Primer5.0 software to design and obtain primers through artificial optimization. The primers include forward primers and a reverse primer, the nucleotide sequence of the forward primer is: 5'-ATGGTGAATGTGGGATTGGA-3', and the nucleotide sequence of the reverse primer is 5'-TCACACCATATATGTCATGC-3'. Using the synthesized cDNA as a template, PCR amplification was carried out.
[0041] The PCR amplification system is a 20 μL system, including: Premix ExTaq 10 μL, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, tobacco cell cDNA 1 μL, ddH 2 O 8 μL.
[0042]The PCR amplification reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and 35 ...
Embodiment 2
[0044] The role of embodiment 2 KUP7 gene in promoting potassium ion uptake and transport
[0045] The T-vector connected with the KUP7 gene described in Example 1 and the expression vector P416 (p416 yeast free type shuttle expression vector, TEF constitutive promoter, CYC1 terminator, CEN6ARSH4 replication origin, and the screening marker in yeast is URA3, The screening marker in Escherichia coli is Amp. Excerpted from Functional Expression of aω-3Fatty AcidDesaturase Gene from Glycine max in Saccharomyces cerevisiae)
[0046] Carry out double enzyme digestion (restriction sites: Sma Ⅰ and Xho Ⅰ), recover the target gene and expression vector P416, and then connect with ligase, and transfer the ligated recombinant yeast expression vector into the competent cells of Escherichia coli DH5α , carry out PCR amplification and enzyme digestion on the transformed Escherichia coli single colony to verify whether the construction is successful, and transfer the successfully constructe...
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