Tobacco KUP8 protein and encoding gene and application thereof
A technology that encodes genes and tobacco, applied in the field of genetic engineering, can solve problems such as lack of absorption capacity, reduced survival rate, enhancement, etc., and achieve the effect of promoting potassium ion absorption and transport
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Embodiment 1
[0039] The acquisition of embodiment 1 KUP8 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'-ATGGATCGGCAAACAGGAAG-3', and the nucleotide sequence of the reverse primer is 5'-TCAAGCCTCATAAAGCATAC-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 KUP8 gene in promoting potassium ion uptake and transport
[0045] The T-vector connected with the KUP8 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, the selection marker in yeast is URA3, The screening marker in Escherichia coli is Amp. It is recorded in Functional Expression of aω-3Fatty Acid Desaturase Gene from Glycine max in Saccharomyces cerevisiae) to carry out double enzyme digestion (restriction site: SmaI and XhoI), and recover the target gene and expression vector P416, Then connect with ligase, 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 construct the successful recombinant The yeast exp...
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