Tobacco cadmium transporter gene NtNRAMP6A and application thereof
A technology of transgenic plants and genes, applied in the field of genetic engineering, can solve the problems of affecting the biological yield and quality, affecting the growth of tobacco roots and the formation of chloroplasts, etc.
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Embodiment example 1
[0017] Example 1: Cloning of tobacco NtNRAMP6A gene
[0018] After sowing tobacco K326, when 5-6 true leaves were cultivated in the greenhouse, the fresh leaves were taken to extract total RNA using the plant total RNA extraction kit of Axygen Company, and cDNA was synthesized by reverse transcription using the reverse transcription kit of TaKaRa Company.
[0019] The NRAMP gene (accession number: XM_016625392.1) in the Genbank database was used as the reference sequence, and Primer 5 was used to design the forward and reverse primers. The nucleotide sequence of the forward primer is SEQ NO: 3: 5'-ATGGCGGCGAACTCGACCCCA-3', and the nucleotide sequence of the reverse primer is SEQ NO: 4: 5'-TCAATTAGTGGTCCTCTGCTGA-3'.
[0020] Tobacco NRAMP6A gene was amplified using KOD Fx NEO high-fidelity enzyme from NEB Company using the cDNA template of tobacco 'K326'. The reaction volume is 50 μL: 2×PCR Buffer 25 μL, dNTPs 10 μL, KOD Fx NEO 1 μL, forward primer 2 μL, reverse primer 2 μL, t...
Embodiment example 2
[0024] Example 2 Transformation and phenotypic determination of cadmium-sensitive yeast
[0025] 1. Yeast Competent Preparation
[0026] 1) The yeast strain is a cadmium-sensitive strain of Δycf1 (MATa; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YDR135c::kanMX4). The yeast strain (Δycf) was picked and streaked on YPDA solid medium, and incubated upside down at 30° C. until the bacteria grew to an appropriate size. A single colony was inoculated into a centrifuge tube containing 3 mL of YPDA liquid medium, and the bacteria were shaken at 30°C and 250 rpm for 8–12 hours. Pipette 5μL of bacterial liquid into a 250mL conical flask containing 50mL YPDA liquid medium. Continue to shake at 30°C and 250rpm for 16-20 hours. During this period, measure the OD value. Stop shaking the bacteria when the OD600 reaches 0.15-0.3.
[0027] 2) At room temperature, the cells were collected by centrifugation at 700g for 5 min, the supernatant was discarded, and 100 mL of YPDA liquid medium was added to r...
Embodiment example 3
[0040] Implementation case 3 Tobacco NRAMP6A gene editing and cadmium determination
[0041] 1) SgRNA design and vector construction of NRAMP6A gene
[0042] The specific sgRNA was designed as: GAGCTTGGATATGCAAAGC, and the sgRNA was connected to the vector by the gateway method to obtain a recombinant vector, and a recombinant CRISPR / Cas9 vector was obtained.
[0043] 2) Genetic transformation and mutation detection
[0044] The constructed recombinant CRISPR / Cas9 vector was transferred to Agrobacterium-competent GV3101 by heat shock. By obtaining positive clones, the recombinant CRISPR / Cas9 vector was transferred into cultivated tobacco K326 through tobacco genetic transformation experiment, and grew on the resistant medium. The resistant shoots are transferred to the rooting medium. After the roots grow, a small amount of water will be added to harden the seedlings for 2-3 days; cultured in an artificial climate chamber. The genomic DNA was extracted and sequenced to iden...
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