Application of poplar salt tolerant gene PtoeIF5A1
A salt-tolerance and gene-encoding technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as unclear biological functions of eIF5A
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Embodiment 1
[0036] Example 1, construction of pBSK-Ω-Flag-PtoeIF5A1 vector
[0037] 1. The leaves of the tower-shaped Populus tomentosa CV-BJHR01 seedlings with a growth time of 3 months were selected, and the total RNA was extracted after liquid nitrogen quick freezing and reverse transcribed into cDNA.
[0038] 2. Through the analysis of the Populus tomentosa genome library (http:∥genome.jgi-psf.org / Poptr11 / Poptr11.home.htm1), obtain the full sequence of the predicted PtreIF5A1 gene of Populus tomentosa, and design primers in its non-coding region to The cDNA of the tower-shaped Populus tomentosa CV-BJHR01 (hereinafter referred to as Populus tomentosa cDNA) obtained in step 1 was used as a template for nested PCR amplification.
[0039] The nested PCR system is as follows:
[0040] The first round of PCR:
[0041] System: Populus tomentosa cDNA 2.0 μL, 10 μmol / L upstream and downstream primers 1 μL each, 2.0 U / L Phusion DNA polymerase 0.2 μL, 2.5 mmol / L dNTPs mixture 1.6 μL, 5×HF Buff...
Embodiment 2
[0061] Embodiment 2, flower bud soaking method transforms Arabidopsis thaliana
[0062] The method of transgenic Arabidopsis adopts the flower bud soaking method of people such as Clough, and concrete process is as follows:
[0063] 1. Pick the single colonies of the recombinant bacteria obtained in Example 1 and the control recombinant bacteria into 5ml LB (containing Kan50μg / ml, Gent25μg / ml), and culture at 28°C for one day.
[0064] 2. Transfer the cultured bacterial solution to 200ml LB containing the same antibiotic at a volume ratio of 1:100, and continue to culture to OD 600 =1.0, centrifuge at 5000g room temperature for 10min, resuspend the cell pellet in 200ml 1 / 2MS medium (containing 50g / l sucrose, 200μl / l Silwet L-77), respectively, to obtain recombinant bacteria and control recombinant bacteria suspension.
[0065] 3. Invert the Arabidopsis thaliana to be transgenic, soak the inflorescence above the rosette leaf in the recombinant bacteria or control recombinant b...
Embodiment 3
[0067] Embodiment 3, the screening of transgenic Arabidopsis
[0068] 1. Treat transgenic Arabidopsis and control Arabidopsis T0 generation seeds with 70% ethanol water solution by volume percentage for 1 min, wash with sterilized water for 2 min, then treat with sodium hypochlorite for 13 min, and then wash with sterile water for 5 times, each time After 2 minutes, the treated seeds were placed at 4°C for vernalization for 2-3 days.
[0069] 2. Spread the vernalized seeds evenly on 1 / 2MS screening medium (10g / l sucrose, 50μg / ml Kan, 8g / l agar, pH=5.7), and move them into a 22°C incubator (16hr light, 8hr dark , the light intensity is 80-100Lux) to germinate.
[0070] 3. About 6 days later, the transgenic Arabidopsis seedlings had green leaves and longer roots, while the control Arabidopsis could also germinate on the 1 / 2MS screening medium, but the seedlings had yellower leaves and shorter roots than the transgenic seedlings at the same time. Transfer the seedlings of trans...
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