Cloning vectors are used to clone and amplify target gene fragments in host cells. This type of vector contains a replication signal sequence, which can replicate in host cells to obtain high-copy DNA molecules, but cannot be expressed because it does not contain a promoter sequence or the promoter sequence is not recognized by the host cell RNA polymerase. NAC transcription factors have many functions, such as participating in plant secondary growth, playing a role in cell division and plant senescence, participating in hormone regulation and signal transduction, participating in mineral element nutrition and crop quality improvement, etc. At the same time, NAC is also involved in plant defense responses to biotic stress and plays a role in abiotic stress.
A common strategy for cloning vectors is TA cloning. This strategy utilizes the property of adding an A to the 3' end of the PCR product when performing PCR reactions with most thermostable DNA polymerases, and connects the PCR product to a vector with a 3'T sticky end to form a recombinant. This experiment takes the pMD18-T vector as an example. This vector is a special vector for efficient cloning of PCR products. It is modified based on the pUC18 vector. The EcoRV recognition site is inserted between the XbaI and SalI recognition sites at the multiple cloning site of the pUC18 vector. After digestion with EcoRV, "T" is added to the 3' ends on both sides. This strategy greatly improves the efficiency of ligation and cloning of PCR products.
1. Primer design
Design a forward primer before the start codon of the soybean NAC gene and a reverse primer after the stop codon.
2. PCR amplification and purification
Using soybean cDNA as a template, Taq DNA polymerase was used for PCR amplification, as shown in the reaction system in Table 1. Mix the mixture gently and centrifuge briefly. Collect the components at the bottom of the tube and set the PCR machine according to the reaction program in Table 2.
Table 1. Reaction System for PCR Amplification
| Component | 2X Taq Mix | Forward / Reverse Primer (10 µmol/L) | Soybean cDNA | ddH2O |
| Volume | 25 µL | 1 µL | 5 µL | 19 µL |
Table 2. Reaction Procedure for PCR Amplification
| PCR Program (35 Cycles) | 94 °C | 55 - 65 °C | 72 °C |
| Time | 30 s | 1 min | 1 min |
After amplification is completed, use 0.8% agarose gel for electrophoresis, cut the gel and recover it, and the purified product will be used immediately or temporarily stored at -20°C.
3. Ligation reaction
Mix the prepared soybean NAC gene fragment and pMD18-T vector. The reaction system is shown in Table 3. Flick the mixture to mix and centrifuge briefly to collect the ingredients at the bottom of the tube. The ligation reaction was carried out for 4 h at 16°C.
Table 3. Ligation System of pMD18-T-NAC
| Ligation System | NAC Purification Product | pMD18-T Vector | Solµtion I |
| Volume | 4.5 µL | 0.5 µL | 5 µL |
4. Heat shock transformation
Add 10 µL of the ligation product to a 1.5 mL tube in an ice bath, then add 50 µL of DH5α competent cell suspension, gently pipette with the pipette tip to mix, and let stand on ice for 30 minutes. The transformation product was heat-shocked in a 42°C water bath for 90 s, and immediately placed on ice to cool for 3 min. Then add 800 µL LB liquid culture medium to the tube, and incubate at 37°C with shaking at 180 r/min for 50 minutes until the bacterial solution is slightly turbid.
Take 20-100 µL of the above bacterial solution and spread it on the plate. After the bacterial solution is completely absorbed by the culture medium, incubate it overnight at 37°C.
5. Screening
Uniform white colonies can be seen on Amp-resistant plates cultured overnight. Use a pipette tip to pick 3 to 10 colonies, add them to the LB liquid culture medium containing Amp resistance, and culture them at 37°C, shaking at 180 r/min for 3-5 h until the bacterial liquid becomes turbid. Take 1 µL of each bacterial solution for PCR verification.
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