Construction of Tomato PIP Subcellular Localization Vector

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Expression of some eukaryotic proteins in prokaryotic host cells is efficient and cost-effective. However, many eukaryotic proteins synthesized in bacteria have low or inactive protein activity due to incorrect folding methods or low folding efficiency. In the post-genomic era, in order to study the cellular localization and function of a certain gene, it is more necessary to use eukaryotic expression vectors to construct a eukaryotic expression system to complete scientific research work. Research shows that 70% to 90% of water movement in the body is carried out through PIP aquaporins. In many plants, aquaporins are expressed and abundantly found in vascular bundles and nearby tissues.

Principle

This experiment takes the construction of the pUC-SPYNE vector as an example. The vector has a 35S promoter upstream of the multiple cloning site. This promoter is the promoter of cauliflower virus. It is a strong promoter that can stably activate the expression of downstream genes in plant cells and is widely used in transgenic plants.

Subcellular localization refers to the specific location within a cell where a certain protein or expression product exists, for example, in the nucleus, cytoplasm, or on the cell membrane. There is a GFP sequence in the pUC-SPYNE vector sequence, which is green fluorescent protein. It emits green fluorescence under the laser irradiation of a scanning coaggregation microscope, so that the position of the protein can be accurately located.

GFP actually labels the substance to be studied, which is equivalent to the function of a reporter protein. To use GFP, a fusion protein vector must be constructed and efficiently expressed after transfection. Since a fusion protein is being constructed, all sequences from the start codon to the stop codon must be cloned when cloning the target gene. In this way, if a GFP signal is seen in a certain part of the cell under a fluorescence microscope, it means that the protein fused to GFP also exists in that part, thus achieving the purpose of determining the subcellular localization of a certain substance.

Procedures

1. Digestion and recovery of pMD18-T-PIP and pUC-SPYNE

Take two 250 µL microcentrifuge tubes, add the reagents according to the enzyme digestion system in Table 1 and Table 2, mix well, centrifuge briefly, and digest overnight at 37°C.

Table 1. Enzyme Digestion System of pMD18-T-PIP

Component	10X 2.1 Buffer	pMD18-T-PIP	Xba I	Xho I	ddH₂O
Volume	5 µL	1-2 µg	1 µL	1 µL	Make up to 50 µL

Table 2. Enzyme Digestion System of pUC-SPYNE

Component	10X 2.1 Buffer	pUC-SPYNE	Xba I	Xho I	ddH₂O
Volume	5 µL	1-2 µg	1 µL	1 µL	Make up to 50 µL

After the reaction is completed, use 0.8% agarose gel for electrophoresis and serve as a negative control when loading the sample. After verifying that the enzyme digestion is correct, cut the gel and recover it, and the purified product will be used immediately or temporarily stored at -20°C. The obtained fragments were PIP gene fragment and pUC-SPYNE vector fragment respectively

2. Ligation reaction

Add the following reagents in sequence to a 250 µL microcentrifuge tube according to Table 3, mix and centrifuge briefly. The ligation reaction was carried out for 1h or overnight.

Table 3. Ligation System of pUC-SPYNE-PIP

Component	10X Ligation Buffer	PIP fragment	pUC-SPYNE fragment	T4 Ligase	ddH₂O
Volume	2 µL	5 µL	1 µL	0.5 µL	Make up to 20 µL

3. Heat shock transformation

Add 20 µL of the ligation product to a 1.5 mL tube in an ice bath, then add 50 mL of DH5a competent cell suspension, pipe gently with the pipette tip to mix, and let stand on ice for 30 min. The transformation product was heat-shocked in a 42°C water bath for 90 s and immediately cooled on ice for 3 min. Then add 800 µL LB liquid culture medium to the tube, and incubate at 37°C, 180 r/min with shaking for 50 min 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.

4. Screening

Uniform white colonies can be seen on Amp-resistant plates cultured overnight. Use a pipette tip to pick 5-10 colonies, add them to LB liquid culture medium containing 1 mL of Amp resistance, and culture them at 37°C with shaking at 180 r/min for 3-5 h until the bacterial liquid becomes turbid. Take 1 µL of each bacterial liquid for PCR verification. If the verification is correct, sequencing verification can be performed.

Note:

When selecting restriction sites, first analyze the restriction sites on the cloned fragments. The restriction sites should be selected to avoid restriction sites on the cloned fragments.
PCR verification may be inaccurate during screening. Sequencing verification or double digestion verification can ensure the correctness of the connection.

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