Parts
This year, we contributed three basic plasmid vectors and four recombinant plasmid vectors, aiming to explore gene interactions and help plants synthesize more flavonoids with the help of different vectors.
We successfully relied on Escherichia coli (DH5α) to construct four plasmid vectors for gene overexpression and in vitro realization of gene interactions.
| Part Name | Short Description | Status | Length(bp) |
| PAL-PCY | PAL code sequenc, PCY-35S-His | Induction of PAL overexpression | 11698 |
| TBH-PCY | TBH code sequenc, PCY-35S-His | Induction of TBH overexpression | 10273 |
| TBH-PJG | TBH code sequenc, PJG4-5 | Activation of the PAL promoter to induce gene expression | 7178 |
| PAL-PlacZi | PAL cis-acting elements on promoters, PlacZi2u | As a mediator of LAc reporter gene coloration | 10357 |
We integrated the CDS sequence of the cucumber PAL gene into our PCY-35S overexpression vector with His tag by homologous recombination, and the recombinant plasmid is shown below（Fig.1）.
After we transformed PCY-35g integrating the CDS sequence of the PAL gene into E. coli DH5α, we picked single colonies for colony PCR. and chose the ones that were successfully transformed for amplification and plasmid extraction, and then transformed them into Agrobacterium to infest plants. The following figure shows the colony PCR results（Fig.2）.
To further confirm that the constructed plasmids were correct, we sent them for sequencing. Below are the sequencing results（Fig.3）.
we integrated the CDS sequence of cucumber TBH gene into our PCY-35g overexpression vector with His tag by homologous recombination, and the following figure shows the recombinant plasmid（Fig.4）.
After we transformed PCY-35g integrating the CDS sequence of TBH gene into E. coli DH5α, we picked single colonies for colony PCR. and chose the successful ones for amplification and plasmid extraction, and then transformed them into Agrobacterium to infest plants. The following figure shows the colony PCR results (yellow box part)（Fig.5）.
To further confirm that the constructed plasmids were correct, we sent them for sequencing. Below are the sequencing results（Fig.6）.
After we transformed PJG integrating the CDS sequence of TBH gene into E. coli DH5α, we picked single colonies for colony PCR. and chose the ones that were successfully transformed for amplification and plasmid extraction, and then transformed them into yeast cells, which in turn were subjected to color development experiments. The following figure shows the colony PCR results (red box part)（Fig.8）.
To further confirm that the constructed plasmids were correct, we sent them for sequencing. Here are the sequencing results（Fig.9）.
we integrated the cis-acting regulatory elements on the promoter of the cucumber PAL gene into our PlacZi vector by homologous recombination, and the recombinant plasmid is shown in the figure below (Fig.10).
To further confirm that the constructed plasmids were correct, we sent them for sequencing. Below are the sequencing results (Fig.12).
In order to construct plasmids that can be transferred into plants for interactions and functional validation, we utilized three basic plasmid vectors.
| Part Name | Short Description | Status | Length(bp) |
| PCY-35S-His | Artificially modified plasmids | Induced gene overexpression | 9544 |
| PJG4-5 | Artificially modified plasmids | Activation of downstream gene promoters to induce reporter gene expression | 6449 |
| PlacZi2u | Artificially modified plasmids | Carrying the LAc reporter gene | 8203 |
The vector contains kanamycin sulfate resistance gene (KanaR), which is used for selection of E. coli; the vector also provides multiple restriction enzyme sites for insertion of exogenous genes; it also contains replicon for E. coli, which ensures stable replication of the vector in E. coli; the core of the vector is the CaMV 35S constitutive promoter, which has various cis-acting elements, including -343 to -46bp upstream of the transcription start site, the transcriptional enhancement region, -343 to -208 and -208 to -90bp, the transcriptional activation region, and -208 to -90bp. The core part of this vector is the CaMV 35S constitutive promoter, which has various cis-acting elements, and the upstream of its transcriptional start site, -343～-46bp, is the transcriptional enhancement region, -343～-208 and -208～-90bp are the transcriptional activation region, -90～-46bp is the transcriptional activation region, and -90～-46bp is the transcriptional activation region. -90 to -46bp is a transcriptional enhancement region, -343 to -208 and -208 to -90bp are transcriptional activation regions, and -90 to -46bp is a region that further enhances the transcriptional activity. The action of this vector can help the gene to realize overexpression (Fig.13).
This vector contains an ampicillin resistance gene (AmpR) for selection against E. coli; it also provides multiple restriction sites for insertion of exogenous genes; and it contains a replicon for E. coli, which ensures stable replication of the vector in E. coli; in addition to that, this vector contains tryptophan- and In addition, the vector also contains tryptophan and uracil on PlacZi for screening of second-deficiency plates. The characteristics of this vector ensure the smooth progress of the experiment (Fig.14).
This vector contains the ampicillin resistance gene (AmpR) for selection against E. coli; it also provides multiple restriction enzyme sites for insertion of exogenous genes; and it contains a replicon for E. coli, which ensures stable replication of the vector in E. coli; in addition to that, this vector contains uracil - and PJG In addition, the vector also contains uracil- and PJG-tryptophan for the screening of two-deficient plates. The characteristics of this vector ensure the smooth progress of the experiment (Fig.15).
