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1. Promoters

1.1 The first batch of Promoters and primers

1.2 The second batch of Promoter primers

1.3 Gibson assembly simulation of Promoters in SnapGene software

SnapGene is used for simulating assembly. For the second batch of identified promoters, we initially added homologous arms in the opposite direction and made subsequent revisions to the primers. It is worth mentioning that the AtMRP3 promoter was used for both forward and reverse reporter vector construction. GFP and GUS vector maps, using AtMRP3 as an example:

2. GUS & GFP vector construction

(8 main steps)

1. Plant DNA extraction
2. PCR amplification
3. Vector preparation
4. Homologous recombinant and clone transformation
5. Colony PCR verification
6. sequencing
7. Agrobacteria Transformation
8. PCR verification

Vector construction result summary:
The construction of our vector can be divided into three stages Chronologically:

- From 6.13 to 7.3, design the experimental technical route and implementation plan, learn and explore the experimental conditions for promoter amplification and vector construction.
- From 7.3 to 7.17, the first round of experimental testing, successfully construct a total of 3 different recombinant vectors for 2 promoters: AtRCD1-GUS, AtSRO1-GUS, AtRCD1-GFP.
- From 7.24 to 8.20, the second round of promoter vector construction experiment, successfully construct a total of 9 different vectors for 7 promoters:

DP1 and DP2 are AtRCD1-GFP carriers, and the stripes are correct.

DS1 and DS2 construct result graphs for AtRCD1-GUS carriers, and the band situation is unknown.

OS1 and OS2 build result graphs for AtSRO1-GUS vectors with correct stripes.

We will send the above 6 tubes of bacterial liquid for sequencing, return the sequencing results.

We get successful construction of these 3 vectors.

Promoter GUS-PCR2, GFP-Ntccx2, GFP-NtNRAMP2, GFP-NtNRAMP3, GFP-NtNRAMP5, GFP-NtNRAMP6, GFP-PCR2 homologous recombination results The electrophoresis results are shown in the figure up. Each group of bacterial liquids was selected from three tubes for testing. The sequencing results showed that the homologous recombination of the tested bacterial liquids was all successful. The correctly sequenced bacterial liquids were extracted by shaking plasmids .

All those sequencing result mapping are done by our supervisor Wanjun Chen.

3. Plant cadmium response transient transformation experiment

After seeking help from Teacher Wang Xiaojuan to construct the carrier, we obtained four recombinant carrier experimental materials: NtNRAMP2-GUS, NtNRAMP5-GUS, NtNRAMP6-GUS, and NtCCX2-GUS on August 11th. Combined with the tobacco cadmium response instantaneous transformation experiment we designed earlier, we carried out instantaneous transformation design and verification

Periwinkle Instantaneous Test

Because the tobacco seedlings cultivated in the laboratory are very valuable, we purchased periwinkle seedlings online in advance to ensure that the team members fully mastered the technical essentials of transient transfection, and used periwinkle petals for instantaneous injection. The following are the test results of periwinkle transient transfection

The potted plants of Changchun flowers purchased online were pretreated before the experiment and used for bacterial injection

Instantaneous transformation test results of periwinkle

Among them, PBS is a blank control, GUS-Super promoter is a positive control, and AtRCD1-promoter is one of our test promoters.

From the results of the petal transient transfection experiment, the blank control was not stained blue, and the positive control was stained blue, indicating that the team members successfully injected the soil pus bacteria solution containing a strong promoter into the Changchun petals, completing the infection; at the same time, the blank control was not stained blue, indicating that there was no obvious pollution in our experimental operation. The test promoter AtRCD1-promoter did not show a blue reaction, indicating that the promoter, based on this transient transfection exp ethod, cannot be expressed in Changchun petals.

Design of tobacco instantaneous experiment scheme

We designed the ideal experiment according to the mind map

However, we did not have enough tobacco seedlings that met the conditions for instantaneous injection, so we simplified the experimental plan based on laboratory conditions and finally obtained the following experimental results:

clean water
Cadmium (50ng/μL)
blank comparison
positive control
Conclusion Strong response, strong specificity Strongly responsive, weakly specific,
this promoter may be responsive to
drought in addition to cadmium
Strong response, strong specificity
-Observe the results of pAtPCR2-GFP vector transient transfection using a fluorescence microscope.

Summary: After cadmium treatment, the EGFP reporter gene is expressed albeit weak. Super Promoter represents original unedited EGFP vectors with mannopine synthase promoter which constitutive express EGFP genes. In the second row, it shows after cadmium treatment, the EGFP gene could be spotted near the white arrow while without cadmium treatment, no EGFP signals were detected. Also, we used a UV-flashlight as the light source while taking those photos. Because chlorophyll is auto-fluorescent, the photos of the whole leaves appear to be red. Please also see Part:BBa_K4799989 for more information on GUS result of pAtPCR2 promoter.

Declaration of Contributions:pAtPCR2 literature investigation, primer design was done by student member Yuhan Gu (Johnny), Johnny was also responsible for overseeing the functional verification process, summarizing the part pages and pAtPCR-related results. The pAtPCR2-GUS, pAtPCR-GFP vector construction was led by teacher Wanjun Chen, with Qiao Qiao , Youyou Yu, Chenyue Wang, Ao Li, Siyuan Su, Youran Yao contributed to PCR amplification, vector preparation, homologous recombination, E. Coli and Agrobacteria transformation. Instructor Wanjun Chen and Xiaojuan Wang performed pAtPCR2 for both GFP and GUS transient verification, with the student Xinye Jian, and Hongxi Zhang participating in tobacco plant breeding, plasmid extraction, and transient infiltration. All GFP photos were taken by Xinye Jian and Wanjun Chen.

4. Experimental results of luminescent vector of pAtPCR2

Declaration of Contributions:Vector preparation for luminescent vector construction was performed by instructor Xiaojuan wang in the Shenzhen laboratory, with instructor Wanjun Chen participating in a few plasmid extraction, and PCR verification experiments. Student Xinye Jian prepared the tobacco plant needed to do transient transfection. Student Hongxi Zhang performed the transient infiltration of the engineered Argobacteria. Luminescent plant photos were taken by instructor Lichuan Chen.

After conducting experiments in multiple stages, we believe that in a limited time, we have screened out many very promising plant cadmium response regulatory switches, such as NtNRAMP2, NtNRAMP6, and NtNRAMP5. Due to time constraints, we cannot continue to conduct more functional verification experiments for promoters.We requested help from the teachers at BGI. As the experimental difficulty of reconstructing the luminescent carrier itself is greater, we hope that they can conduct subsequent luminescence tests based on our existing experimental results. Finnally, with their great result, our promoter PCR2 can really respond to cadmium.