Team:GDSYZX/Results

• 1 Sequencing and comparison of gene HQT synthesis
• 2 Cloning, recovery and purification of target gene HQT and promoter GluD-1
  • 2.1 HQT electropherogram
  • 2.2 GluD - 1 electropherogram
• 3 Screening of positive clones-colony PCR & sequencing
  • 3.1 Colony PCR electrophoresis
  • 3.2 Sequencing results
• 4 Western Blot detects the expression of the target protein
  • 4.1 35s::HQT
  • 4.2 GluD-1::HQT
• 5 Extraction and detection of target product

RESULTS

1 Sequencing and comparison of gene HQT synthesis

After obtaining the genes synthesized by Tianyi Huiyuan Company, we conducted gene sequencing and compared it with the previously designed gene sequences. The result showed that the HQT gene sequences synthesized by tianyi Huiyuan Company were consistent with those designed by us, which was in line with our expectation.

fig.1

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Cloning, recovery and purification of target gene HQT and promoter GluD-1

2.1 HQT electropherogram

The fig.2 is the DNA gel electrophoresis of the HQT gene after PCR. From it we can see that there is clearly a bright band between 1000bp and 2000bp, which is basically the same size as our target gene (the length of the HQT gene is 1320bp ), so we speculate that the band amplified by PCR is the HQT gene.

fig.2

2.2 GluD - 1 electropherogram

The fig.3 is the DNA gel electrophoresis of the GluD-1 promoter after PCR. From it we can see that there is clearly a bright band between 750bp and 2000bp, which is basically the same size as our target gene (the length of the HQT gene is 1228bp ), so we speculate that the band amplified by PCR is the GluD-1 promoter.

fig.3

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3 Screening of positive clones-colony PCR & sequencing

Colony PCR can help us eliminate false positive colonies and screen out colonies that can be sequenced.

3.1Colony PCR electrophoresis

3.1.1 35s::HQT

The fig.4 shows the electrophoresis of the colony PCR with the promoter of P35S::HQT vector. The gene we designed to amplify is HQT. The length of the HQT gene is 1320bp. The electropherogram shows that the first, second, third, and fifth wells have bands between 1000-2000bp (the size of HQT is about 1320). Therefore, this result indicating that the colony may contain our target gene. Next, these colonies were expanded and cultured to extract plasmids for later sequencing verification.

fig.4

3.1.2 GluD-1::HQT

The fig.X shows the electrophoresis of the colony PCR with the promoter of PGluD-1::HQT vector. The gene we designed to amplify is GluD-1. The length of the GluD-1 gene is 1228bp. The electropherogram shows that all wells have bands between 1000-2000bp. Therefore, this result indicating that the colony may contain our target gene. Next, these colonies were expanded and cultured to extract plasmids for later sequencing verification.

fig.5

3.2 Sequencing results

Sequencing results showed that colonies 1 and 2 picked from P35S::HQT positive colonies contained the correct HQT gene sequence (fig.6), and colonies 1 and 3 picked from PGluD-1::HQT positive colonies contained the correct GluD-1 gene sequence (fig.7). （Because we use the GluD-1 gene to replace the 35S promoter on the P35S::HQT vector, we do not need to sequence the HQT gene.）

3.2.1 35s::HQT

fig.6

3.2.2 GluD-1::HQT

fig.7

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4 Western Blot detects the expression of the target protein

4.1 35s::HQT

The results of Western Blot showed that a large amount of HQT expression could be detected in rice protoplasts transfected with 35s::HQT(fig.8). And the expression level of HQT increased with the extension of the expression time. The expression level was the highest at 15h, and then the expression level began to decline. It may be due to the death of some cells during the cell culture time. We selected the protoplasts cultured for 15 hours and their culture medium for HPLC detection.

fig.8

4.2 GluD-1::HQT

The results of Western Blot showed that the target band could not be detected in the rice protoplasts transfected with GluD-1::HQT, which indicated that the HQT gene was not expressed (or the expression level was too low to be detected), which further proved that GluD-1 promoter was endosperm specificity (fig.9).

fig.9

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5 Extraction and detection of target product

fig.10

We use HPLC to compared the wile type of Oryza sativa L. protoplast, chlorogenic acid standard samples and our product sample obtained from the transfected protoplast (fig.10). The peak value of the product measured in our product sample was basically consistent with the peak value of chlorogenic acid samples. However, no overlapping peak with the protoplast standard sample was found in the wild-type liquid chromatography, which proved that wild protoplasts do not produce chlorogenic acid，and we can determine that the transfected protoplast can produce chlorogenic acid.