Part:BBa_K2947000

4HPAAS

4HPAAS(referring to 4-hydroxyphenylacetaldehyde synthase) protein coding region. It catalyzes L-tyrosine to produce 4-hydroxy-phenylacetaldehyde. It is the first enzyme in the biosynthesis of salidroside.

Usage and Biology

4HPAAS is one gene of the Aromatic acetaldehyde synthases(AASs). AASs, together with tryptophan decarboxylases(TDCs) and TyDCs, encompass a large family of pyridoxal phosphate(PLP)-dependent enzymes broadly referred to as the plant AAAD family.(Facchini et al., 2000; Kaminaga et al., 2006) As their respective names imply, TyDCs, YDCs, and AASs catalyze discrete decarboxylation or decarboxylation-deamination reactions using specific aromatic amino acids as substrates.

Previous research has shown that 4HPAAS functions in the direct conversion of tyrosin to 4-hydroxyphenylacetaldehyde(4-HPAA) in Rhodiola salidroside biosynthesis.(Michel et al., 2017)

1.In order to find a suitable detection time for salidroside, we explored the rule that the expression of 4HPAAS and UGT33 in Arabidopsis protoplasts in respond to the change of time. It was found that the expression of 4HPAAS and UGT33 reached the highest level 10 hours after transfection, so we speculated that this time should be the time with the highest synthesis rate of salidroside, so we chose 12 hours after transfection as the best time to detect salidroside.

A: Western blot was used to detect the expression of 4HPAAS and UGT33 in respond to the change of time; B: We used “i mage J” to analyze the relative expression of 4HPAAS in respond to the change time (set the band gray value of 4hours as 1); C: i mage J software and UGT33s with time (set the band gray value of 4 hours as 1).

2.The protoplast and salidroside standard samples of arabidopsis thaliana transfected with UGT33 and 4HPAAS genes were compared by liquid chromatography. The peak value of the product measured in sample No. 1 was basically consistent with the peak value of salidroside standard samples.

Therefore, we can determine that the transfected protoplast of arabidopsis thaliana can produce salidroside.

MODELING(with UGT33:BBa_K2947001)

detial:https://2019.igem.org/Team:GDSYZX/Model

By Using Image J software to analyze the relative brightness of protein bands, we obtained the data of gray value of two genes/4h varying with time. There is a positive correlation between relative gray value and protein expression, so we use the standardized relative gray value to reflect relative protein expression level. Then we use SPSS software to draw the scatter plot of relative gray value about time to judge the overall trend. (The relative gray value is equal to the following gray value divided by the gray value of 4h)

We found that these scatters showed a quadratic function parabola trend, so we square the time data. Next, we test Pearson correlation coefficient of time, time squared and relative gray value to check whether there is a linear correlation between them.

Then we found that the correlation between UGT33 gene variables was quite high, P value was less than 0.05, with a high significance. The correlation coefficient of 4HPAAS gene was higher than 0.6, P value was higher than 0.05, but less than 0.2. Although the degree was not significant, the correlation coefficient was higher. It can be concluded that the binary linear regression equation can be constructed between the relative gray value of two gene sequences and the square term of time . By using the least square method, we get the regression equation between relative gray value and time.

Then we analyze the fitting degree of the two models and calculate the R-square of the regression equation. We find that the goodness of fit of the two models is good, and the R-square is close to 1. Therefore, both models are better. In the case of significance level 0.05, the F-test of the two models is significant, which shows that all independent variable time has a higher linear significance for relative gray value on the whole, and the p-value of coefficient T test is less than 0.05. It also shows that each independent variable time has a higher linear significance for relative gray value. From the fitting results, the relative gray value of UGT33 and 4HPAAS is proportional to time and inversely proportional to the square of time. From the results calculated from the model, it can be concluded that when Time=12.32, the relative gray value of UGT33 reaches a maximum value of 4.55; when Time=10.68, the relative gray value of 4HPAAS reaches a maximum value of 3.92. Since we measure every four hours, we can assume that when the time reaches about 9-12 hours, the expression reaches a maximum. It can also be seen from the two fitted images that the relative gray value increases with time and then increases and then decreases the parabolic trend. Based on this modeling analysis, we conclude that 12 hours is sufficient for the experiment.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 631
  Illegal BglII site found at 712
  Illegal BglII site found at 1156
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]