Difference between revisions of "Part:BBa K4340601"

 
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PYL8 is a highly efficient ABA inhibitor. It binds to ABA and stops the signaling of ABA.
 
PYL8 is a highly efficient ABA inhibitor. It binds to ABA and stops the signaling of ABA.
 +
We next tested whether the hormone binding domain can inhibit and attenuate the effect of ABA (plant hormone that inhibits germination and plant growth) to plant germination. The pET11a and PYL8 E.coli culture groups, and a group with ABA treatment and PYL8 E. coli culture had a higher germinated percentage than the group without ABA (blank). This indicated that the PYL8 had a positive effect on the seed germination stage both with ABA and without ABA. (Figure 1)
 +
In the PYL8 group results, the percentage of germinated soybeans with PYL8 E.coli culture and ABA was significantly higher than in groups only with ABA. This shows that the PYL8 worked to produce an inhibitor of ABA. Notably, the PYL8 group (only added PYL8 cell culture) is even higher than the group without ABA. This demonstrated that PYL8 can discharge the effect of ABA, and even benefit seed germination squarely. (Figure 2)
 +
 +
==Experiment 1: Hormone-treated germination test==
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Figure 1. The seed germination percentage of soybeans in seven days with all factors.
 +
 +
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Figure 2. The seed germination percentage of soybeans in seven days with factors with PYL8.
 +
 +
Figure 3. The seed germination percentage of soybeans in seven days with NSP4-T2R4.
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 +
To sum up, the efficiency of the plasmids ranked is PYL8> NSP4-T2R4 (slightly higher than) pET11a.
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 +
==Experiment 2: Western Blot and Real-time PCR==
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Figure 4. The protein expression of NSP4-T2R4 and PYL8
 +
 +
Figure 5&6: The real-time PCR result of PYL8 and NSP4-T2R4 compared with BL21 E.coli strain.
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 +
We have conducted a western blot experiment to validate the quality of protein expression of PYL8 and NSP4-T2R4. In the experiment, there is a clear band of PYL8 in both 10ul and 20ul samples at 35 kDa. There is a relatively more blended band of NSP4-T2R4. (Figure 5) Therefore, further experiments would be needed to confirm whether no protein expression is the reason why T2R4 cannot inhibit ABA as in PYL8.
 +
 +
In the real-time quantitative PCR test, we found the RNA expression of both genes, indicating that the PYL8 expressed RNA successfully. (Figure 7)
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 13:53, 10 October 2022


PYL8

PYL8 is a highly efficient ABA inhibitor. It binds to ABA and stops the signaling of ABA. We next tested whether the hormone binding domain can inhibit and attenuate the effect of ABA (plant hormone that inhibits germination and plant growth) to plant germination. The pET11a and PYL8 E.coli culture groups, and a group with ABA treatment and PYL8 E. coli culture had a higher germinated percentage than the group without ABA (blank). This indicated that the PYL8 had a positive effect on the seed germination stage both with ABA and without ABA. (Figure 1) In the PYL8 group results, the percentage of germinated soybeans with PYL8 E.coli culture and ABA was significantly higher than in groups only with ABA. This shows that the PYL8 worked to produce an inhibitor of ABA. Notably, the PYL8 group (only added PYL8 cell culture) is even higher than the group without ABA. This demonstrated that PYL8 can discharge the effect of ABA, and even benefit seed germination squarely. (Figure 2)

Experiment 1: Hormone-treated germination test

Figure 1. The seed germination percentage of soybeans in seven days with all factors.


Figure 2. The seed germination percentage of soybeans in seven days with factors with PYL8.

Figure 3. The seed germination percentage of soybeans in seven days with NSP4-T2R4.

To sum up, the efficiency of the plasmids ranked is PYL8> NSP4-T2R4 (slightly higher than) pET11a.

Experiment 2: Western Blot and Real-time PCR

Figure 4. The protein expression of NSP4-T2R4 and PYL8

Figure 5&6: The real-time PCR result of PYL8 and NSP4-T2R4 compared with BL21 E.coli strain.

We have conducted a western blot experiment to validate the quality of protein expression of PYL8 and NSP4-T2R4. In the experiment, there is a clear band of PYL8 in both 10ul and 20ul samples at 35 kDa. There is a relatively more blended band of NSP4-T2R4. (Figure 5) Therefore, further experiments would be needed to confirm whether no protein expression is the reason why T2R4 cannot inhibit ABA as in PYL8.

In the real-time quantitative PCR test, we found the RNA expression of both genes, indicating that the PYL8 expressed RNA successfully. (Figure 7)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 1000
    COMPATIBLE WITH RFC[1000]