Part:BBa_K4340601

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.

Photo 1. Germination of soybeans with or without added ABA, and either with PYL8, NSP4-T2R4 or empty pET11a vector transformed E. coli.

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 3. The protein expression of NSP4-T2R4 and PYL8.

Figure 4: The real-time PCR result of PYL8 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. (Figure 3)

In the real-time quantitative PCR test, we found the RNA expression of both genes, indicating that the PYL8 expressed RNA successfully. (Figure 4)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 305
  
• 1000
  COMPATIBLE WITH RFC[1000]