Part:BBa_K4340600

T2R4 with NSP4 peptides

T2R4 is one of the ABA binding proteins and adding NSP4, the secretion peptide is helpful to secrete more proteins. We 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)

NSP4-T2R4, however, had the same function as the pET11a vector control, (Figure 2) which means that NSP4-T2R4 might not have an outstanding effect on increasing the speed of germination of soybeans.

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 NSP4-T2R4.

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

Figure 2: The real-time PCR result of NSP4-T2R4 compared with BL21 E.coli strain.

We have conducted a western blot experiment to validate the quality of protein expression of NSP4-T2R4. In the experiment, there is a relatively more unclear band of NSP4-T2R4. (Figure 1) Therefore, further experiments would be needed to confirm whether no protein expression is the reason why T2R4 cannot inhibit ABA.

However, in the real-time quantitative PCR test, we found the RNA expression of NSP4-T2R4 genes, indicating there could be some protein expression problem with T2R4, but not RNA. (Figure 2)

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 346
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 48
  
• 1000
  COMPATIBLE WITH RFC[1000]