Difference between revisions of "Part:BBa K4340600"

Revision as of 10:12, 11 October 2022

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.

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)

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

Sequence and Features