Specific primers, enzymes and cDNA were reacted in PCR to obtain gene fragments ligated to the pEASY vector. The sequencing results showed that we successfully cloned PtoCOMT. And the length of this gene is 1098bp.

The gene was successfully attached to the PBI121 vector by seamless cloning technology. The recombinant PBI121-PtoCOMT-GFP vector was transformed into Agrobacterium tumefaciens and then transformed by leaf disc method. And transgenic plants were obtained.

To ascertain the direct involvement of PtoCOMT in melatonin biosynthesis, transgenic populus with overexpression of PtoCOMT were generated. We used Melatonin ELISA Kit to detect melatonin in wild-type and transgenic plants. A standard curve needs to be constructed based on the standards in the kit at concentrations of 0,1,2,4,8,16 pg/ml. The contents of melatonin in transgenic populus increased by 1.5-fold compared to that in wild-type populus. Therefore, we found that COMT was not efficient in catalyzing the synthesis of melatonin, so we wanted to develop a method to improve the synthesis efficiency.

We identified positive selection sites of PtoCOMT by bioinformatics analysis. These sites were then mutated to construct a mutant library. The results showed that mutations at positions 48, 49, 89, 92 position have high degree of significance. And we used alphafold to predict protein structure and it turns out that the structure does change at these sites. So we sort of figured out a way to design proteins.