In-silico construction of potential cis-acting elements from PD_CbNPR1 with T7 minimal promoter

Abstract

A hybrid promoter is a synthetic promoter engineering technique that has a function as a tool for cis-acting elements validation. The hybrid promoter is assembled by combining the minimal promoter [MP] with fragments containing cis-acting elements. PD_CbNPR1 from Capsicum annuum cv. Berangkai has several potential cis-acting elements for the regulation of plant resistance gene expression but has not been validated in the in-vitro assay. The potential elements are the W-Box motif which has three variations, then the WLE1 and RAV1AAT which have one variation motif. Sequences containing the cis-acting elements of PD_CbNPR1 were synthesized as fragment g-block. The MP used was the T7 Promoter in the pET28a[+] plasmid. The g-block fragment will be fused in the upstream region of the T7 Promoter. Before constructing the hybrid promoter in-vitro, it is necessary to construct the T7 promoter with cis-acting elements in-silico. The software used in this research was the plasmid editor ApE vers.2.0.36. The in-silico construct of the T7 promoter with cis-acting elements aimed to obtain plasmid construction designs that will be assembled in-vitro. The constructs obtained in this study consisted of six variations, in the form of a circular plasmid contains the potential cis-acting element. These constructs are called constructs A [WBox1-WBox2-WBox3-WLE1-RAV1AAT + pET28a[+]], B [WBox1 + pET28a[+]], C [WBox2 + pET28a[+]], D [WBox3 + pET28a[+]], E [WLE1 + pET28a[+]], and F [RAV1AAT + pET28a[+]]. The six constructs will be used in further study to regulate gene expression, as a tool to validate the cis-acting elements’ role in-vitro.