Construction of a recombinant vector containing SAI gene using Gateway-based RNAi system: a preliminary study in the development of high sucrose sugarcane

Abstract

Recombinant plasmid construction that produces hairpin molecules for gene knockdown is not an easy method. Gateway cloning system is a technology to transfer DNA fragments utilizing clonase and specific sites. This study aimed to construct RNAi expression vector containing soluble acid invertase (SAI) gene using Gateway system to produce high-sugar content sugarcane. PCR was conducted to generate specific SAI fragment using cDNA template from two sugarcane varieties: Bululawang and PSJK922. Of the two specific primer pairs designed, only one pair amplified the SAI 161 bp gene fragment. Amplification occurred in both cDNA samples. TA (Thymine-Adenine pair) cloning was performed with pCR8/GW/TOPO plasmid and a clonase reaction was carried out to insert the specific fragments into the pHELLSGATE 8 hairpin plasmid. The results showed that two of the four colonies analyzed by associated restriction enzymes had plasmid pHELLSGATE8::hpSAI that contained a hairpin component of the SAI fragment. One attempt to construct the hairpin RNA using Gateway system in this study indicates the easiness of the system when compared to traditional cloning. The DNA sequence in the hairpin portion of this plasmid will be analysed to confirm SAI gene fragment, and it will be followed by transformation of sugarcane and SAI characterization.