Cloning and Characterization of Invertase gene from various

sugarcane varieties

Wilhelmus Terang Arga Sanjaya, Dwi Andreas Santosa, Giyanto

Master Program on Animal Production Science and Technology
Departemen of Soil Science and Land Resources
Faculty of Agriculture, Bogor Agricultural University, Bogor 16680
Bogor e-mail: terangarga@gmail.com, and dsantosa@indo.net.id

Abstract

Sugarcane is a very important food crop that supplies 65% of the world's sugar
needs. In Indonesia, sugarcane demand for sugar industries continues to grow along
with the continued decline in productivity and the quality of sugarcane as raw
material. In the past five decades, the amount of sugarcane yield has dropped by 75%,
making Indonesia the largest importer of sugar cane in the world up to 4.5 million
tons in 2018. Increasing sugarcane yield is one of the keys to increase sugarcane
productivity. Genetic engineering to control the activity of invertase enzymes can be
used as an approach to increase sucrose accumulation in sugarcane. The approach will
not only reduce sucrose hydrolysis during the planting period but also during the
post-harvest process. However, genetic information related to the invertase enzyme in
sugarcane in Indonesia is still very minimal. This study aims to isolate the sequence of
invertase genes from various sugarcane plants and characterize their activities. This
research was conducted through several stages consisting of sampling various
sugarcane varieties in Indonesia, callus induction, RNA isolation, cDNA synthesis,
invertase gene cloning into cloning vectors, sequencing and then invertase subcloning
genes to test expression activity qualitatively and quantitatively. A comprehensive
analysis of the invertase gene family was carried out, including gene structures,
phylogenetic relationship, and 3D protein structures. In this study, two targeted
sequences were confirmed to be able to express the invertase enzyme through
qualitative testing, including INVC9-PSJT941 and INVC9-PS882 sequences. Both have
activity up to 27.82 ± 0.84 and 26.77 ± 0.93. Based on bioinformatics approach, two
invertase gen was indicated as a part of cell wall invertase subfamily. These results can
be a very important reference in developing technology to control invertase enzyme
activity, especially to knock out gene through RNAi technology and genome editing
using CRISPR.

Keywords : β-fructofuranosidase, cell wall invertase, RNAi, sucrose accumulation,

genome editing