Transgene Insertion Stability and Aluminum Tolerance Candidate Gene Expression in T3 Generation of Transgenic Tobacco

Abstract

An aluminum (Al) tolerance gene candidate, called GERLP gene, has been successfully isolated from an Indonesian local rice cv. Hawara Bunar, and transformed into tobacco plants under CaMV 35S strong promoter. The genetic stability of the gene insertion and its expression in the transgenic plant needs to be verified. This study was conducted to analyze the genetic stability of the GERLP gene insertion and the expression of the GERLP gene and two Al related genes in T3 generation of the transgenic tobacco. Putative transgenic seeds were selected on hygromycin containing MS media for 28 days. Hygromycin resistant plants were then verified with PCR analysis using primers developed from the GERLP gene sequence. The expressions of the GERLP gene and two Al related genes were analyzed using qPCR technique. The result showed that 24 lines out of 26 T2 lines were able to grow on selective medium with ratio of 3:1 for hygromycin resistant to sensitive. The other two lines showed consistently complete germination on selective medium until T3 generation, which indicates that both lines were homozygous for hpt gene. Since the hpt gene is located near left border downstream the GERLP gene in the recombinant plasmid construct, it suggests that both lines were also homozygous for the GERLP gene.. Genetic stability analysis in T2 and T3 transgenic tobacco plants revealed that the transgenic tobacco plants carried both GERLP and hpt genes. The gene expression analysis shows that the increasing expression of the GERLP gene in the transgenic tobacco is followed by the increase of the STOP1 and ALMT1 gene expressions, which indicates that there is a regulation relationship between these three genes. This finding suggests that the GERLP gene expression regulates the expression of the STOP1 and ALMT1 genes.

Strictosidine Synthase Coding Gene Expression towards Quinine Biosynthesis and Accumulation: Inconsistency in Cultured Cells and Fresh Tissues of Cinchona ledgeriana

“…The composition of the reaction mixture was 5 µL 1X SensiFAST SYBR® High-ROX Mix, 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), 1 µL cDNA template (50 ng) and 10 µL ddH 2 O. The results were analyzed for relative expression, following the protocols of Livak and Schmittgen (2001) and Ratnasari et al (2016).…”