Construction of hpRNA expression vector for silencing a gene in Rhizopus oryzae

Abstract: Depending on the previous research on LDHA gene silencing in Rhizopus oryzae CCUG 28959 through introduction of siRNA, a integral vector was constructed by inserting two copies of LDHA gene (by PCR cloning) in a fashion that it can express hpRNA in the transformed fungi, which will trigger the post transcriptional degradation of targeted mRNA through RNA degradation pathway which is known to be quelling in fungi.The vector was successfully designed with the LDHA gene, transformed in to the host organism, and also transferred to its progeny. This helps in maintaining stability of the transformed cell lines. This created vector will be advantageous at this point when compared to the use of siRNA for gene silencing, which is not a stable way. In the future, this vector can be used for down regulating other genes of interest in R. oryzae and can also be used for studying its effect on other metabolic pathways.In this study, Hygromycin resistance to the R. oryzae CCUG 28959 was shown at levels up to 1000 μg/ml, which has not been reported previously.