Molecular cloning and characterization of five genes from embryogenic callus in Miscanthus lutarioriparius

Abstract

The regeneration from embryogenic callus of higher plants in tissue culture is regulated by explants types and developmental stage and also regulated by some genes. In Miscanthus lutarioriparius, five candidate genes were selected to decide the differential expression between embryogenic and non-embryogenic calli, including MlARF-GEP (guanine nucleotide-exchange protein of ADP ribosylation factor), MlKHCP (kinesin heavy chain like protein), MlSERK1 (somatic embryogenesis receptor-like kinases 1), MlSERK2 (somatic embryogenesis reportor-like kinases 2), and MlTypA (tyrosine phosphorylation protein A) with Genbank accession numbers KU640196–KU640200. Multiple sequence alignment analysis showed that five genes were highly conserved among members of their gene families respectively. Phylogenetic relationship analysis showed that five genes were closest with homologous genes of Zea mays and Sorghum. The qRT-PCR results showed significant differences of five genes expression pattern between two different callus types, the relative expression in embryogenic callus was detected to exceed in non-embryogenic callus. Furthermore, simple sequence repeats (SSR) marker statistics results via Chi-square showed a significant correlation between MlSERK1 genotype and induction of embryogenic callus in M. lutarioriparius. This study may lay the foundation of the molecular mechanism on the embryogenic callus induction of M. lutarioriparius and perhaps provide some gist for further study on genetic manipulation.