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.

Bioengineering and Molecular Biology of Miscanthus

“…In the study of Zhao et al, full-length ORF cDNA sequences of three genes, M1ARF-GEP, M1SERK1 and M1SERK2 (GenBank access numbers KU640196, KU640198 and KU640199), obtained from kU640199, were cloned by amplification of degenerate primers designed for sorghum and maize sequences [62]. However, for Zhao et al, based on the resulting cDNA, the other two genes accept only partial open frames for reads in the cDNA sequence and are referred to as M1KHCP and M1TypA (GenBank access numbers KU640197 and KU640200) [62]. The length of the complete M1ARF-GEP ORF sequence is 5385 bp and encodes a protein of 1794 amino acids.…”

“…It contains an open reading frame measuring 1872 bp, which encodes a protein of 624 amino acids. The length of 5 -UTR and 3 -UTR is 9 and 75 base pairs, respectively [62]. The length of the MlSERK2 cDNA sequence is 2119 bp.…”

“…It contains an ORF of size 1878 bp, which encodes 626 amino acids. Zhao et al determined that the length of the incomplete ORF MlTypA sequence is 1962 bp and encodes a protein of 654 amino acids [62].…”

“…Compared to other monocots (B. distachyon, O. brachyantha, O.sativa, S. italica, S. bicolor and Z. mays) and dicots (A. thaliana, B. napus, M. domestica and R. communis), the sequence and length of the MlARF-GEP protein are quite conservative [62,63]. It is determined by multiple comparison analysis [62].…”

“…Compared to M1ARF-GEP, the highest identity is 99% (SbARF-GEP). The phylogenetic tree constructed by the MEGA 5.1 program shows that MIARF-PVA is divided into monocotyledonous and dicotyledonous groups [62]. According to Zhao et al, this means that M1ARF-GEP evolved irrespective after differentiation of monocots and dicots.…”