Molecular characterization and expression analysis of ribosomal L18/L5e gene in Pennisetum glaucum (L.) R. Br

Abstract

Ribosomal L18/L5e (RL18/L5e) is a member of the ribosomal L18/L5e protein family, which has an essential function in translation of mRNA into protein in the large ribosomal subunit. In this study, RL18/L5e was isolated and sequenced from local Pennisetum glaucum (L.) R. Br. cultivar which is known to adapt to environmental stress. The obtained cDNA for PgRL18/L5e was 699 bp in length, with an open reading frame of 564 bp. The deduced protein sequence contained 187 amino acids and comprised an RL18/L5e domain, which shared high sequence identity with orthologous proteins from Viridiplantae. The obtained PgRL18/L5e cDNA contained two exons of 154 and 545 bp, respectively, and an intron of 1398 bp. Secondary and 3D structures of the deduced PgRL18/L5e protein were predicted using in silico tools. Phylogenetic analysis showed close relationships between the PgRL18/L5e protein and its orthologs from monocot species. Multiple sequence alignment showed high identity in the RL18/L5e domain sequence in all orthologous proteins in Viridiplantae. Moreover, all orthologous RL18/L5e proteins shared the same domain architecture and were nearly equal in length. Quantitative real-time PCR indicated a higher transcript abundance of PgRL18/L5e in shoots than in roots of 3-day-old seedlings. Moreover, the expression of PgRL18/L5e in seedlings under cold and drought stress was substantially lower than that in untreated seedlings, whereas the highest expression was shown under heat stress. This study provides insights into the structure and function of the RL18/L5e gene in tolerant crops, which could facilitate the understanding of the role of the various plant ribosomal proteins in adaptation to extreme environments.

Keywords: Gene expression; Pennisetum glaucum; Phylogenetics; Ribosomal L18/L5e; cDNA.

Fig. 1

The structure of PgRL18/L5e gene. (A): Structure of the PgRL18/L5e gene based on the alignment with the genomic sequence of chromosome 2 of Pennisetum glaucum. The two exons are shown in light blue, the intron is shown as the white arrow, and the 3ʹ and 5ʹ UTR are shown in light pink. (B): The nucleotide sequence and deduced protein sequence for PgRL18/L5e cDNA. The amino acid sequence (light blue) is written above the nucleotide sequence, and the RL18/L5e domain is highlighted in dark blue. The start and stop codons are highlighted in yellow.

Fig. 2

Conservation of the PgRL18/L5e protein sequence analyzed by ConSurf. ConSurf scores the most conserved amino acid as 9 and the most variable as 1.

Fig. 3

Secondary structure of the deduced PgRL18/L5e protein as predicted by NetSurfP-2.0. The relative surface accessibility is shown as ; the sequences denoted in red are exposed, and those denoted in blue are buried (threshold at 25%). Secondary structure is illustrated as follows: Helix, Strand, Coil. The thickness of the line denotes the probability of disordered residues.

Fig. 4

(A): The predicted 3D structure of the sequence from aa 62 to 182 of the deduced PgRL18/L5e protein generated by SWISS-MODEL. The N-terminal is indicated in blue, and the C-terminal is shown in red. (B): The alignment between ribosomal L5 protein (5xy3.1.G) from the SWISS-MODEL template library and PgRL18/L5e protein.

Fig. 5

Phylogenetic analysis of PgRL18/L5e and homologous proteins. The maximum likelihood tree was built using MEGA 7.0.26. A hypothetical homologous protein from Chara braunii was used as an outgroup.

Fig. 6

Relative expression of PgRL18/L5e gene in Pennisetum glaucum seedlings analyzed by quantitative real-time PCR. (A): The expression levels of PgRL18/L5e in seedlings treated during germination with four types of stress: heat, salt, cold, and drought. The y-axis corresponds to the relative expression level. (B): The tissue-specific expression levels of PgRL18/L5e in three different tissues— root, seeds, and shoots—from 3-day-old germinated seedlings.