Isolation, cloning, and characterization of a cuticle collagen gene, Mi-col-5, in Meloidogyne incognita

Abstract

Cuticle collagens form a major part of the nematode cuticle and are responsible for maintaining the overall shape of the animal and its protection from the external environment. Although substantial research on cuticle collagen genes has been carried out in Caenorhabditis elegans, their isolation and characterization in plant parasitic nematodes have been limited to a few genes only. In this study, a cuticle collagen gene, Mi-col-5, was isolated from root-knot nematode, Meloidogyne incognita. A partial segment of 402 bp was first cloned and analyzed on Gbrowse followed by subsequent cloning of the 1047 bp long full cDNA specifying the open reading frame. The deduced amino acid sequence showed 92% sequence identity with that of Mj-col-5. However, a transmembrane helix was predicted in Mi-col-5 which was not present in Mj-col-5. The conserved pattern of cysteine residues in Mi-col-5 suggested that it belonged to group 2 of nematode cuticle collagens but with a longer carboxy terminal region as was the case with Mj-col-5. Domain prediction revealed the presence of a nematode cuticle collagen N terminal domain and a pfam collagen domain along with collagen triple helix repeats. A phylogenetic tree based on the amino acid sequences showed evolutionary relationship of Mi-col-5 with cuticle collagens genes of other nematodes. 3D models for Mi-col-5 were predicted with the best confidence score of -2.78. Expression of Mi-col-5 transcript was found to be maximum in egg masses followed by adult females and J2s suggesting its role in the early stages of the development of the nematode during its life cycle.

Keywords: Cuticle collagens; Egg masses; Meloidogyne incognita; Mi-col-5.

Fig. 1

Restriction digestion of pGEMT clones with EcoRI releasing a 402 bp partial fragment of Mi-col-5. Lane M 1 Kb ladder. lanes 1–4 positive pGEMT clones. lanes 5–7 negative plasmids

Fig. 2

Amplification of full Mi-col-5 gene from adult female cDNA. Lane M, 1 Kb plus ladder. lane 1 adult female cDNA

Fig. 3

Conserved pattern of cysteine residues classifies Mi-col-5 in group 2 of cuticular collagen genes of nematodes. The positions of Gly-X-Y domains along three cysteine containing domains are represented here. This system of classification of cuticular collagen genes was proposed by Johnstone (2000) on the basis of conserved patterns of cysteine residues. In C. elegans, group 2 has 38 members

Fig. 4

Secondary structure prediction of deduced amino acid sequence of Mi-col-5. Comparison of Mi-col-5 and Mj-col-5 secondary structure predictions. Helices, extended strands, turns, and coils are represented by longest (blue), second longest (red), second shortest (green), and shortest (violet) vertical lines, respectively (a), Deduced amino acid sequence of Mi-col-5 is represented by upper cases and lower cases represent the corresponding secondary structural characteristics such as random coils (c), extended strand (e), α helices (h), and β turns (t) (b)

Fig. 5

TMHMM predictions for Mi-col-5 (a), Mj-col-5 (b). Blue, red, and pink colors represent the portions of the amino acids inside, in the transmembrane helix and outside the cell, respectively

Fig. 6

Clustal Omega analysis of the Mi-col-5 and Mj-col-5 amino acid sequences. Area under the black box shows the transmembrane helix region of Mi-col-5 and its difference with Mj-col-5

Fig. 7

Folding predictions for Mi-col-5. Predicted folded and unfolded regions of Mi-col-5 (a), amino acid sequence for Mi-col-5 showing ordered segments in green and disordered segments in red (b)

Fig. 8

Phylogenetic tree constructed by maximum likelihood method using MEGA 6 showing evolutionary relationship of Mi-col-5 with cuticle collagens of other plant parasitic, animal parasitic, and free living nematodes

Fig. 9

Molecular modeling of Mi-col-5 using I-TASSER server. Predicted 3D models of Mi-col-5 representing model 1 and model 3, respectively, (a) and (c), Ramachandran plots for predicted model 1 and model 3, respectively, (b) and (d)

Fig. 10

Expression of Mi-col-5 in different developmental stages of M. incognita