IN SILICO MUTATION PROFILING OF RBCL GENE AND CLASSIFICATION OF SELECTED EDIBLE MEMBERS OF THE CUCURBITACEAE FAMILY

Olorunsola, Honey AAnu and Covenant University, Theses (2022) IN SILICO MUTATION PROFILING OF RBCL GENE AND CLASSIFICATION OF SELECTED EDIBLE MEMBERS OF THE CUCURBITACEAE FAMILY. Masters thesis, Covenant University Ota.

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Abstract

The Cucurbitaceae, with about 965 genera in 95 species is ranked next to the Solaneceae; the largest family of fruits and vegetables. Its members are among the world’s most significant plant foods. As at 2022, the whole genome sequence of 18 species of this family have been published. Ribulose biphosphate carboxylase large (rbcL) is central to the global carbon cycle and is one of the single barcode genes of individual plants that have phylogenetic utility. However, there has not been a documented detailed investigation into the rbcL gene mutation of this family. This research aimed at providing information on Single amino acid substitution (SAAS) of the rbcL gene of selected cucurbits and gaining insight into their classification. The studied populations are entries of Citrullus lanatus, Citrullus colocynthis, Cucurbita pepo, Cucurbita maxima, Cucurbita moschata, Cucumis sativus, Cucumis melo, Telfaria occidentalis, Momordica charantia, Lagenaria siceraria, and Coccinia grandis. Five amino acid sequence entries of these members (including their reference sequence) were retrieved from the RefSeq database of the National Centre for Biotechnology Information. The sequences of each species were compared with their reference sequence using MUSCLE algorithm of the MEGA11 to check for the presence of SAASs in the gene. The type and effect of mutation on protein stability were predicted using the PPVED v1.0 and I-MUTANT v.3.0 online server, respectively. The rbcL genomic DNA isolation, amplification, purification, and sequencing of Telfairia occidentalis were carried out. Multiple sequence alignment of the translated amino acid sequence alongside the references of the other plant species was carried out using MUSCLE algorithm of MEGA11. A phylogenetic tree was constructed using Maximum-likelihood method. The gene sequences of six species were conserved while four species had the presence of SAASs. F108A was recorded in Cucumis sativus, D10X and M228I were recorded in Citrullus lanatus, T36- and A343X were recorded in Lagenaria siceraria, and I44X and Q375X were recorded in Cucumis melo. A343X and F108A were predicted to be functional mutations with a decrease in the stability of the protein binding site. T36- is well established to be a deleterious mutation. Only Citrullus lanatus had non- functional mutations. In total, 45 entries of 10 species were screened and only five entries of four species had mutations (10.9%). The phylogenetic tree revealed that T. occidentalis is positioned within the Cucurbitaceae family, although, it had the lowest bootstrap confidence compared to other members. To authenticate this positioning, rbcL sequences of ten species belonging to four closely related families within the Order Cucurbitales were added to those of the Cucurbitaceae, and a new tree was constructed. Telfairia occidentalis was centrally positioned between the Cucurbitaceae and the close relatives. The newly constructed tree also revealed that T. occidentalis was not only the closest to the root of the tree, but it was also closer to the relatives than to Cucurbitaceae. Conclusively, this study validated the conservation of rbcL gene sequence amongst the populations screened. It also demonstrated that T. occidentalis classification within the Cucurbitaceae cannot be ascertained but should be further investigated using other molecular markers. Keywords: RbcL, Phylogeny, Cucurbitaceae, Mutation, Telfairia occidentalis.

Item Type:	Thesis (Masters)
Uncontrolled Keywords:	RbcL, Phylogeny, Cucurbitaceae, Mutation, Telfairia occidentalis
Subjects:	Q Science > QH Natural history > QH301 Biology
Divisions:	Faculty of Medicine, Health and Life Sciences > School of Biological Sciences
Depositing User:	nwokealisi
Date Deposited:	02 Sep 2022 10:18
Last Modified:	02 Sep 2022 10:18
URI:	http://eprints.covenantuniversity.edu.ng/id/eprint/16148

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