Molecular cloning and characterization of large subunit ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcL) gene in Chinese malunggay (Sauropus androgynus (L.) Merr.)

Date

12-2012

Degree

Bachelor of Science in Agricultural Biotechnology

College

College of Agriculture and Food Science (CAFS)

Adviser/Committee Chair

Antonio C. Laurena

Abstract

Genomic DNA from Chinese malunggay (Sauropus androgynus (L.) Merr.) was extracted using Mini Bead Beater and yielded good quality DNA with high concentration. PCR-based approach was used to clone and then sequence the full length sequence of the gene encoding for the large subunit of Rubisco. Primers rbcL 5?F and rbcL 3? ZC amplified a 1.5 kpb band. The 1.5 kpb PCR product was used as template for established primers AR-1 and AR-2 to amplify the nested sequence of about 500 bp, as a confirmation that the full length rbcL was the one amplified. The long and nested rbcL sequences were successfully cloned using pGEM®-T Easy Vector Cloning System. After the essential steps in the cloning process ? ligation, transformation, plating, and sub-culturing, the recombinant plasmids were extracted, and was sent to an outside service facility for DNA sequencing. A full length sequence of the rbcL gene in S. androgynus, with 1, 506 bp, was obtained. There is observed high homology of 70% in the DNA sequence of S. androgynus rbcL compared to the rbcL gene sequences of five other species, namely malunggay, corn, pea, tomato, and papaya. There is also observed high conservation of amino acid sequence of the Rubisco enzyme but there is questionable identity of amino acids from positions 245 up to 351 in S. androgynus compared to other five rbcL plant sequences. The position of the nested rbcL sequence was found out to be located at the 112th to the 594th base of the full length rbcL DNA sequence. Varying restriction maps and restriction sites for the two long rbcL clones were generated using Vector NTI. Using BLAST, it was found out that the nested rbcL is most homologous to closely related species of Sauropus androgynus, while the long and xiv full length rbcL was found out to be most homologous to plant species of the Moringa family and close relatives of this family. Codon usage of the rbcL of S. androgynus was most similar to dicot species. This study is the first to clone and sequence the full length rbcL gene of S. androgynus. Since the rbcL gene is also one of the gene sequences used as a barcode, this study provided the first rbcL barcode for. S. androgynus.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2012 A127 /A75

Document Type

Thesis

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