Selection and validation of reference genes for quantitative RT-PCR of Lakatan banana (Musa acuminata Colla) fruit ripening

Date

6-2016

Degree

Bachelor of Science in Agricultural Biotechnology

College

College of Agriculture and Food Science (CAFS)

Adviser/Committee Chair

Eureka Teresa M. Ocampo

Abstract

This study was conducted to identify the reference gene that can be used for real time PCR (or qPCR) analysis of gene expression at different banana fruit ripening stages particularly green mature, climacteric, 70% yellow and 100% yellow . The RNA at different ripening stages of banana (Lakatan variety) fruit was extracted. First strand cDNA was synthesized and used in regular reverse transcriptase PCR to validate the primers in isolating the candidate reference genes namely 25s, EF1, ACT 11, L2 and TUB, and the banana ripening-specific gene MaMADS2. After validation, real time PCR was performed, with amplification products shown to be positive using melt peaks and melt curves. Among the candidate genes, ACT 11 was determined as the reference gene for MaMADS2 gene since it produced a constant and the most similar amplification cycle under the different ripening conditions. ACT 11 gene amplified at 26th cycle which was comparable to the cycle number at which MaMADS2 amplified. Calculations for relative quantitation of MaMADS2 using ACT 11 as reference gene showed 2-fold increase at climacteric stage. There was no MaMADS2 expression at 100% yellow stage. This study identified a suitable reference gene for measuring real time gene and quantitative gene expression in fruit ripening in Lakatan banana. This reference gene can also be used in gene expression studies of other climacteric fruit.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2016 A127 /E28

Notes

Major in Crop Biotechnology

Document Type

Thesis

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