Screening for flower-specific cDNA sequences in Sago palm (metroxylon sagu) via a differential display technique

Bong, Shiaw Kong (2004) Screening for flower-specific cDNA sequences in Sago palm (metroxylon sagu) via a differential display technique. Masters thesis, Universiti Malaysia Sarawak, UNIMAS.

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Abstract

Differential display is rapid and economical method compared to traditional differential screening of cDNA libraries or construction of subtracted cDNA libraries for the identification of differentially expressed genes. This technique is used in the present study to identify genes that are specifically expressed in flower tissues of sago) Sago palm maturation period is a major obstacle in the development of this plant as a commercial crop. Previously, it has been found that the flowering of sago palm is a direct indicator for the maximum level of starch content in the trunk. This study focuses on the isolation of specific genes that can be expressed only in the flower tissue. Characterization of these genes can lead to the discovery of the regulatory gene for maturation in sago palm. A nonradioactive differential display' technique, which takes advantage of chemiluminescent technology, has been adopted for this study. This adaptation has proven to be successful compared to other nonradioactive techniques. The results were more convincing and reproduceable. A random primer was used to amplify the cDNA generated from mRNA of different tissues and the differentially expressed cDNA bands were displayed in the lwniniscent detection film. Two differentially expressed bands, S 1 and S2 were selected from the cDNA fingerprints. The bands were then excised from the cDNA fingerprints and reamplified using the same random primer. These differentially expressed bands were then analyzed by blot analysis to determine their specificity. They showed positive results in all tI blotting experiment including cDNA blotting and Northern blotting. The blotting experiments ' also utilized the chemiluminescent detection method. The S 1 and S2 bands w.ere then cloned into a pPCR-Script Amp SK(+) cloning vector before it was transformed into a E. coli and stored in glycerol stock for further analysis. cDNA sequencing of the S 1 bands showed high.

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