Examination of Cold-Plaque Screening Technique as a Means to Isolate Low Abundance Genes from Oil Palm (Elaeis Guineensis) Flowers
Genes that are present at low abundance in cells are difficult to clone by using standard molecular biology techniques such as conventional differential screening. In plants, many of these low abundance genes encode transcription factors or proteins involved in signal tranduction. Therefore in th...
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| Format: | Thesis |
| Language: | English English |
| Published: |
1999
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/8408/1/FSMB_1999_12_IR.pdf http://psasir.upm.edu.my/id/eprint/8408/ |
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| Summary: | Genes that are present at low abundance in cells are difficult to clone by using standard
molecular biology techniques such as conventional differential screening. In plants, many
of these low abundance genes encode transcription factors or proteins involved in signal
tranduction. Therefore in this study, a cold-plaque screening technique was used as a
means to enrich for low abundance genes from an oil palm male flower cDNA library.
When a total of 441 non-hybridising plaques ('cold' plaques) were isolated, 123
clones (opcp population) were found to contain inserts with a minimal size of 500 basepairs
and were independent clones. Initial screening of these clones by reverse northern
analysis with the same probes used during differential screening and an additional probe
from female flower of 6 em showed that these 0PCP clones could be categorised into five
subpopulations based on their tissue-specificity and expression levels. 61.8% of the 123
clones were expressed at high abundance with all the three probes (Subpopulation A)whilst 4.1% of the clones were lowly-expressed in both male and female flower tissues
(Subpopulation B). 7.3% of the clones were expressed at medium abundance but were
male-predominant (Subpopulation C) while 11.4% of the clones were expressed at low
abundance and were male-predominant (Subpopulation D) and 15.4% of the clones did
not show any detectable expression with any of the probes used (Subpopulation E).
Partial sequencing of all clones from subpopulation B, C, D and E as well as eight
clones from subpopulation A showed that opcp72 (subpopulation D) is a putative UIP2
(Unusual Floral Organ (UFO) binding protein) homolog, opcpl44 (subpopulation A)
encodes elongation factor-la, opcp327 (subpopulation E) encodes a putative RLK 5
(Receptor-like Protein Kinase) homolog and opcp441 (subpopulation A) is a putative
fructose 1, 6-bisphosphate aldolase gene.
Expression studies on ten opcp clones with two representative clones from each
subpopulation showed similar expression profiles where hybridisation signals were
detected at the early flower development with higher signals in the meristem tissues but
no detectable hybridisation signals in 3.5 em and 6 em male flower, one of the stages used
to make the male flower cDNA library. Southern hybridisation of genomic DNA for clone
opcp72, opcpl44 and opcp327 showed that these genes are low copy genes.In conclusion the use of cold-plaque screening techniques can result in the isolation of a
variety of clones whose expression ranges from low abundance (undetectable in the
Northern blots), to those that are lowly expressed during the stages of floral development
used to construct the oil palm male-flower cDNA library. |
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