The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR
The RT-PCR technique was used to isolate partial gene fragments for the chalcone synthase (CRS), flavanone 3-hydroxylase (F3H) and phytoene synthase (PSY) genes from 0. taka. The RT-PCR products were amplified by degenerate primers specifically designed for the genes and the templates (total RNA)...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2000
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| Online Access: | http://psasir.upm.edu.my/id/eprint/9091/ http://psasir.upm.edu.my/id/eprint/9091/1/FSAS_2000_21%20IR.pdf |
| _version_ | 1848841051660353536 |
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| author | Ong, Wai Kean |
| author_facet | Ong, Wai Kean |
| author_sort | Ong, Wai Kean |
| building | UPM Institutional Repository |
| collection | Online Access |
| description | The RT-PCR technique was used to isolate partial gene fragments for the chalcone
synthase (CRS), flavanone 3-hydroxylase (F3H) and phytoene synthase (PSY) genes
from 0. taka. The RT-PCR products were amplified by degenerate primers
specifically designed for the genes and the templates (total RNA) were prepared
from the leaves, open flowers and flower buds of 0. taka. A 6S0bp RT -PCR product
was successfully amplified from the three different total RNA templates when the
degenerate primers for CHS were used. Similarly, a 543bp RT-PCR product was
obtained when the degenerate primers for PSY were used on the three different total
RNA templates. Only the total RNA preparation from flower buds gave a 503bp RTPCR
product when the degenerate primers for F3H were used. The deduced amino acid sequence for the 650bp DNA fragment was found to have a
high homology to other CHS sequences in the Genebank, averaging at 66%.
Additionally, this sequence also has an exceptionally high homology to previously
reported bibenzyl synthase (BibSyl) sequences, with an average percentage of 85%.
The 503bp fragment has on average 76% homology to the F3H sequences reported
for other plant species. As for the 543bp fragment, it has an avemge of 76%
homology to other PSY gene sequences published at Genebank. The results indicate
that the CHS and PS Y genes are expressed in all the tissues tested whereas the F3 H
gene is only expressed at the flower bud stage in 0. taka.
These gene fragments were labelled with DIG and used as probes to screen a
genomic DNA library constructed from partially digested genomic DNA of O. taka.
Currently, the F3H probe (503bp DNA fragment) has led to the isolation of a
genomic clone with an insert size of around 11kb. The genomic clone was restricted
into 2 fragments with BamH I and subcloned into the pUC 18 vector separately. The
characterisation of the clone is underway. |
| first_indexed | 2025-11-15T07:37:06Z |
| format | Thesis |
| id | upm-9091 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English English |
| last_indexed | 2025-11-15T07:37:06Z |
| publishDate | 2000 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-90912024-03-25T00:33:27Z http://psasir.upm.edu.my/id/eprint/9091/ The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR Ong, Wai Kean The RT-PCR technique was used to isolate partial gene fragments for the chalcone synthase (CRS), flavanone 3-hydroxylase (F3H) and phytoene synthase (PSY) genes from 0. taka. The RT-PCR products were amplified by degenerate primers specifically designed for the genes and the templates (total RNA) were prepared from the leaves, open flowers and flower buds of 0. taka. A 6S0bp RT -PCR product was successfully amplified from the three different total RNA templates when the degenerate primers for CHS were used. Similarly, a 543bp RT-PCR product was obtained when the degenerate primers for PSY were used on the three different total RNA templates. Only the total RNA preparation from flower buds gave a 503bp RTPCR product when the degenerate primers for F3H were used. The deduced amino acid sequence for the 650bp DNA fragment was found to have a high homology to other CHS sequences in the Genebank, averaging at 66%. Additionally, this sequence also has an exceptionally high homology to previously reported bibenzyl synthase (BibSyl) sequences, with an average percentage of 85%. The 503bp fragment has on average 76% homology to the F3H sequences reported for other plant species. As for the 543bp fragment, it has an avemge of 76% homology to other PSY gene sequences published at Genebank. The results indicate that the CHS and PS Y genes are expressed in all the tissues tested whereas the F3 H gene is only expressed at the flower bud stage in 0. taka. These gene fragments were labelled with DIG and used as probes to screen a genomic DNA library constructed from partially digested genomic DNA of O. taka. Currently, the F3H probe (503bp DNA fragment) has led to the isolation of a genomic clone with an insert size of around 11kb. The genomic clone was restricted into 2 fragments with BamH I and subcloned into the pUC 18 vector separately. The characterisation of the clone is underway. 2000-05 Thesis NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/9091/1/FSAS_2000_21%20IR.pdf Ong, Wai Kean (2000) The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR. Masters thesis, Universiti Putra Malaysia. Plant gene isolation - Case studies English |
| spellingShingle | Plant gene isolation - Case studies Ong, Wai Kean The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR |
| title | The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR |
| title_full | The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR |
| title_fullStr | The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR |
| title_full_unstemmed | The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR |
| title_short | The isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using RT-PCR |
| title_sort | isolation and partial characterisation of the chalcone synthase, flavanone 3-hydroxylase and phytoene synthase gene fragments from oncidium taka by using rt-pcr |
| topic | Plant gene isolation - Case studies |
| url | http://psasir.upm.edu.my/id/eprint/9091/ http://psasir.upm.edu.my/id/eprint/9091/1/FSAS_2000_21%20IR.pdf |