Difference between revisions of "Shamita P"
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I decided to study at least one of the CBF transcription activators not found in blueberry as well as one frost tolerance gene found to be downstream of CBF. I searched the NCBI database to obtain mRNA sequences of my genes of interest. Of the several genes that I input into the Vaccinium database (conducting tBLASTx against the scaffolds), I found that there were two genes in particular with promising results. The first of these was the ''EguCBF1c'' in Eucalyptus, whose match against Scaffold 00009 had an E score of 10^(-31). When I submitted ''TaCBF14'' gene from common wheat for analysis in the same manner, the top hit was also Scaffold 00009 with an E score of 10^(-17). | I decided to study at least one of the CBF transcription activators not found in blueberry as well as one frost tolerance gene found to be downstream of CBF. I searched the NCBI database to obtain mRNA sequences of my genes of interest. Of the several genes that I input into the Vaccinium database (conducting tBLASTx against the scaffolds), I found that there were two genes in particular with promising results. The first of these was the ''EguCBF1c'' in Eucalyptus, whose match against Scaffold 00009 had an E score of 10^(-31). When I submitted ''TaCBF14'' gene from common wheat for analysis in the same manner, the top hit was also Scaffold 00009 with an E score of 10^(-17). | ||
| − | The tBLASTx translated my mRNA query into a protein and then matched it with all proteins constructed by all reading frames of the nucleotide sequence of the scaffolds. For this reasons, it's the longest search conducted in the BLAST database. Using the amino acid sequences that were output and their corresponding nucleotide matches to the scaffold, I was able to approximate where in the scaffold my genes were located. | + | The tBLASTx translated my mRNA query into a protein and then matched it with all proteins constructed by all reading frames of the nucleotide sequence of the scaffolds. For this reasons, it's the longest search conducted in the BLAST database. Using the amino acid sequences that were output and their corresponding nucleotide matches to the scaffold, I was able to approximate where in the scaffold my genes were located. Both ''CBF'' genes Eucalyptus and Common wheat produced hits in the same region of the scaffold, at approximately 488,000 bp. |
I submitted scaffold 00009 to be searched for SSRs using default parameters that favored lengthy di- or tri- nucleotide repeats. Vaccinium.org returns an excel file with the location and length of SSRs along with primers engineered to amplify the regions containing the SSR. See below. | I submitted scaffold 00009 to be searched for SSRs using default parameters that favored lengthy di- or tri- nucleotide repeats. Vaccinium.org returns an excel file with the location and length of SSRs along with primers engineered to amplify the regions containing the SSR. See below. | ||
| + | [[Image:Excel_SSR.jpg]] | ||
| − | + | Choosing SSRs in the vicinity of my genes, I found 4 lengthy di-nucleotide repeats around 488,000 bp and one tri-nucleotide repeat near 210,000 bp (not pictured). The excel file does not always contain primers for every SSR match, so those positions are of no use to us. For primers it does provide, I chose ones that produced PCR products that were less than 300 bp. | |
| + | |||
| + | When mapping this to the Word File which contained the entire scaffold 00009, I found my SSR match (pg 183) to be about 4 pages away from the 11 combined hits found (pg 187) from the gene search on the scaffold. | ||
Revision as of 02:51, 28 February 2012
Cold Tolerance of the Northern highbush blueberry (Vaccinium corymbosum)
General background on cold tolerance
Winter Acclimation and Cold Hardiness of the Blueberry: Primarily geared towards individuals who wish to cultivate blueberries, but provides some good general background information on the cold tolerance associated with regional varieties.
Additionally, Polashock et al (2010) provides substantial background information on genetic basis of cold tolerance. In summary, they discuss that the purpose behind studying these genes is to understand how modifying cold-tolerance in blueberry might prevent massive crop loss due to freezing temperatures during a winter frost. The overall acclimation to cold occurs in two steps, the first of which is induced by a shorter photo-period (less sunlight), and the second of which is induced by lower temperatures. Polashock et al targeted a host of genes in a family of transcription factors called CBF (C-repeat binding factor). These TF appear to bind a conserved region CCGAC within promoters that activate a host of downstream genes involved in cold acclimation. Using this gene as a starting point, I decided to search for candidate genes downstream of CBFs in other species that were being activated in cold conditions.
Searching the CBF (C-repeat binding factor) genes
The exciting thing about CBFs is that they are found in many species of plants. So, if there are genes downstream of this TF in those plant species, they might be good targets for study in blueberry as well. I explored various papers discussing cold tolerance genes in Eucalyptus, Arabidopsis, and common wheat. Although common wheat is a monocot, I felt like it would be worth exploring because like blueberry, it is an important crop and might also have invested interest in its frost tolerance.
Starting with the paper above by Polashock et al, I obtained a list of the following genes from the following papers:
Cold Acclimation/Freezing Tolerance in Blueberries
Polashock et al (2010)
-COR6.6
-COR78
-COR15A etc..
Frost Tolerance in Temperate Cereals
Galiba et al (2009)
-FR2
-TaCBF14
-TaCBF15
Cold Tolerance in Eucalyptus Species
Navarro et al (2009)
-EguCBF1c
-EguCBF1d
Cold Tolerance signaling in Arabidopsis
Lissarre et al, 2010
-ICE1
-ICE2
I decided to study at least one of the CBF transcription activators not found in blueberry as well as one frost tolerance gene found to be downstream of CBF. I searched the NCBI database to obtain mRNA sequences of my genes of interest. Of the several genes that I input into the Vaccinium database (conducting tBLASTx against the scaffolds), I found that there were two genes in particular with promising results. The first of these was the EguCBF1c in Eucalyptus, whose match against Scaffold 00009 had an E score of 10^(-31). When I submitted TaCBF14 gene from common wheat for analysis in the same manner, the top hit was also Scaffold 00009 with an E score of 10^(-17).
The tBLASTx translated my mRNA query into a protein and then matched it with all proteins constructed by all reading frames of the nucleotide sequence of the scaffolds. For this reasons, it's the longest search conducted in the BLAST database. Using the amino acid sequences that were output and their corresponding nucleotide matches to the scaffold, I was able to approximate where in the scaffold my genes were located. Both CBF genes Eucalyptus and Common wheat produced hits in the same region of the scaffold, at approximately 488,000 bp.
I submitted scaffold 00009 to be searched for SSRs using default parameters that favored lengthy di- or tri- nucleotide repeats. Vaccinium.org returns an excel file with the location and length of SSRs along with primers engineered to amplify the regions containing the SSR. See below.
Choosing SSRs in the vicinity of my genes, I found 4 lengthy di-nucleotide repeats around 488,000 bp and one tri-nucleotide repeat near 210,000 bp (not pictured). The excel file does not always contain primers for every SSR match, so those positions are of no use to us. For primers it does provide, I chose ones that produced PCR products that were less than 300 bp.
When mapping this to the Word File which contained the entire scaffold 00009, I found my SSR match (pg 183) to be about 4 pages away from the 11 combined hits found (pg 187) from the gene search on the scaffold.
