Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata
Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To const...
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| Format: | Article |
| Language: | English |
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Hanrimwon Publishing Company
2015-06-01
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| Series: | The Plant Pathology Journal |
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| Online Access: | http://www.ncbi.nlm.nih.gov/pmc/articles/4453991 |
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| author | Jin-Xin Gao Jing Jing Chuan-Jin Yu Jie Chen |
| author_facet | Jin-Xin Gao Jing Jing Chuan-Jin Yu Jie Chen |
| author_sort | Jin-Xin Gao |
| collection | DOAJ |
| description | Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 6.39 ×10⁵ transformants/3 μg pGADT7-Rec. The titer of the primary cDNA library was 2.5×10⁸ cfu/mL. The numbers for the cDNA library was 2.46×10⁵. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a “bait” to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway. |
| first_indexed | 2024-12-12T16:06:33Z |
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| id | doaj.art-b40a6e7913254dad842a60b8ec104512 |
| institution | Directory Open Access Journal |
| issn | 1598-2254 |
| language | English |
| last_indexed | 2024-12-12T16:06:33Z |
| publishDate | 2015-06-01 |
| publisher | Hanrimwon Publishing Company |
| record_format | Article |
| series | The Plant Pathology Journal |
| spelling | doaj.art-b40a6e7913254dad842a60b8ec1045122022-12-22T00:19:18ZengHanrimwon Publishing CompanyThe Plant Pathology Journal1598-22542015-06-0131210811410.5423/PPJ.OA.01.2015.0001PPJ.OA.01.2015.0001Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunataJin-Xin Gao0Jing Jing1Chuan-Jin Yu2Jie Chen3School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, P. R. ChinaSchool of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, P. R. ChinaSchool of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, P. R. ChinaSchool of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, P. R. ChinaCurvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 6.39 ×10⁵ transformants/3 μg pGADT7-Rec. The titer of the primary cDNA library was 2.5×10⁸ cfu/mL. The numbers for the cDNA library was 2.46×10⁵. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a “bait” to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.http://www.ncbi.nlm.nih.gov/pmc/articles/4453991Brn1Curvularia lunatapathogenesis-related proteinVelBY2H library |
| spellingShingle | Jin-Xin Gao Jing Jing Chuan-Jin Yu Jie Chen Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata The Plant Pathology Journal Brn1 Curvularia lunata pathogenesis-related protein VelB Y2H library |
| title | Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata |
| title_full | Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata |
| title_fullStr | Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata |
| title_full_unstemmed | Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata |
| title_short | Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata |
| title_sort | construction of a high quality yeast two hybrid library and its application in identification of interacting proteins with brn1 in curvularia lunata |
| topic | Brn1 Curvularia lunata pathogenesis-related protein VelB Y2H library |
| url | http://www.ncbi.nlm.nih.gov/pmc/articles/4453991 |
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