Amplification and bacterium expression of high-molecular-weight glutenin subunit 1Bx14 gene from wheat
HMW-GS 1Bx14 was candidated as a good subunit for dough quality. HMW-GS 1Bx14 coding sequence can be amplified and expressed in vitro for characterizing its function. An improved PCR method was presented for amplification of HMW-GS 1Bx14 which is a fragment of large (about 2.4 kb) and contain GC-ric...
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| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2007-01-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/1008-9209.2007.01.0045 |
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| Summary: | HMW-GS 1Bx14 was candidated as a good subunit for dough quality. HMW-GS 1Bx14 coding sequence can be amplified and expressed in vitro for characterizing its function. An improved PCR method was presented for amplification of HMW-GS 1Bx14 which is a fragment of large (about 2.4 kb) and contain GC-rich regions. This experiment adapted following three stratages to optimize PCR system for successful amplification: i) designing a couple of primers with high Tm; ii) Adopting two-temperature PCR; iii) Improving PCR buffer for beneficial long amplicon; adding some organic solvents (DMSO, Lycine) and BSA for resistant to form secondary structure and lengthen in polymerase half-life, respectively. Furthermore, it is very difficulty to express HMW-GS 1Bx14 in E. coli because of the bias of codon usage. The Rosetta (DE3) plysS bacterium was used to conquer the effection of rare codon and express HMW-GS 1Bx14 gene successfully. It will be benefit for further studying on the function of 1Bx14 gene. |
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| ISSN: | 1008-9209 2097-5155 |