Amplification and bacterium expression of high-molecular-weight glutenin subunit 1Bx14 gene from wheat
小麦高分子量谷蛋白14亚基基因的PCR体系及原核表达研究

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.