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

    小麦高分子量谷蛋白14亚基(HMW-GS 1Bx14)被认为可能对面团加工品质有很大贡献的亚基.为了验证其功能,本研究欲通过PCR方法扩增14亚基基因,并使其在大肠杆菌中大量表达,为下一步用掺粉实验验证其功能奠定基础.但是由于HMW-GS 1Bx14基因由2388 bp个碱基组成,GC含量较高,并且在基因中包含约1.6 kb的重复区,所有这些都使常规PCR难以成功扩增出HMW-GS 1Bx14 基因.本研究采取以下三种措施:①设计一对高Tm值的特异引物;②采用二段法PCR反应程序;③改良PCR反应的缓冲体系、添加有机溶剂(DMSO,甜菜碱等)和Taq酶保护剂(BSA等);最后成功地扩增出目的片段.此外由于不同生物间对密码子使用存在很大差异,因此HMW-GS 1Bx14基因很难在大肠杆菌中得到有效大量的表达.本研究选用万能菌株Rosetta(DE3)plysS作为表达用菌,从而克服了不同生物间密码子使用偏爱性的问题,成功的使HMW-GS 1Bx14得到表达,为下一步研究HMW-GS 1Bx14的基因功能奠定了基础.

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-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.