¦¡-sulfur Gene In Wheat - Rye Translocation Line Materials R59 Cdna Cloning, Expression, Purification And Ftir Study

Objective: To clone the gene encodingα-thionin in wheat-rye translation strain R59, and to express the gene in E.coli BL21(DE3) , then fourier transform infrared (FTIR) microspectroscopy has been used to investigate the effects of C-terminal acidic protein on the secondary structure ofα-thionin in the absence of signal peptide during the prokaryotic expression process. Methods: cDNA encodingα-thionin was amplified by RT-PCR from the total RNA of R59 seedling leaf, and the recovered fragment was cloned and sequenced. The primers bearing restriction enzyme site of Nco I and Hind III were designed and were ultilized to amplify full-length of ORF , signal peptide/ C-terminal acidic protein-truncated fragement (SC,△SC,S△C,△S△C) of R59-thionin gene. The target fragments were then ligated into the Nco I/ Hind III -digested pET-32 a (+) to produce the prokaryotic expression vector pET-32a(+)/SC,pET-32a(+)/△SC ,pET-32a(+)/S△C and pET-32a(+)/△S△C . After recombinant plasmids were identified by restriction endonuclease digestion analysis and DNA sequencing, they were transformed into E.coli BL21(DE3), and then were induced by IPTG to express the target proteins. SDS-PAGE and Western blotting were utilized to identify the fusion protein. The effects of C-terminal acidic protein during the expression process was detected by using fourier transform infrared microspectroscopy. In addition, the soluble protein obtained from pET-32a(+)/△S△C was further purified by Ni2+-NTA column. Following the treatment of enterokinase, the target protein was finally purified by the means of high performance liquid chromatography (HPLC). Results:α-thionin gene contains an open-reading frame (ORF) of 408 nt encoding a 136 amino acid residue, which shares 78% ~ 100% homologies with wheat, barley and ryes at amino acid level. Restriction endonuclease digestion analysis and sequencing data revealed that the target gene has been successfully inserted the expression vector pET-32 a (+). SDS-PAGE analysis revealed that no matter whether or not C-terminal acidic protein existed, the heterogenous gene couldn't be expressed in high yield in E.coli BL21(DE3) in the case of the presence of signal peptide. When signal peptide was trunctated, the introduction of C-terminal acidic protein gave rise to the formation of inclusion body, however, the absence of C-terminal acidic protein greatly enhanced the solubility of the heterogenous protein. FTIR result showed that apart from the aggregate complex absorbing at 1630 cm-1 , there still occurredα-helix structure in inclusion body, which is responsible for the 1654 cm-1 absorption peak; Gaussian curve-fitting was done on the Fourier self-deconvolution spectra within Amide I region of intact cells. The experimental data revealed that the relative contens of aggregate absorbing at 1629±1cm-1 was gradually increasing with induction time, which is consistant with the results of SDS-PAGE. Simutaneously, the formation of aggregate gave rise to the increase ofα-helix, as well as the decrease ofβ-turn and random coil in the case of pET-32a(+)/△SC. It was not the case for pET-32a(+)/△S△C, however, where random coil experienced the increase in the relative average fractions, whileβ-turn andβ-sheet at 1629±1cm-1 behaviored in different ways. Conculsion: The gene fragement having signal peptide couldn't expressed successfully in E. coli BL21(DE3). When signal peptide was lost, the absence of C-terminal acidic protein greatly enhanced the solubility of the fusion protein. The target protein could be purified by using Ni2+-NTA affinity chromatography. FTIR results revealed thatβ-sheet and random coil are most likely to transform into aggreate andα-helix with the introduction of C-terminal acidic protein. These studies will be helpful for further research on the function and oxidative refolding ofα-thionin.