| Protein disulfide isomerase (PDI; EC 5.3.4.1) of which the retention in the ER is mediated by its KDEL sequence at the carboxyl terminus, is an abundant oxidoreductase enzyme in the endoplasmic reticulum (ER) and participates in protein folding, assembly, and post-translational modification. PDI is a key protein in regulating the formation of proteins disulfide in wheat. DsbA, an abundant enzyme in the lumen of endoplasmic reticulum and with the strongest oxidizing ability in the Dsb family, is strongly oxidizing and involved directly in the oxidation of thiol groups of proteins.Low-molecular-weight glutenin subunit (LMW-GS) is essential in determining dough extensibility and gluten strength. The dough quality of bread wheat and durum wheat are closely related to allelic variation of LMW-GS. The strength of wheat gluten is controlled by glutenin polymeric proteins which are composed of glutenin subunits by disulfide bonds. In this study, a cDNA fragment was isolated from the wheat cultivar"Shaan 253"viaRT-PCR and the sequence features were analyzed with bioinformatics. The expression vector was constructed and transformed into the host bacteria Escherichia coli BL21(DE3), then expressed protein induced by IPTG was purificated. Meanwhile, dicistronic expression plasmid vector containing LMW and DsbA genes was constructed by splice overlap extension (SOE) PCR, then coexpressed LMW and DsbA in the E. coli BL21(DE3) and the micro-mixing test was conducted using a 10 g farinograph by oxidation/reduction reaction in this study. The results were as follows:1. The complete nucleotide sequence of cDNA fragment with a length of 1539 bp was isolated (GenBank accession No: HQ911363) from"Shaan 253". Sequence analysis shown that the similarity between HQ911363 and published PDI gene sequence can be reached 99.81%. Amino acid sequence analysis showed that the protein encoded by HQ911363 contained typical isomerase catalytic site -CGHC- and endoplasmic reticulum retention signal peptide -KDEL-.Sequence alignment analysis showed that the amino acid sequence is highly conservative both in the redox activity sites and endoplasmic reticulum retention signal peptide.2. The result of phylogentic tree which was constructed based on Triticum aestivum L.(BAH20801and CAI30632), T. turgidum L.(CAC21228),Hordeum vulgare L.(AAA70345), Zea mays L.(AAX09971), Brassica carinata L.(ABB17025), Medicago sativa L.(ACJ85784), Nicotiana tabacum L.(CAA72092) and deduced amino acid sequence of HQ911363 and with MEGA 4.0 indicated that the HQ911363 and related proteins of Triticum aestivum L.(BAH20801and CAI30632), T. turgidum L.(CAC21228) were most close in the phylogenetic tree, which showed highly homology and least related to Nicotiana tabacum L.(CAA72092).3. The positive cloning vector was subcloned by F2 and R2. Amplification product and vector pET-32a(+) were digested by endonuclease BamH I and Hind III, then the two restriction products were linked by T4 DNA ligase. The recombinant plasmids were transformed into E.coli BL21(DE3) and identified by colony PCR and double endonuclease, which showed that the products size about 1500bp was in accord with anticipation. The result of sequencing indicated that PDI gene was amplified and the recombinant plasmid was constructed successfully.4. The positive clones of recombinant plasmid were transformed into expression host strain E.coli BL21(DE3) competent cells, The fusion protein was observed at approximately 72 kDa with the induction of 1mM IPTG at 37°C for 8 h, which was in accordance with the predicted molecular weight by DANMAN software. Then the expressed protein induced by IPTG was purificated by ?KTA purifier 100 protein chromatography system. The fusion protein was successfully expressed and purified by the dual tests of SDS-PAGE and Western-blot.5. 3 LMW-GS genes were abtained (GenBank accession No:JF439428, JF439429 and JF439430). Three amino acid mutations were found among these deduced amino acid sequences: the first occurred at position 53 in the start of the repetitive domain, the second at the position 108 in the end of the repetitive domain, the last at the position 229 in the C-terminal II. The phylogenetic tree of LMW-GS was depicted by MEGA 4.1 Solftware and constructed by the neighbour-joining method. These proteins were clustered into LMW-m type encoded by Glu-B3 locus, which was consistent with the result of amino acid sequence analysis.6. As these deduced amino acid sequences were very similar, LMW/DsbA (JF439428) coding mature proteins was amplified with primer pair F3/R3. After 6 h induction with 0.6 mM IPTG at 25°C, the recombinant cells were harvested and whole cell lysates were analyzed by 12% SDS-PAGE, which revealed a target band, about 52 kDa fused with histidine-tagged thioredoxin (Trx), at the expected position. Western blot analysis showed that a band with molecular weight of about 52 kDa was positively stained. Solubility analysis revealed that coexpressing DsbA improved the solubility of expressed LMW-GS.7. The result of farinograph tests showed that the incorporation of LMW-GS resulted in significant increase in DvT and decreased DS significantly (p < 0.05). All the ST, BDT, FQN values showed slight increase, and MTI decreased but not statistically significant. |
