Cloning, Molecular Identification And Prokaryotic Expression Of Two Hmw-gs Genes From Chinese Wheat (triticum Aestivum L.) Landrace Banjiemang

The wheat germplasm resources are abundant in China. Being important geneticresources of quality and yield improvement, the Chinese wheat（Triticum aestivum L.）landraces have varieties of resistance genes and excellent characters. The high molecularweight glutenin is one of the important storage proteins in the wheat grain endosperm, andboth the composition and the allele variation of high molecular weight glutenin subunits(HMW-GS) directly affect the quality properties of wheat flour. Exploring allele variations ofHMW-GS in landraces and materials available in wheat quality improvement can lay thegenetic basis and material basis for wheat quality breeding. The study presented here usedBanjiemang, a Chinese wheat landrace, as material, and analysed the HMW-GS compositionby SDS-PAGE experiment. The open reading frames (ORFs) of x-type and y-type subunitgenes on Glu-B1loci of Banjiemang were cloned using designed specific primers. Themultiple sequence alignment and phylogenetic tree based on the amino acid sequencesdedecud from the cloned genes were conducted. At the same time, the cloned sequences wereexpressed in bacterial. The cloned target genes were confirmed by LC-ESI-MS.Finally, theSDS-sendimentation volume of Banjiemang was tested using the trace SDS-sedimentationmethod. The results are as follows:1. The HMW-GS composition in Banjiemang was detected by SDS-PAGE. Therewere4HMW-GS bands in Banjiemang, null on Glu-A1loci,2on Glu-B1loci and2onGlu-D1(1Dx2+1Dy12) loci.2. Two sets of primers specific to x-type and y-type HMW-GS genes were designed toamplify the target genes using the gDNA of Bangjiemang as template. Two fragments ofabout2.4kb and2.1kb were obtained. The obtained fragments were then purified, cloned,sequenced and analysed. The sequence homology search and alignment revealed two novelgenes belonging to HMW-GS gene family. The two genes, designated as1Bx23.1and1By15.1, were deposited in Genbank with accession number KJ579439and KJ579440, respectively.The ORF of1Bx23.1and1By15.1was2367bp and2106bp long (without the stopcodon), and encoded proteins of789and702amino acid residues, respectively. Both1Bx23.1and1By15.1shared the typical primary structure of HMW-GS, namely a signal peptide,conserved N-terminal, central repetitive domain and conserved C-terminal. There existed4(3in the N-terminal and1in the C-terminal) and7(5in the N-terminal,1in the repetitivedomain near the C-terminal and1in the C-terminal) cysteine residues in1Bx23.1and1By15.1subunits, respectitively. The number and position of cysteine residues of1Bx23.1and1By15.1subunits were identical to typical Bx-and By-type subunits, respectitively.Homology search showed that1Bx23.1had the highest identity of99%with1Bx23subunitwhile1By15.1had the highest identity of95%with1By15subunit.1Bx23.1subunitpresented a tandem repeat motif of hexapeptide (PAQGQQ) deletion at position553incorrespondence to1Bx23subunit;1By15.1subunit presented15amino acid residues(PAQGQQGQYPASQQQ) deletion at position365and a repeat motif of hexapeptide(SGQGQQ) deletion at position586in correspondence to1By15subunit. There were12(1inN-terminal,10in the repetitive domain,1in C-terminal) and19(2in N-terminal,16in therepetitive domain,1in C-terminal) amino acid residue substitutions taking place in1Bx23.1and1By15.1subunits compared to other HMW-GS, respectitively.3. The bands represented the x-type and y-type subunits on Glu-B1loci in Banjiemangwere manually excised from the gel followed by LC-ESI-MS analysis. The peptide sequencesof the subunits identified by MS matched well with the amino acid sequences deduced from1Bx23.1and1By15.1genes.4. Two sets of PCR primers were designed for mutant ORFs amplification with signalpeptides removed and NdeI and EcoRI restriction enzyme sites introduced into5’ terminal ofupstream and dowmstream primers, respectitively. The constructed bacterially expressionvectors were transformed into E. coli BL21(DE3) competent and the positive clones wereinduced by IPTG. Finally, the expressed proteins were analysed by SDS-PAGE. InSDS-PAGE analysis of the protein extracts prepared from the induced bacterial cultures, theelectrophoretic mobilities of bacterially expressed proteins were co-migrated with theauthentic subunits on Glu-B1loci from the seed endosperm of Banjiemang. The resultsdemonstrated that the target genes expressed successfully in E. coli.5. A phylogenetic tree was constructed based on the multiple sequence alignment ofamino acid sequences of HMW-GS on Glu-1locus. In the phylogenetic tree,1Bx23.1wasclustered with x-type subunits and closely related to1Bx23subunit, whereas1By15.1wasclustered with y-type subunits and closely related to1By15subunit. 6. The SDS-sedimentation volume of Banjiemang was5.90ml, which was significantlyhigher than that of the high-quality cultivar Xinong979, indicating the high quality ofBanjiemang.