Cloning Of B-hordein Genes And Upstream Regulating Sequences From Hull-less Barley And Their Expression

Seed endosperm storage proteins in higher plants are the main resources ofnitrogen for germinating and plant proteins for human and animals. Barley prolamins(also called hordeins) are the major storage proteins in the endosperm and account for50-60％of total proteins. Hordeins are classically divided into three groups:sulphur-rich (B,γ,-hordeins), sulphur-poor (C-hordeins) and high molecular weight(HMW, D-hordeins) hordeins based on the size and composition. B-hordeins andC-hordeins are two major groups and each respectively account for about 70-80％and10-12％of the total hordein fraction in barley endosperm. Genetic analysis showedthat B-, C-, C-,γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on thechromosome 1H (5). Hor2 locus is rich in alleles that encode numerousheterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantityand distribution are significant factors affecting malting, food and feed quality ofbarley. To understand comprehensively the structure and organization of B-hordeingene family in hull-less barley and explore the developmental control mechanisms ofHor2 locus gene expression and eventually to better exploitation in crop grainquality improvement, we isolated and cloned B-hordein genes and promotors ofhull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expressionfounction in bacteria expression system and explore their spatial and temporalexpression pattern by quantitative real time PCR. Our results are as followed,1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barleycultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularlycharacterized by PCR, based on three B-hordein genes published previously(GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmedthat the six clones all contained a full-length coding region of the barley B-hordeingenes. Eleven clones all contain an in-frame stop codon and they are probablypseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitivedomain, and C-terminal domain. The number of the repeats was largerly variable andresulted in polypeptides in different sizes or structures among the genes. Twelve suchrepeated motifs were found in Z07-2 and Z26, and they are close to those of the wildbarleys, and it is most probably caused by unequal crossing-over and/or slippageduring replication as suggested for the evolution of other prolamins. The relatednessof prolamin genes of barley and wheat was assessed in the phylogenetic tree based ontheir polypeptides comparison. Our phylogenetic analysis suggested that the predictedB-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wildbarleys and the two most similar sequences of LMW-GS of T. aestivum formedanother subfamily. This result indicated that the members of the two subfamilys havea distinctive difference. In addition, we found the B-hordeins with identicalC-terminal end sequences were clustered into a same subgroup (except BXQ053,BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein genesubfamilies possibly can be classified on the basis of the conserved C-terminal endsequences of predicted polypeptide and without the limit of SDS-PAGE proteinbanding patterns.2. The specific primers were designed according to the published sequences ofbarley B-hordein genes from Z09 and Z26. Using total DNA isolated from them asthe templates, eight clones (designated Z09Pand Z26P) of upstream sequences of theknown B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequencesanalysis shows that the putative TATA box was present at position -80 bp andCAAT-like box at position -140 bp. Besides, a putative Endosperm Box includingan Endo sperm Motif (EM) and a GCN4-Like Motif was found at position -300 bp insix clones, and another Endosperm-like box was found at positon -560 bp.While the Endosperm Box or Endosperm-like box was not found in Z09P-2and Z26P-3. This may indicate that gene expression drived by the two promtors wasprobably controlled by different trans-acting factors and the genetic controlmechanism of corresponding gene expression may be diverse.3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifyinggene expression fountion. Protein SDS-PAGE analysis showed that only thetransformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs producedproteins related to B-group hordeins of barley, and the mounts of proteins induced by3 mM IPTG and 3 h were higher than other conditions. This established a base forisolating and putifying B-hordein and further exploring their effects on barley grainquality.4. The gene-specific primers of B-hordein genes from Z09 and Z26 were usedfor the quantification of B-hordein gene expression. Theα-tubulin gene fromHordeum vulgate subsp, vulgate (GenBank accession number U40042) was used as acontrol gene. The result shows the transcription of the B-hordein genes in Z09 wasfound 7 days after flowering, while the transcription of the B-hordein genes in Z26was found 4 days after flowering, but at a very low level, and it suggested that theB-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordeingenes in Z09 expressed at so a lower level than Z26 that it can not detected. Inaddition, B-hordein genes in Z26 accession showed higher expression levels thanthose in Z09 in four developing stages. Furthermore, a progressive increase in theexpression levels of the B-hordein genes between 12 and 18 days after anthesis wasobserved in both Z09 and Z26. It implies that the B-hordein allelic variants encodedby Hor2 locus exist the differential expression in mRNA levels of during barleyendosperm development.