Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Comparison between responses to gametophytic and sporophytic recurrent selection in maize (Zea mays L.)

Summary Experiments were conducted to determine the chromosomal location of the gene conditioning overproduction of a methionine-rich, 10-K zein in maize kernels of line BSSS53. In addition, the chromosomal location of the structural gene encoding the overproduced protein was determined. Whereas the structural gene, designated Zps10/(22), was found to be located on the long arm of chromosome 9 near the centromere, the locus regulating overproduction of the zein protein was mapped to the short arm of chromosome 4. This regulatory gene has been designated Zpr10/(22). Regulation of 10-K zein production by Zpr10/(22) is, therefore, via a trans-acting mechanism.     

Isolation and sequence of a gene encoding a methionine-rich 10-kDa zein protein from maize

We have isolated the gene encoding a methionine-rich 10-kDa zein protein from a lambda EMBL3 maize genomic 'mini' library of the inbred line BSSS-53 and determined its nucleotide sequence. The sequence matches perfectly with a cDNA clone from the inbred line W22 (which has the same restriction fragment length polymorphism as many inbred lines tested) indicating that we have isolated a functional storage protein gene that is very conserved in maize. This comparison also excludes any splicing of any precursor mRNA and therefore any presence of introns. A number of potential regulatory sequences have been located in the flanking regions. The 10-kDa-zein gene represents the last size class in the zein multigene family to be characterized. Its structure allows us now to re-examine the relationship of all the zein proteins and also to compare the structure of a new class of storage proteins that are rich in methionine, an essential amino acid in livestock fodder.     

Heterogeneity of zein mRNA and protein in maize

Zein, the prolamine fraction of maize, is localized in the endosperm in membrane-bound structures called protein bodies, which have polyribosomes on their surfaces. These polysomes or the mRNA fraction isolated from them will direct the synthesis of zein-like proteins in vitro. The in vitro products consist primarily of two molecular weight classes but show considerable charge heterogeneity when analyzed by isoelectric focusing. Although the molecular weight classes are very similar for different inbred lines, the isoelectric focusing patterns differ.Results given here suggest that the extensive charge heterogeneity of zein proteins does not result from the presence of a large number of totally distinct mRNAs. Zein proteins synthesized in vitro fall into several families related by sequence homologies in their mRNAs. In Illinois High Protein (IHP) the major zein mRNAs can be classified into three families based on their binding to cloned complimentary DNA copies of IHP zein mRNA. Each of three other lines we have studied (W22, Oh43, and W64A) has zein mRNAs that are related to those of IHP. Among these four lines the molecular weights of the members of a given family are generally similar, but the number of members in a family and their isoelectric points differ.     

Variation of Storage Proteins and Isozymes within Maize Inbred Lines

Seed storage proteins of ten maize inbred lines were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, fourteen isozyme loci representing nine isozyme systems were analysed. Salt-soluble protein fraction contained a large number of proteins (30 – 40 bands) of different sizes with genotypic differences among the ten inbred lines. The methionine-rich 10 kD zein showed differential expression in the ten inbred lines with different migration rates on the SDS-PAGE. This polypeptide was completely absent in the inbred line G221D. Among nine of the inbred lines, eight of 14 isozyme loci were polymorphic and six were monomorphic resulting in seven unique fingerprints.     

Separation of alcohol-soluble proteins (zeins) from maize into three fractions by differential solubility

The prolamin of maize (Zea mays L.), zein, was extracted from endosperm meal with 60% (v/v) 2-propanol/1% (v/v) 2-mercaptoethanol either directly or subsequent to extraction with 90% (v/v) 2-propanol. The zein extracted with 90% 2-propanol was essentially made up of 20 to 24 kilodalton polypeptides (α-zein) while that extractable with 60% 2-propanol/1% 2-mercaptoethanol contained, in addition to α-zein, 17 to 18 kilodalton methionine-rich polypeptides and a 27 kilodalton proline-rich polypeptide. While zein was separated into three fractions by differential solubility in 90% 2-propanol and 30% 2-propanol/30 millimolar sodium acetate (pH 6) using two different fractionation protocols. Each of the three solubility fractions (SF1, SF2, and SF3) had a unique polypeptide composition. Based on results obtained from two inbreds, K55 and W64A, the SF1 constituted 75 to 80% of the total zein and included as major components 20 to 24 kilodalton polypeptides and a minor 10 kilodalton polypeptide. The SF2 made up 10 to 15% of the total zein and included exclusively 17 to 18 kD methionine-rich polypeptides. A 27 kilodalton proline-rich component constituted the SF3 and contributed 5 to 10% to total zein.     

Tissue-specific zein synthesis in maize kernel

Zein may account for as much as 10% of the total protein in the mature embryo of maize inbred W64A. This protein exhibited an electrophoretic pattern on SDS gels similar to that of the endosperm. Like the endosperm system, the synthesis of zein components in the embryo was controlled by the opaque-2 and floury-2 mutations. However, unlike zein synthesis in the endosperm, zein synthesis in the embryo could not be increased by nitrogen fertilizer. Variations in amino acid composition were observed between the zein components of the embryo and those of the endosperm.     

Asymmetric localization of seed storage protein RNAs to distinct subdomains of the endoplasmic reticulum in developing maize endosperm cells

Plant storage proteins are synthesized and stored in different compartments of the plant endomembrane system. Developing maize seeds synthesize and accumulate prolamin (zein) and 11S globulin (legumin-1) type proteins, which are sequestered in the endoplasmic reticulum (ER) lumen and storage vacuoles, respectively. Immunofluorescence studies showed that the lumenal chaperone BiP was not randomly distributed within the ER in developing maize endosperm but concentrated within the zein-containing protein bodies. Analysis of the spatial distribution of RNAs in maize endosperm sections by in situ RT-PCR showed that, contrary to the conclusions made in an earlier study [Kim et al. (2002) Plant Cell 14: 655-672], the zein and legumin-1 RNAs are not symmetrically distributed on the ER but, instead, targeted to specific ER subdomains. RNAs coding for 22 kDa alpha-zein, 15 kDa beta-zein, 27 kDa gamma-zein and 10 kDa delta-zein were localized to ER-bounded zein protein bodies, whereas 51 kDa legumin-1 RNAs were distributed on adjacent cisternal ER proximal to the zein protein bodies. These results indicate that the maize storage protein RNAs are targeted to specific ER subdomains in developing maize endosperm and that RNA localization may be a prevalent mechanism to sort proteins within plant cells.     

富含蛋氨酸玉米醇溶蛋白在大肠杆菌中的表达

目的:克隆玉米中富含蛋氨酸的10kD玉米醇溶蛋白,证明植物源目的基因在大肠杆菌中能够表达.方法:从玉米胚乳中克隆高蛋氨酸基因zein,通过PCR扩增zein片段,连接pGEX - 4T -1原核表达载体,转入大肠杆菌中,IPTG诱导后,HPLC测定蛋氨酸含量.结果:经PCR扩增出467bp条带,Blast分析同源性99%,经IPTG诱导进行SDS - PAGE检测,发现在36kD处出现一条明显的条带.诱导后菌体总蛋氨酸含量比正常菌体提高了9.6%.结论:证实了植物源10kD玉米醇溶蛋白在大肠杆菌中能够表达.     

Binding of a nuclear factor to a consensus sequence in the 5' flanking region of zein genes from maize

The genomic organization of the zein structural genes and of regulatory loci influencing their expression suggests that control of zein gene expression will involve interactions between cis elements in the flanking DNA sequences and products from trans-acting genes. The interaction between fragments from the 5' flanking region of a zein gene and specific, double-stranded oligonucleotides with crude nuclear extracts from maize endosperm have been studied by nitrocellulose filter binding, gel retention and DNase I footprinting assays. Specific binding of a nuclear factor was observed and the exact position of the protein binding site was determined. The 22-nt binding site included 14 bp of a 15-bp sequence conserved in all zein genes.     

Cloning of double stranded DNAs derived from polysomal mRNA of maize endosperm: isolation and characterisation of zein clones.

Starting from polysomal RNA of developing maize endosperm and applying the cloning procedure of integrating dsDNA into the Pst I site of plasmid pBR 322, clones containing sequences complementary to endosperm mRNAs were obtained. 25 per cent of these clones were identified as containing zein specific DNA sequences which hybridized either with the zein mRNA coding for the 22 000 Mr protein or with the zein mRNA coding for the 19 000 Mr protein. The zein-specific DNA inserts of the recombinant plasmids were further characterized by restriction enzyme analysis.