Isolation and identification of mRNA for the high-molecular-weight storage proteins of wheat endosperm

The prolamin storage proteins of the wheat endosperm contain a sub-class of high-molecular-weight (HMW) polypeptides which have been implicated in determining breadmaking quality. Membrane-bound polysomes isolated from developing wheat endosperms contain mRNA for these HMW components. Although unfractionated polyadenylated RNA derived from the polysomes did not direct the synthesis of these components in an in-vitro wheat-germ system, it did when incubated with a rabbit reticulocyte lysate system. Identification of the translation products as HMW prolamins was based on their large incorporation of [³H]leucine and [³H]glycine relative to [³H]lysine, their mobility on polyacrylamide-gel electrophoresis and the observation that the changes of mobility in response to change in wheat genotype were the same as those observed for the authentic protein. The mRNA was fractionated by electrophoresis and density-gradient centrifugation. The mRNA for the HMW prolamins was found to have a relative molecular mass of about 1.6·10⁶.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - poly(A)⁺RNA polyadenylated RNA - SDS sodium dodecyl sulphate     

Starch and prolamin level in single and double high-lysine barley mutants

At maturity the high-lysine barley (Hordeum vulgare L.) Ris0 mutants 1508, 527 and 29 kernels contained about 20% less starch and twice as much free sugars as the parent varieties Bomi and Carlsberg II. An enhanched effect on starch reduction and free sugar accumulation was observed during kernel development when the single mutants 527 and 29 are combined with the mutant 1508. At maturity, kernels of the double mutants 527/1508 and 29/1508 contained, respectively, 68 and 43% less starch than Bomi. The double mutant 29/1508 kernel had a slightly lower prolamin content than mutant 1508 which is the most prolamin-deficient single mutant. In the double mutant 527/1508, however, an almost complete suppression of prolamin synthesis was observed during kernel development. The percentage of lysine in the seed proteins of the double mutants was about the same as in the most extreme single mutant 1508. Based on the additive effect of the individual high-lysine genes in the double mutants, it is concluded that the influences of these genes on prolamin and starch synthesis are independent.     

水稻10kD醇溶蛋白基因克隆,序列分析及对植物百脉根的转化

应用ＰＣＲ 技术,从水稻基因组中扩增１０ ｋＤ 水稻醇溶蛋白基因的编码区,得到一特异的０．５ ｋｂ 的片段。对该片段进行酶切分析和全序列测定,结果表明： 该片段与Ｍａｓｕｍ ｕｒａ Ｔ．等的报道相比, 其核苷酸序列及推测的氨基酸序列的同源率分别为９５％ 和９３％ 。就其分子数计算,甲硫氨酸及半胱氨酸含量分别占１８．２％ 和９％ , 含硫氨基酸总数为２７．２％ , 比同类的１０ ｋＤ玉米醇溶蛋白的含硫氨基酸总数还要高。将该基因置于ｒｂｃ Ｓ启动子调控下,动员入农杆菌中,转化豆科植物百脉根（ＬｏｔｕｓｃｏｒｎｉｃｕｌａｔｕｓＬ．）, 在含有卡那霉素的抗性培养基上筛选抗性植株。利用ＰＣＲ 方法检测１０ ｋＤ 醇溶蛋白基因整合到百脉根基因组中     

Evolutionary relationship of the members of the sulphur-rich hordein family revealed by common antigenic determinants

Summary Five monoclonal antibodies raised against an enriched C hordein fraction have been characterized in detail and were found to be specific for the members of the sulphur-rich hordein family. Two antibodies specific for B hordein polypeptides were identified, one of which reacted predominantly with CNBr cleavage class III polypeptides. 1 hordein was recognized by two antibodies, of which one also reacted with 2 hordein and several members of the CNBr cleavage class II B hordein polypeptides. One antibody recognized 3 hordein but cross-reacted at higher antibody concentration with almost all of the B and C hordein polypeptides. The specificity of the monoclonal antibodies was confirmed by Western blotting of one- or two-dimensionally separated hordein from the B hordein-deficient mutant hor2ca and its wild-type Carlsberg II and the 3 hordein-deficient genotype Nevsky. The identification of the hordein-specific monoclonal antibodies was further supported by immune precipitation of in-vitro transcribed and translated 2 hordein, and hor2ca and Carlsberg II mRNA translation products. The monoclonal antibodies were used to screen for mutants in hordein synthesis. Two mutants, one deficient in 1 hordein synthesis and a second in 2 or closely related B hordein polypeptides were identified. A model is proposed for the evolution of the sulphur-rich hordein loci Hor5 and Hor2.     

Temporal and spatial expression analysis of gamma kafirin promoter from Sorghum (Sorghum bicolor L. moench) var. M 35-1

Different cis acting elements of gamma kafirin gene from Sorghum bicolor var. M 35-1 were amplified and cloned using different combination of the primers. The amplified promoter was replaced with CaMV35S promoter of vector pCMBIA-1304 and resultant vector contained beta-glucuronidase (gus) gene under the control of amplified gamma-kafirin promoter. The resulting fusants were then transformed in to different explants of sorghum via particle bombardment. The regulation of uid gene expression was analyzed to find out the minimum required 5' regulatory sequence and cis acting elements for the efficient expression. However no gus expression was detected in leaves of micropropagated plants, scutellum and calli at any stage of growth. The expression of gus, with pKaf gus-P4 gene construct, was detected in immature embryos and endosperm 20 days after pollination (DAP). The result suggest that at least three motifs (two GCN4 and one prolamin box) besides TATA and CATC boxes are required for the efficient expression of the kafirin gene of sorghum. The study shows that PCR based isolation of different motifs and regions can be used as an alternate to deletion analysis for observing the role of various motifs and their importance in the gene expression and regulation.     

Immunochemical studies on barley seed storage proteins

The antigenic relationships between the prolamins of barley, rye and wheat have been studied by examining the specificity of an antibody to C hordein in a quantitative study using a laser nephelometer. The antibody reacts weakly with B hordein and strongly with 75-kdalton and 40-kdalton -secalins from rye and ₃ some -gliadins from wheat. Absorption experiments and immunodiffusion tests indicate that there are shared antigenic determinants for most of the prolamins. All the species with reacting prolamins belong to the sub-family Festucoideae of the Gramineae. The prolamins of maize, pearl millet and sorghum, species of the sub-family Panicoideae, do not react. The results confirm the known lack of homology between the prolamins of the two sub-families and also indicate the presence of relationships not yet established between C hordein, the 75-kdalton and 40-kdalton -secalins and also ₃ gliadin.Abbreviations HMW high molecular weight - PAGE polyacnylamide-gel electrophoresis - PBS phosphate-buffered saline - RLS relative light scattering - SDS sodlum dodecyl sulphate     

Specificity of an antibody to a subunit of high-molecular-weight storage protein from wheat seed and its reaction with other cereal storage proteins (prolamins)

An antiserum to subunit 2 from the high-molecular-weight (HMW) subunits of the glutenin fraction of Triticum aestivum cv. Highbury was shown to react with related subunits from other cultivars of wheat. The reaction was measured quantitatively by laser nephelometry in polyethylene glycol phosphate-buffered saline after dissolving the HMW fraction in 0.1 M acetic acid; urea used to dissolve the HMW prolamins inhibited the reaction, in some cases at the low concentration of 0.06 M. A study of the comparative reactions of other cereal prolamins was made. D hordein, the homologous HMW protein of barley, showed less reaction, which was more inhibited by urea than the wheat subunits. Some -gliadins from the wheat cultivars Chinese Spring and Cheyenne reacted more strongly than the injected fraction and there was less inhibition by urea. A-, - and ₃ of wheat also reacted with the antiserum while a secalin of rye of M_r 40000 gave a weak reaction.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - PBS phosphate-buffered saline - PE pyridylethylated - SDS sodium dodecyl sulphate     

Linkage of the hordein loci Hor1 and Hor2 with the powdery mildew resistance loci Ml-k and Ml-a on Barley chromosome 5

Summary The linkage relationship among the loci Hor1, Hor2, Ml-k and Ml-a on the short arm of chromosome 5 was studied by progeny testing the F₂ generation of two crosses. The loci Hor1 and Hor2 code for polypeptides of the storage protein hordein (prolamin) and the loci Ml-k and Ml-a determine the resistance reaction with some powdery mildew fungi cultures. The order of the loci is Ml-k, Hor1, Ml-a, and Hor2, the first named being nearest the centromere. The recombination percentage between Hor1 and Hor2 was determined in the F₁ and F₂ generations in both crosses, the combined estimate being 7.4±0.9 per cent. The recombination percentage estimated between Ml-k and Hor1 was 4.0±1.3, between Hor1 and Ml-a, 5.3±1.1, and between Ml-a and Hor2, 6.1±1.2. The estimates involving the Ml- loci were all probably a little too high.     

Cloning and characterization of a cDNA encoding a rice 13 kDa prolamin

Summary A cDNA library constructed from mRNAs obtained from developing rice endosperm was screened with a cDNA clone (RM7) of highest frequency of occurrence (1.8%). The translati) product directed by the mRNA which was hybrid-released from RM7 cDNA in a wheat germ cell-free system showed a molecular size of 13 kDa when coexisting with the protein body fraction of developing maize endosperm. A polypeptide sequence composed of 156 amino acids was deduced from the nucleotide sequence. By comparison with the 19 N-terminal amino acids obtained from Edman degradation of the isolated rice 13 kDa prolamin fraction, the signal sequence was determined as consisting of 19 amino acids. The deduced polypeptide is rich in hydrophobic amino acids such as Leu and Val, and also in Gln, but lacks Lys. Hence, the amino acid composition is consistent with that of rice 13 kDa rolamin. By homology with previously reported cereal prolamins, only a single octapeptide sequence, Gln-Gln-Gln-CysCys-Gln-Gln-Leu, which was observed in 15 kDa and 27 kDa zein, B- and -hordein, /- and -gliadin, and -secalin was conserved in the rice 10 kDa and 13 kDa prolamin. No repetitive sequences and/or sequences homologous to other cereal prolamins, except the above octapeptide, were observed for the mature 13 kDa prolamin polypeptide. The signal sequence region of the 13 kDa prolamin, however, shows homology of more than 65% in both the nucleotide sequence and the amino acid sequence with rice 10 kDa prolamin and maize zein.