Modification of amino acid composition of endosperm proteins from in-vitro-selected high lysine mutants in rice

Summary Endosperm protein mutants in rice may be recovered by biochemical selections with inhibitory levels of lysine and threonine. Among the phenotypes recovered from in vitro selections are lines with increased protein and percent lysine in the protein. This work was designed to identify changes in proteins of rice mutants and to further our understanding of the mechanisms of lysine plus threonine selections in rice. Among the most obvious amino acid changes in mutants was a higher lysine level in all protein solubility fractions and a decrease in tyrosine. Methionine and glutamate are reduced in some protein fractions. However, methionine is significantly higher in the mutant than the control in the glutelin fraction. Several other aspartate pathway amino acids are higher in the mutant than the unselected controls. Separation of proteins in SDS-PAGE gels showed shifts in the protein profiles in the mutants, including a decrease in the major 30 kDa low lysine globulin component, and an increase in several high-molecular-weight components, approximately 60–100 kDa. Increases in the lysine content of proteins of different solubility classes and different proteins within classes are detailed.     

Variability of the protein and amino acid content in the rice grain]

The amino acid composition of raw protein of ten regional varieties of rice harvested in 1972 and 1973 was investigated. The amino acid composition was measured in the Hd-1200e analyzer. Coefficients of variations in the content of protein and invididual amino acids were determined in relation to the rice variety. Consistent correlation between the lysine concentration in the protein and the protein content in the grain was established. The influence of climatic conditions on the protein accumulation in the rice grain was noted.     

Enhancement of essential amino acid contents in crops by genetic engineering and protein design

The importance and urgency of providing humans and animals with quality proteins are reflected in the growing scientific and industrial interest in augmenting the nutritive value of the world's protein sources. Such nutritive value is determined by the protein content in 'essential amino acids', those that cannot be synthesized de novo and that must be supplied from the diet. It is the object of this review to discuss recent advances in the genetic modification of crops that aim to provide enhanced quantities of essential amino acids.     

Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.     

Screening of Lysine-rich Plant Species and Identification of the Purified Proteins

The nutritional quality of seed proteins from cereals, such as wheat and rice, is comparatively low due to its deficency in lysine and some essential amino acids. In this research extensive varieties of plant seed samples were collected and screened by analysis of amino acid composition. Three lysing-rich species which contain more than 6.7% of lysine in total seed proteins were found. 31 kinds of proteins were purified from a species which contains 7.9% of lysine using the modified methods of IEF and SDS electrophoresis. One protein with PI 6.1 and 18 kD was identified which contains 11.4% of lysine and was rich in threonine, valine and isoleucine. This is the first example of the protein which could complement several limiting amino acids of wheat or rice. Further research on the structural gene encoding this protein would have great potential value for improvement of protein quality of these cereals.     

温光对稻米蛋白质及氨基酸含量的影响

研究水稻结实期的温、光条件对稻米蛋白质与氨基酸含量的影响,其结果表明：同一品种作为早季栽培时,稻米氨基酸含量比作晚季栽培时低,分期播种时,以结实期日平均气温为２４℃时蛋白质含量为最高;不同海拔高度种植时,以８００ｍ处蛋白质含量为最高;结实期人工高温处理,稻米蛋白质和氨基酸含量降低,低温（２１／２０℃）处理时,粳稻的氨基酸含量增加而籼稻则有所降低,籼稻氨基酸形成的适温在２４￣２６℃之间,弱光下,糙米     

Dietary influence on protein level in milk and milk yield in dairy cows

Diet can influence the yield of milk protein more than it can influence milk protein content. Providing sufficient dietary crude protein in forms that will maximize amounts of amino acids required for milk protein synthesis to the gastrointestinal tract, in forms that can be digested and absorbed, will provide for optimal milk protein yield and content. Maximizing ruminal microbial protein synthesis is an important part of this strategy. The rest of the strategy involves providing sufficient amounts of the remaining required amino acids as ruminally protected proteins, or amino acids in forms that can be digested in the gastrointestinal tract. A diet deficient in protein will reduce milk protein content 1 to 2 g kg⁻¹ and may substantially reduce yields of milk and milk protein. A diet containing high amounts of readily fermentable carbohydrates may increase milk protein content 1 to 2 g kg⁻¹, and may increase yields of milk and protein, but may also result in digestive and metabolic upsets. Diets containing supplemental fat will increase yield of milk protein, but not as extensively as the increase in yield of milk, because milk protein content is usually reduced 1 to 2 g kg⁻¹. The increased efficiency of milk fat and lactose synthesis is likely to be the reason for this depression in milk protein content. A means of overcoming this problem is a continuing research challenge.     

Effect of various kinds of dietary protein and supplementation with limiting amino acids on growth,haemolymph components and uric acid excretion in the silkworm, Bombyx mori

Effects of various kinds of dietary protein on growth of the silkworm, Bombyx mori, were determined using semi-synthetic diets. Also, the ingestion, digestion and utilization of dry matter and of nitrogen were measured. Nutritive effects of dietary proteins and supplementation of limiting amino acids on haemolymph protein and amino acids pattern were also investigated. Larval growth was largely dependent on the dietary proteins. When the larvae were reared on a diet containing weakly nutritive proteins such as gluten and zein, haemolymph protein was decreased and uric acid excretion was markedly accelerated. The free amino acid composition of the haemolymph manifested characteristic patterns according to the kinds of dietary protein.The supplementation of gluten and zein with their limiting amino acids resulted in a rise of haemolymph protein and a drop in uric acid excretion. The amino acid patterns in the haemolymph were greatly changed according to supplementation.     

Free and bound amino acids and proteins in developing grains of rice with enhanced lysine/proteins

 Free amino acids were determined in developing seed of a rice mutant with enhanced grain lysine. This phenotype frequently has enhanced protein. Some free amino acids of developing seed are inversely related to the level of total amino acids in proteins of the mature grain. Amino acids that were enhanced in protein, including aspartic acid, threonine, methionine and lysine, were notably lower in the free amino-acid pool. Our conclusion is that mutant-developing grains process aspartate amino acids more rapidly than the controls. Conversely, arginine, valine and glutamic acid/glutamine accumulate as free amino acids with mutant/control ratios of 1.39, 1.29 and 1.12, respectively. Glutamic acid/glutamine in proteins of mature seeds is lower in the mutant than the control. ³H-lysine incorporation showed enhanced isotope incorporation into at least four proteins. One mutant protein was less actively labelled than analogous controls. The ³Hlysine pattern indicates processing modifications in this useful rice mutant. Received: 14 October 1996/Accepted: 8 November 1996     

Transgenic approaches to improve the nutritional quality of plant proteins

Summary Plant proteins, when used as dietary protein, are generally incomplete in nutrition due to their deficiency in several essential amino acids, for example, lysine and tryptophan in cereals and methionine and cysteine in legumes. Attempts to breed crops with increased levels of lysine and methionine have been less than satisfactory. Modern biotechnology offers alternative approaches for rectifying this nutrition deficiency. In the past decade, several transgenic strategies aimed at modifying the amino acid composition of plant proteins and enhancing the content of specific essential amino acid(s) for nutrition improvement have been developed and tested. These include synthetic proteins, modification of protein sequences, over-expression of heterologous or homologous proteins, and metabolic engineering of the free essential amino acid pool and protein sink. The progress and potential of these approaches and studies are reviewed. As plant proteins are the primary source of all dietary protein consumed by humans and animals and are inexpensive to produce in comparison with meat, improving their quality will make a significant contribution to our future food needs. The research and development in this area of interest is making promising progress towards this endeavor.     

Relationship between relative protein value and somein vitro indices of protein quality

Besides amino acid composition of a protein, their bioavailability is an important determinant of the protein quality. In view of the observations over the last decade or two, implicating the small peptide uptake by the mammalian intestine as a major route of protein absorption, a few animal and plant proteins were subjected to sequential enzymatic digestionin vitro with pepsin, pancreatin + trypsin and erepsin and the release of amino acids as small (including dipeptides) and large peptides and free amino acids, was determined. The relative protein values of α-lactalbumin, egg whites, casein, gluten, zein and protein isolates of soyabeans and groundnuts was determined using rat growth method. It was observed that relative protein value were positively correlated with the essential amino acid index of protein, quantity of essential amino acids released as small peptides and the dipeptide content of enzymatic digests, while there was a negative correlation between relative protein value and essential amino acid content of large peptide fraction.     

Changes in salt-soluble proteins of rice during grain development

Salt-soluble proteins, albumin and globulin, were prepared from dehulled rice (Oryza sativa L., line IR1541-76-3) during grain development. Albumin and globulin progressively increased during grain development up to about 12 days after flowering (DAF) and then decreased slightly during grain desiccation. Free amino N was maximum at 10 DAF. Total protein and glutelin-prolamin (by difference) continued to increase up to 20 DAF. Aminogram of total protein and globulin showed a progressive decrease in lysine and threonine among the essential amino acids. Albumin showed a similar trend except for the lesser change in lysine content. Disc gel electrophoresis showed a maximum of four major and six minor protein bands for albumin and only one major and three minor bands for globulin. Sodium dodecyl sulfate-gel electrophoresis revealed three major polypeptide subunits for albumin with MW of 11 000, 8 500 and 16 000, and two for globulin with MW of 20 000 and 12 000.     

Higher membrane fluidity mediates the increased subcutaneous fatty acid content in pigs fed reduced protein diets

The production of pork with moderate amounts of intramuscular fat (IMF) without an increase in subcutaneous fat is highly desirable for the meat industry. Several studies indicate that dietary protein reduction during the growing–finishing period of pigs enhances IMF content, but its consequence on carcass fat deposition is still contradictory. In this study, we hypothesized that the effects of reduced protein diets (RPD), corrected or not with the limiting amino acid lysine, on subcutaneous fat deposition from pigs with distinct genotypes are mediated by adipose membranes biophysical properties. In total, 36 crossbred (Large White×Landrace×Pietrain – a lean genotype) and purebred (Alentejana breed – a fatty genotype) male pigs were randomly assigned to the control group, the RPD group or the reduced protein diet equilibrated for lysine (RPDL) group, allowing a 2×3 factorial arrangement (n=6). Backfat thickness and total fatty acid content were higher in Alentejana relative to crossbred pigs. Although dietary treatments did not change backfat thickness, RPD and RPDL increased total fatty acids content of subcutaneous fat. In order to understand this effect, adipose tissue membranes isolated from pig’s subcutaneous fat were assayed for glycerol permeability and fluidity, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probes. The glycerol transport across adipose membranes was not mediated by aquaglyceroporins and remained unchanged across dietary groups. Regardless of lysine correction, RPD increased membrane fluidity at the hydrocarbon region (lower DPH fluorescence anisotropy) in both genotypes of pigs. This result was associated with a lower ratio between oleic acid and linoleic acid on membrane’s fatty acid composition. Adipose membrane’s cholesterol content was independent from genotype and diet. Taken together, the present study shows that dietary protein reduction is successful in maintaining backfat thickness, although a negative side effect was observed on total fatty acids in subcutaneous fat, which may be due to changes in the fluidity of adipose membranes.     

Human–Human Hybridomas from Lung Cancer Patients Secrete Anti-DNA Antibodies

The effects of modification of the arginine/lysine ratio of dietary protein on the cholesterol kinetics were studied in male rats. Single amino acids (lysine to soybean protein and arginine to casein) were added to approximate the arginine/lysine ratio in different proteins. After acclimation to these diets for 30 days, rats were administered intravenous [¹⁴C]cholesterol and oral [³H]cholesterol. Analysis of the die-away curve of [¹⁴C]cholesterol showed an apparent independence of cholesterol kinetics to the dietary manipulations, but there was a moderate reduction of the size of the slowly exchangeable pool and of the biliary concentration of cholesterol when lysine was added to soybean protein. Addition of amino acids neither influenced cholesterol absorption nor the fecal excretion of the radioactivities from labeled cholesterol. The results indicate that manipulating the arginine/lysine ratio of dietary protein by adding single amino acids is not necessarily effective in ameliorating cholesterol metabolism in rats, although the arginine addition caused a significant reduction of serum cholesterol and triglyceride.     

A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

异育银鲫幼鱼对饲料中赖氨酸的利用及需要量研究

以添加晶体氨基酸的半精制饲料饲喂异育银鲫幼鱼,通过69d的生长实验来确定其赖氨酸需要量。饲料以白鱼粉为主要蛋白源,饲料中的总赖氨酸含量分别为1.82%、2.32%、2.82%、3.32%3、.82%、4.32%和4.82%7个水平。实验在室内循环水养殖系统中进行,每种饲料随机3个重复。实验结果表明,异育银鲫能够利用饲料中的晶体赖氨酸、蛋氨酸。在投喂后3h,其血浆中的游离赖氨酸、蛋氨酸含量最高。当饲料中赖氨酸含量为3.32%时,异育银鲫的终末尾均重、特定生长率和鱼空壳占体重的百分比最高,肝体指数最低。当饲料中赖氨酸含量为3.82%时,异育银鲫的干物质表观消化率显著高于其他组(PPP>0.05)。血红蛋白含量以赖氨酸含量为2.82%的饲料组最高,4.82%组最低;随着饲料中赖氨酸含量的升高,异育银鲫红细胞数下降,血清脲氮含量升高,且血清脲氮含量具有组间显著性差异(P<0.05)。根据折线法,由异育银鲫的特定生长率同饲料中赖氨酸水平的相关性得出其赖氨酸需要量为3.27%,占饲料蛋白的8.52%。       

Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins

Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins.     

A novel calmodulin-like protein gene in rice which has an unusual prolonged C-terminal sequence carrying a putative prenylation site.

A rice cDNA encoding a novel calmodulin-like protein was identified. It has 38 additional amino acids at the C-terminus of a complete, typical calmodulin (CaM) sequence of 149 amino acids. The four C-terminal amino acid residues form a CAAL motif which could be a site for protein prenylation and may subsequently cause the protein to become membrane associated. RT-PCR analysis confirmed that such a combined protein gene truly exists in rice. Sequence analysis of its genomic counterpart showed that there is an intron located at junction of the normal CaM sequence and the 38 C-terminal amino acids. This introduces a potential stop codon for normal CaM if an alternative splicing mechanism is involved. Southern blot analysis of rice genomic DNA revealed that there is only one locus for this gene. The northern blot analysis showed that this gene is highly expressed in rice roots, shoots and flowers. The distribution of this protein demonstrates the functional importance of this novel CaM-like protein in rice.     

Isolation of the major basic nuclear protein and its localization on chromosomes of the dinoflagellate,Oxyrrhis marina

Basic nuclear proteins were extracted from isolated nuclei of Oxyrrhis marina. The HPLC pattern of the extract showed a single major peak, which consisted of a major band with an apparent molecular mass of 23 kDa on an SDS-PAGE gel. We designated this protein as Np23 because of its apparent molecular mass. The amino acid composition of this protein revealed its extremely basic nature with a high lysine content. Polyclonal antibodies were raised against Np23. Immunofluorescence microscopy showed that Np23 was localized within the nucleus of dividing and non-dividing cells as well, and immuno-electron microscopy showed that the protein was localized only on chromosomes. These data established that Np23 is the major basic chromosome protein of Oxyrrhis marina.