Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

High Lysine and High Tryptophan Transgenic Maize Resulting from the Reduction of Both 19- and 22-kD α-zeins

The major maize seed storage proteins, zeins, are deficient in lysine and tryptophan content, which contribute to the poor nutritional quality of corn. Whether through the identification of mutations or genetic engineering, kernels with reduced levels of zein proteins have been shown to have increased levels of lysine and tryptophan. It has been hypothesized that these increases are due to the reduction of lysine-poor zeins and a pleiotropic increase in the lysine-rich non-zein proteins. By transforming maize with constructs expressing chimeric double-stranded RNA, kernels derived from stable transgenic plants displayed significant declines in the accumulation of both 19- and 22-kD α-zeins, which resulted in higher lysine and tryptophan content than previously reported for kernels with reduced zein levels. The observation that lysine and tryptophan content is correlated with the protein levels measured in transgenic maize kernels is consistent with the hypothesis that a pleiotropic increase in non-zein proteins is contributing to an improved amino acid balance. In addition, a large increase in accumulation of free amino acids, consisting predominantly of asparagine, asparate and glutamate, was observed in the zein reduction kernels.     

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F₁ hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T₆ transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.     

优质蛋白玉米02基因控制赖氨酸超量积累的分析

以３８个ＱＰＭ（或０２）和对照普通玉米为实验材料,进行０２基因控制赖氨酸超量积累的生化和遗传分析。主要实验结果如下：（１）ＱＰＭ玉米０２基因为隐性的单基因遗传,它控制着胚乳、雄穗和幼苗期叶片中赖氨酸的超量积累,一些修饰因子和遗传背景对胚乳物理性状产生影响;（２）ＱＰＭ玉米、普通玉米的胚较之胚乳,或者ＱＰＭ玉米胚乳较之普通玉米胚乳都含有较多的天门冬氮酸、甘氨酸、赖氨酸和精氨酸,含有较少的脯氨酸、谷氨酸、亮氨酸和苯丙氨酸;（３）两种玉米之间,在胚乳蛋白质含量及胚乳可溶性蛋白、醇溶蛋白、谷蛋白的赖氨酸含量方面没有什么不同;（４）已经育成一批ＱＰＭ或０２玉米自交系,并配制出几个强优势杂交组合。     

Temporal profiling of essential amino acids in developing maize kernel of normal,opaque - 2 and QPM germplasm

Maize, an important cereal crop, has a poor quality of endosperm protein due to the deficiency of essential amino acids, especially lysine and tryptophan. Discovery of mutants such as opaque-2 led to the development of nutritionally improved maize with a higher concentration of lysine and tryptophan. However, the pleiotropic effects associated with opaque-2 mutants necessitated the development of nutritionally improved hard kernel genotype, the present-day quality protein maize (QPM). The aim of present study was to analyze and compare the temporal profile of lysine and tryptophan in the developing maize kernel of normal, opaque-2 and QPM lines. A declining trend in protein along with tryptophan and lysine content was observed with increasing kernel maturity in the experimental genotypes. However, opaque-2 retained the maximum concentration of lysine (3.43) and tryptophan (1.09) at maturity as compared to QPM (lysine-3.05, tryptophan-0.99) and normal (lysine-1.99, tryptophan-0.45) lines. Opaque-2 mutation affects protein quality but has no effect on protein quantity. All maize types are nutritionally rich at early stages of kernel development indicating that early harvest for cattle feed would ensure a higher intake of lysine and tryptophan. Two promising lines (CML44 and HKI 1105) can be used for breeding high value corn for cattle feed or human food in order to fill the protein inadequacy gap. Variation in lysine and tryptophan content within QPM lines revealed that differential expression of endosperm modifiers with varying genetic background significantly affects nutritional quality, indicating that identification of alleles affecting amino acid composition can further facilitate QPM breeding program.     

Nutritional Evaluation of Genetically Modified Maize Corn Performed on Rats

The aim of this study was to determine the composition and nutritional value of conventional and transgenic, so-called Roundup Ready (RR) maize with an introduced gene of glyphosate resistance. Crude protein, crude fibre, ash, fat, starch, sugar, amino acids, fatty acid and macroelement levels were determined by chemical analysis. In both maize lines a low level of Ca (0.15g.kg m 1 DM) and of the essential amino acids lysine and tryptophan (2.6 and 1.7g.kg^{m 1} DM, respectively) were observed. In the biological experiment carried out on rats the tested maize lines were the only dietary sources of nitrogen, thus, the experimental diets contained 9% CP in dietary dry matter. In the feeding experiment no significant differences in the protein efficiency ratio (PER) were observed between groups receiving conventional or transgenic maize (1.51 and 1.41, respectively). Also almost equal results were obtained in the balance experiments. Both maize lines revealed a high nitrogen digestibility (84.9 and 84.5%, respectively) and the net protein utilization amounted to 63.5 and 63.2%, respectively. From these results can be concluded that regarding nutrient composition and utilisation, genetically modified (RR) maize is equivalent to isogenic maize.     

High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi

Because of the limited lysine content in corn grain, synthetic lysine supplements are added to corn meal-based rations for animal feed. The development of biotechnology, combined with the understanding of plant lysine metabolism, provides an alternative solution for increasing corn lysine content through genetic engineering. Here, we report that by suppressing lysine catabolism, transgenic maize kernels accumulated a significant amount of lysine. This was achieved by RNA interference (RNAi) through the endosperm-specific expression of an inverted-repeat (IR) sequence targeting the maize bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (ZLKR/SDH). Although plant-short interfering RNA (siRNA) were reported to lack tissue specificity due to systemic spreading, we confirmed that the suppression of ZLKR/SDH in developing transgenic kernels was restricted to endosperm tissue. Furthermore, results from our cloning and sequencing of siRNA suggested the absence of transitive RNAi. These results support the practical use of RNAi for plant genetic engineering to specifically target gene suppression in desired tissues without eliciting systemic spreading and the transitive nature of plant RNAi silencing.     

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.     

Overexpression of serine acetyltransferase in maize leaves increases seed‐specific methionine‐rich zeins

Maize kernels do not contain enough of the essential sulphur‐amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)‐amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S‐assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S‐assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell‐specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12‐fold higher SAT activity without negative impact on growth. S‐assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10‐kDa δ‐zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40‐fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high‐Met zein accumulation. Moreover, SAT overcomes the shortage of S‐amino acids that limits the expression and accumulation of high‐Met zeins during kernel development.     

Evidence for the genetic control of lysine catabolism in maize endosperm

A split pollination was used to produce normal (Su su su O2 o2 o2) and high lysine double mutant sugary opaque-2 (su su su o2 o2 o2) endosperms on the same ear of sugary opaque-2 maize plants. Amino acids were determined in the vascular sap of the ear peduncle. Lysine content in the sap was compared with lysine stored in both normal and sugary opaque-2 endosperm during kernel filling. Lysine content in the ear peduncle sap could account for all lysine found in both endosperms. Preformed lysine is highly catabolized in the normal endosperm, but not in the high lysine sugary opaque-2 endosperm. The rate of lysine breakdown appears to be an important mechanism by which the high lysine mutant controls lysine level in maize endosperm.     

Effect of sucrose accumulation on zein synthesis in maize starch-deficient mutants

Starch-deficient maize (Zea mays) mutants, brittle-2 (bt2), brittle-1 (bt), and shrunken-2 (sh2), which accumulated large quantities of sucrose, had less than normal amounts of zein (the major storage protein) in the endosperm. Reduction of zein synthesis in the starch-deficient mutants was negatively correlated with the accumulation of sucrose and low osmotic potential in the developing endosperms. When radioactive amino acids were injected into the shank below ears that segregated for the starch-deficient mutant and normal kernels at 28 days post-pollination, mutant kernels absorbed only ca 22–36% of the labelled amino acids found in their normal controls. Thus, a low osmotic potential in the mutant endosperm may favour water movement but reduce solute movement. The inability of amino acids to move into the mutant endosperms, therefore, in part explains the reduction of zein accumulation in starch-deficient mutant endosperms.     

Maternal effects influencing DNA endoreduplication in developing endosperm of Zea mays.

A large proportion of the nuclei in developing endosperm of Zea mays L. undergoes endoreduplication. Nuclear preparations of the entire endosperm from maize kernels of inbred lines, their reciprocal hybrids, and in some cases, F2 and F3 endosperm tissue were evaluated using flow cytometry. Data relative to DNA endoreduplication patterns, percentage of nuclei undergoing endoreduplication, and mean DNA content per nucleus were obtained. The patterns of endoreduplication and extent of DNA amplification differ among some inbreds. In all experiments, the endoreduplication patterns show that the F1 endosperm is more similar to the maternal parent than to the paternal parent. F2 endosperms reveal little difference in endoreduplication patterns among individuals within an F2 family and no more variation than the F1 endosperms. In contrast, F3 endosperms showed greater variation among their endoreduplication patterns. These results indicate a maternal effect on endoreduplication; that is, the genotype of the maternal parent's nuclear genome exerts control over the endoreduplication activities of endosperm tissue.     

Genetic manipulation of lysine catabolism in maize kernels

In plants, lysine catabolism is thought to be controlled by a bifunctional enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). Lysine is converted to saccharopine, through condensation with alpha-ketoglutarate, by LKR, and subsequently to glutamate and alpha-aminoadipate-delta-semialdehyde by SDH. To investigate lysine catabolism in maize kernels, we generated transgenic plants with suppressed LKR/SDH activity in either endosperm or embryo. We found that the suppression of LKR/SDH in endosperm induced an increase in free lysine in developing endosperm, which peaked at 32 days after pollination. At later stages of kernel development, most of the free lysine was found in the embryo along with an elevated level of saccharopine. By combining endosperm LKR/SDH suppression with embryo LKR/SDH suppression through crosses, the saccharopine level in embryo was reduced and resulted in higher lysine accumulation in mature kernels. These results reveal new insights into how free lysine level is regulated and distributed in developing maize kernels and demonstrate the possibility of engineering high lysine corn via the suppression of lysine catabolism.     

Serial extraction of endosperm drillings (SEED)—A method for detecting transgenes and proteins in single viable maize kernels

We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. This technique could be highly useful for several monocots and dicots. Our method involves first sampling the endosperm with a hand-held rotary grinder so that the embryo is preserved and capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the transgene can be detected in the first extract by SDS-PAGE with visualization by total protein staining or immuno-blot detection. The second extract can be purified and used as template DNA in PCR reactions to detect the transgene. This method is particularly useful for screening transgenic kernels in breeding experiments and testing for gene silencing in kernels.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of thehα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the high-molecular-weight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves or embryos of plants grown from seeds expressing this protein in endosperm. Genomic Southern-blot analysis is consistent with results from SDS-PAGE and indicates that the transgene integration sites are complex and are different in the four events studied. Using the presence of this protein as a phenotypic marker, we studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and self-pollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 subunit. These data, together with PCR analysis for the transgene, suggest that the transgene is inefficiently transmitted through pollen in all four events.     

Transgenic sorghum with improved digestibility of storage proteins obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Development of transgenic plants with modified seed storage protein composition and increased nutritive value is one of the most promising areas of genetic engineering. This task is especially important for sorghum—a unique drought tolerant cereal crop that is characterized, however, by a relatively poor nutritive value in comparison with other cereals. It is considered that one of the reasons of the low nutritive value of the sorghum grain is the resistance of one of its seed storage proteins, γ-kafirin, located in the outer layer of endosperm protein bodies, to protease digestion. Using Agrobacterium-mediated genetic transformation, we obtained transgenic sorghum plants (Sorghum bicolor (L.) Moench) harboring a genetic construct for RNAi silencing of the γ-kafirin gene. In the T₁ generation, the plants with almost floury or modified endosperm texture of kernels were found. In these kernels, the vitreous endosperm layer has been reduced and/or covered by a thin layer of floury endosperm. In vitro protein digestibility (IVPD) analysis showed that the amount of undigested protein in transgenic plants from the T₃ generation was reduced by 2.9–3.2 times, in comparison with the original non-transgenic line, and the digestibility index reached 85–88% (in comparison with 59% in the original line). In T₂ families, the plants combining high IVPD with vitreous endosperm type were found. In the electrophoretic spectra of endosperm proteins of transgenic plants with increased digestibility, the proportion of 20 kD protein that is encoded by the γ-kafirin gene, was significantly reduced, in comparison with the original non-transgenic line. HPLC analysis showed total amino acid content in two out of the three studied transgenic plants from the T₂ generation was reduced in comparison with the original non-transgenic line, while the lysine proportion increased by 1.6–1.7 times. The mechanisms conditioning improved digestibility of storage proteins in transgenic plants are discussed. The results of experiments demonstrate that it is feasible to develop sorghum lines combining high protein digestibility and vitreous endosperm that has a high breeding value.     

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (±)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (±)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = −0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.