Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.     

Expression of the sorghum 10-member kafirin gene cluster in maize endosperm

Functional analysis of chromosomal segments containing linked genes requires the insertion of contiguous genomic sequences from bacterial artificial chromosomes (BACs) into the genome. Therefore, we introduced a 90-kb large BAC clone carrying a 10-copy tandem array of kafirin storage protein genes from sorghum linkage group J, mixed with a selectable marker gene, directly into maize cells using the particle bombardment method. Transgenic plants were regenerated and seeds from eight different transgenic lines were produced. One such transgenic plant was selected that had the entire kafirin gene cluster on a single continuous DNA fragment spanning more than 45 kb integrated into its genome. When alcohol-soluble proteins from individual T2 and T3 seeds of this event were analyzed, significant levels of kafirin were found in addition to the endogenous zein storage proteins, demonstrating that the large exogenous DNA segment is stably integrated into the maize genome and expressed at high levels in subsequent generations. Therefore, we could provide a new utility of plant transformation by the particle bombardment method for functional genomics of multigene families and the modification of the nutritive quality of cereal grains. Despite a tandem array of highly homologous sequences at the transgenic locus, no gene silencing was observed, probably owing to the effects of co-transformed flanking sequences. The expression studies of the transgenic locus also revealed new features of storage protein gene promoters that differed from previous transient gene expression studies, thereby illustrating the significance of the concentration and configuration of DNA–protein interactions in the regulation of gene expression.     

外源脱水应答转录因子DREB基因在转基因小麦中的诱导型表达与抗干旱生理效果研究

以冬小麦品种8901、5-98、99-92和104等品种的幼穗和幼胚为材料,用基因枪转化含逆境诱导转录因子DREB和bar基因的质粒pBAC128F（7024bp）。经筛选与植株再生,共获得70多个转基因小麦植株及其后代株系。转基因株系经PCR分析和RNA点杂交检测,结果表明外源转录因子DREB基因已稳定整合到转基因植株及其后代株系中,并且在部分后代株系中获得了表达。叶片脯氨酸含量测定表明,有16个转基因株系的脯氨酸含量与非转基因对照相比,增加相当显著,其中10个株系的脯氨酸含量在1100μg/g以上,比对照提高了2倍多。室内抗旱模拟实验表明,转基因株系停止浇水15d后,叶片仍然表现绿色,而对照叶片则失绿、枯干;复水10d后,转基因株系恢复活力,对照则死亡。研究表明,利用逆境诱导型启动子（rd29B）来增强外源DREB基因的表达,能显著改良小麦的抗旱性。     

玉米Ubi－1启动子在可育转基因玉米植株中的表达活性

本工作将玉米泛素基因－1启动子（Ubi-1)与大肠杆菌β－葡萄糖苷酸酶基因（gus,uidA)的编码区融合,通过基因枪粒子轰击方法转化来自水成熟胚盾片组织的I－型愈伤组织,经PPT选择获得可育的玉米转基因植株,并采用组织化学方法分析了Ubi-1启动子驱动的gus基因在不同组织,细胞中的表达活性,发现gus基因在除花药壁以外的其它所试组织中均可以有效表达。Ubi：GUS在花粉,卵细胞中T1代转基因植株未成熟胚中的表达显示该启动子在植株发育的早期阶段即具有活性。对T0代转基因植株的花粉进行GUS组织化学染色,gus基因呈1：1分离,显示外源基因在转基因植株中以孟德尔方式遗传。同时发现,使用玉米本身的启动子Ubi-1可以降低外源基因在转基因玉米中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因种子。     

Commercial production of aprotinin in transgenic maize seeds

The development of genetic transformation technology for plants has stimulated an interest in using transgenic plants as a novel manufacturing system for producing different classes of proteins of industrial and pharmaceutical value. In this regard, we report the generation and characterization of transgenic maize lines producing recombinant aprotinin. The transgenic aprotinin lines recovered were transformed with the aprotinin gene using the bar gene as a selectable marker. The bar and aprotinin genes were introduced into immature maize embryos via particle bombardment. Aprotinin gene expression was driven by the maize ubiquitin promoter and protein accumulation was targeted to the extracellular matrix. One line that showed a high level of aprotinin expression was characterized in detail. The protein accumulates primarily in the embryo of the seed. Southern blot analysis showed that the line had at least 20 copies of the bar and aprotinin genes. Further genetic analysis revealed that numerous plants derived from this transgenic line had a large range of levels of expression of the aprotinin gene (0–0.069%) of water-soluble protein in T₂ seeds. One plant lineage that showed stable expression after 4 selfing generations was recovered from the parental transgenic line. This line showed an accumulation of the protein in seeds that was comparable to the best T₂ lines, and the recombinant aprotinin could be effectively recovered and purified from seeds. Biochemical analysis of the purified aprotinin from seeds revealed that the recombinant aprotinin had the same molecular weight, N-terminal amino acid sequence, isoelectric point, and trypsin inhibition activity as native aprotinin. The demonstration that the recombinant aprotinin protein purified from transgenic maize seeds has biochemical and functional properties identical to its native counterpart provides a proof-of-concept example for producing new generation products for the pharmaceutical industry.     

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.     

利用花青素可视化跟踪表达系统快速筛选表达Cry1Ab/c基因的转基因玉米

以草甘膦抗性基因Epsps为标记基因, 在原核Kan^r基因两侧引入Cre(环化重组酶)基因识别的Lox-P位点, 同时以编码花青素合成转录因子的Bi和Cl基因为可视化选择报告基因, 构建了Bt杀虫蛋白基因Cry1Ab/c的可视化跟踪表达载体pBAC9017。用PDS1000/He基因枪转化玉米(Zea mays)自交系501的幼胚和胚性愈伤组织, 获得147个草甘膦抗性的玉米再生植株。其中106棵植株获得了结实种子, 16棵植株的结实种子有紫红色花青素基因的表达。经PCR检测表明, 外源Cry1Ab/c基因已经整合到玉米的基因组中。转基因植株种子蛋白粗提物用BT-Cry1Ab/1Ac金标免疫检测试纸条和ELISA检测, 结果表明, Cry1Ab/c在部分转基因植株后代中表达。     

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed strength. The Escherichia coli mutant glgC gene (glgC16), which encodes a highly active and allosterically insensitive AGPase, was introduced into maize (Zea mays L.) under the control of an endosperm-specific promoter. Developing seeds from transgenic maize plants showed up to 2–4-fold higher levels of AGPase activity in the presence of 5 mM inorganic phosphate (Pi). Transgenic plants with higher cytoplasmic AGPase activity under Pi-inhibitory conditions showed increases (13–25%) in seed weight over the untransformed control. In addition, in all transgenic maize plants, the seeds were fully filled, and the seed number of transgenic plants had no significant difference compared with that of untransformed control. These results indicate that increasing cytoplasmic AGPase activity has a marked effect on sink activity and, in turn, seed weight in transgenic maize plants.     

Seed-specific expression of a lysine rich protein sb401 gene significantly increases both lysine and total protein content in maize seeds

The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli using microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis and its expression was confirmed by Western blot analysis. Quantification of lysine and protein content in R1 maize seeds showed that, compared to the non-transgenic maize control, the lysine content increased by 16.1% to 54.8%, and total protein content increased by 11.6% to 39.0%. There was no visible morphological change in vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programmes aimed at improving the nutritional value of maize.     

Overexpression of phyA and appA Genes Improves Soil Organic Phosphorus Utilisation and Seed Phytase Activity in Brassica napus

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg^–1 seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.     

Manipulation of sinapine, choline and betaine accumulation in Arabidopsis seed: towards improving the nutritional value of the meal and enhancing the seedling performance under environmental stresses in oilseed crops.

Sinapoylcholine (sinapine) is the most abundant antinutritional phenolic compound in cruciferous seeds. The quaternary ammonium compounds, choline, betaine and N,N-dimethylglycine, reside along a biosynthetic pathway linked to the synthesis of membrane phospholipids and neurotransmitters with various biological functions. In chicken, choline intake is required for optimal egg-laying performance and a choline supplement in diet is positively correlated with weight gains. A key step in sinapine biosynthesis is catalyzed by sinapoylglucose: choline sinapoyltransferase (SCT; EC 2.3.1.91) to form an ester linkage with sinapoylglucose and choline. The objective of this work was to reduce the sinapine content and simultaneously enhance free choline levels in cruciferous seeds. We report here the characterization of an Arabidopsis T-DNA insertion mutant lacking SCT activity in the seed. The sct mutant seeds contain less than 1% of sinapine and a more than 2-fold increase in free choline compared with wild type. We further expressed a choline oxidase (COX; EC 1.1.3.17) gene from Arthrobacter pascens in the Arabidopsis sct mutant and wild-type background using a napin gene promoter to convert free choline into betaine, an effective stress-alleviating compound in plants. Betaine was not detected in WT or sct mutant seeds. The sct+COX seeds contain nearly 2-fold greater levels of betaine relative to WT+COX seeds, demonstrating a positive correlation between endogenous choline and betaine production. In contrast, stable comparable levels of free choline were detected between sct+COX and WT+COX plants suggesting choline homeostasis likely prevent high levels of betaine production in the seed of transgenic COX plants.     

Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.     

Characterisation of plant tocopherol cyclases and their overexpression in transgenic Brassica napus seeds

Tocopherols, collectively known as vitamin E, are only synthesised in photosynthetic organisms. Tocopherol cyclase (TC) catalyses the formation of the chromanol headgroup of the various tocopherol isoforms. TCs from Arabidopsis and maize (Zea mays) were expressed in Escherichia coli and purified. Analysis of the enzymatic properties revealed similarities but also differences between the two enzymes. Overexpression of chimeric TC gene constructs in developing seeds of transgenic rapeseed plants enhanced and modified the relative abundance of individual tocochromanol species in the seed oil, indicating a regulatory function of the enzyme in prenyllipid metabolism.     

Proteomics insight into the biological safety of transgenic modification of rice as compared with conventional genetic breeding and spontaneous genotypic variation

The potential of unintended effects caused by transgenic events is a key issue in the commercialization of genetically modified (GM) crops. To investigate whether transgenic events cause unintended effects, we used comparative proteomics approaches to evaluate proteome differences in seeds from 2 sets of GM indica rice, herbicide-resistant Bar68-1 carrying bar and insect-resistant 2036-1a carrying cry1Ac/sck, and their respective controls D68 and MH86, as well as indica variety MH63, a parental line for breeding MH86, and japonica variety ZH10. This experimental design allowed for comparing proteome difference caused by transgenes, conventional genetic breeding, and natural genetic variation. Proteomics analysis revealed the maximum numbers of differentially expressed proteins between indica and japonica cultivars, second among indica varieties with relative small difference between MH86 and MH63, and the minimum between GM rice and respective control, thus indicating GM events do not substantially alter proteome profiles as compared with conventional genetic breeding and natural genetic variation. Mass spectrometry analysis revealed 234 proteins differentially expressed in the 6 materials, and these proteins were involved in different cellular and metabolic processes with a prominent skew toward metabolism (31.2%), protein synthesis and destination (25.2%), and defense response (22.4%). In these seed proteomes, proteins implicated in the 3 prominent biological processes showed significantly different composite expression patterns and were major factors differentiating japonica and indica cultivars, as well as indica varieties. Thus, metabolism, protein synthesis and destination, and defense response in seeds are important in differentiating rice cultivars and varieties.     

Protein dynamics during seed germination under copper stress in Arabidopsis over-expressing <Emphasis Type="Italic">Potentilla superoxide dismutase</Emphasis>

Copper (Cu), though an essential micronutrient for plants, poses toxicity at higher concentrations possibly by inducing oxidative stress. With the background that enzyme superoxide dismutase (SOD) ameliorates oxidative stress, the present work focused on understanding physiological and proteomic response of Arabidopsis seeds constitutively over-expressing copper–zinc SOD of Potentilla atrosanguinea (PaSOD) during germination in response to varied concentrations of copper sulphate (Cu stress). Transgenics showed higher germination percentage and required less “mean time to germination” under Cu-stress. In response to Cu stress, 39 differentially expressed protein spots were detected by 2-D electrophoresis in proteins of germinating wild type (WT) and transgenic seeds, of which 14 spots appeared exclusively in transgenics. Among the rest 25 protein spots, 14 showed down-regulation, one showed up-regulation, and 10 spots disappeared. MALDI-TOF and subsequent peptide mass fingerprinting analysis revealed that the down-regulated proteins in transgenics were related to oxidative stress, detoxification, germination, intermediary metabolism and regulatory proteins. Up-regulated proteins in WT and down-regulated proteins in transgenic during Cu stress were the same. Changes in key proteins, vis-à-vis alleviation of oxidative stress in transgenic Arabidopsis over-expressing PaSOD possibly alleviated toxicity of Cu-induced stress during seed germination, resulting in higher germination rate and germination percentage.