Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean

Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean. Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed.     

Effect of puromycin on protein and glycerolipid biosynthesis in isolated mucosal cells

The effect of puromycin on phosphatidylcholine and triacylglycerol synthesis was studied in isolated cells of rat intestinal mucosa using radioactive palmitate, glycerol, 2-hexadecylglycerol, and lysophosphatidylcholine as markers. Puromycin caused a 60–65% inhibition of phosphatidylcholine biosynthesis but did not affect the formation of triacylglycerols. Under comparable conditions protein synthesis was inhibited 90–95% and glycoprotein synthesis 60–70%. The utilization of the various lipid precursors indicated that puromycin inhibited the biosynthesis of phosphatidylcholine via both the CDP-choline and the lysophosphatidylcholine pathways, without interfering with triacylglycerol synthesis from either phosphatidic acid or monoacylglycerol precursors. Since both phosphatidylcholines and proteins are involved in the assembly of chylomicrons, it is suggested that the effect of puromycin on chylomicron formation could be due to an inhibition of the biosynthesis of any one or all three of the membrane components: proteins, glycoproteins, and phosphatidylcholines.     

Somatic embryo cycling: Evaluation of a novel transformation and assay system for seed-specific gene expression in soybean

Somatic embryo cycling, a modification of soybean somatic embryogenic suspension culture, was developed as an efficient and rapid method of producing tissue suitable for stable transformation of soybean germplasm by biolistic particle bombardment. Instead of using immature seed explants, cotyledon-staged somatic embryo hypocotyls were placed on auxin-containing medium, where they initiated new somatic embryos primarily from single epidermal cells. By bombarding hypocotyls prior to initiation of subsequent embryo formation, we have effectively transformed soybean somatic embryos with the reporter genes neomycin phosphotransferase,gb-glucuronidase, and a mammalian stearyl CoA delta-9 desaturase, controlled by a seed-specific promoter. These embryos contain significantly reduced levels of saturated palmitic and stearic fatty acids, and significant amounts of monounsaturated palmitoleic acid, which is not normally abundant in soybean seeds. This study demonstrates the effectiveness of somatic embryo cycling for soybean transformation, and for testing expression of genes for seed-specific proteins. Abnormal flower development in recovered plants is a limitation for application of the technique to produce transgenic seed at present.     

Somatic embryo cycling: Evaluation of a novel transformation and assay system for seed-specific gene expression in soybean

Somatic embryo cycling, a modification of soybean somatic embryogenic suspension culture, was developed as an efficient and rapid method of producing tissue suitable for stable transformation of soybean germplasm by biolistic particle bombardment. Instead of using immature seed explants, cotyledon-staged somatic embryo hypocotyls were placed on auxin-containing medium, where they initiated new somatic embryos primarily from single epidermal cells. By bombarding hypocotyls prior to initiation of subsequent embryo formation, we have effectively transformed soybean somatic embryos with the reporter genes neomycin phosphotransferase,gb-glucuronidase, and a mammalian stearyl CoA delta-9 desaturase, controlled by a seed-specific promoter. These embryos contain significantly reduced levels of saturated palmitic and stearic fatty acids, and significant amounts of monounsaturated palmitoleic acid, which is not normally abundant in soybean seeds. This study demonstrates the effectiveness of somatic embryo cycling for soybean transformation, and for testing expression of genes for seed-specific proteins. Abnormal flower development in recovered plants is a limitation for application of the technique to produce transgenic seed at present.     

Identification,validation, and expression of ABC transporters in Podophyllum hexandrum and their role in podophyllotoxin biosynthesis

Podophyllum hexandrum Royle is an important medicinal herb of North-Western Himalayas, and podophyllotoxin, being its major metabolite, has been used extensively in the preparation of several anticancer drugs. Podophyllotoxin accumulates in rhizomes; however, no information exists on the role of ATP-binding cassette (ABC) transporters vis-à-vis podophyllotoxin content. The present study reports identification, validation, and expression analysis of ABC transporter genes from P. hexandrum. Total 252 ABC transporter genes were identified as unigenes out of which 22 were further validated using real time qPCR in different tissues of varying podophyllotoxin content. Differential expression analysis and Pearson’s correlation coefficient revealed two candidate genes PhABC6 and PhABCIII having a positive correlation with the podophyllotoxin content. PhABCIV showed the highest expression in rhizomes (20.53-folds compared to shoots) suggesting its possible role in transport and accumulation of podophyllotoxin.     

Induction and expression of seed-specific promoters in Arabidopsis embryo-defective mutants

In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild-type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1-uidA chimeric gene, representative of the maturation programme and the Em 1-uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em 1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.     

Protein and glycerolipid biosynthesis in isolaged intestinal epithelial cells of normal and bile-fistula rats.

Protein and glycerolipid biosynthesis was studied in isolated epithelial cells of the intestinal mucosa of normal and 1-day bile-fistula rats. In cells from fistula rats, protein synthesis from [1-¹⁴C]leucine was decreased 40–45% and glycoprotein synthesis from [1-¹⁴C]glucosamine 25–30%. Under comparable conditions, the synthesis of phosphatidylcholine from a variety of precursors was decreased 40–45% while no change was observed in the formation of either phosphatidylethanolamine or triacylglycerols. Chylomicron release was inhibited 70–80% in the cells from the bile-fistula rats. In vitro addition of either lysophosphatidylcholine or choline to the cells from the bile-fistula animals resulted in greatly increased phosphatidylcholine and protein biosynthesis and an effective release of chylomicrons. It is suggested that this stimulation of incorporation of label is due to a net synthesis of phosphatidylcholine required for membrane and lipoprotein repairs in the fistula cells. These results provide further evidence, that, in the rat, biliary phosphatidylcholine may play an essential role in the formation and clearance of chylomicrons from the intestinal mucosa.     

Asparagine biosynthesis in soybean nodules

Two auxin-induced endo-1,4-β-glucanases (EC 3.2.1.4) were purified from pea (Pisum sativum L. var. Alaska) epicotyls and used to degrade purified pea xyloglucan. Hydrolysis yielded nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and heptasaccharide (glucose/xylose, 4:3) as the products. The progress of hydrolysis, as monitored viscometrically (with amyloid xyloglucan) and by determination of residual xyloglucan-iodine complex (pea) confirmed that both pea glucanases acted as endohydrolases versus xyloglucan. K_m values for amyloid and pea xyloglucans were approximately the same as those for cellulose derivatives, but V_max values were lower for the xyloglucans. Auxin treatment of epicotyls in vivo resulted in increases in net deposits of xyloglucan and cellulose in spite of a great increase (induction) of endogenous 1,4-β-glucanase activity. However, the average degree of polymerization of the resulting xyloglucan was much lower than in controls, and the amount of soluble xyloglucan increased. When macromolecular complexes of xyloglucan and cellulose (cell wall ghosts) were treated in vitro with pea 1,4-β-glucanase, the xyloglucan component was preferentially hydrolyzed and solubilized. It is concluded that xyloglucan is the main cell wall substrate for pea endo-1,4-β-glucanase in growing tissue.     

Design of tandem genes cluster and expression vector for biosynthesis of soybean isoflavones

A tandem gene cluster CHS-CHI-IFS (rIFS) for secondary metabolites of plant isoflavones was constructed by using the chalcone synthase (CHS), chalcone isomerase (CHI), and isoflavone synthase (IFS) (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"EU526827","term_id":"170783761","term_text":"EU526827"}}EU526827, {"type":"entrez-nucleotide","attrs":{"text":"EU526829","term_id":"170783765","term_text":"EU526829"}}EU526829, {"type":"entrez-nucleotide","attrs":{"text":"EU526830","term_id":"170783767","term_text":"EU526830"}}EU526830) in a single recombination event with the pET22b vector. The resulting expression vector pET-rIFS was heterogeneously expressed. The highlights of the vector include ease of handling, high efficiency and universal application among diverse plant species. To the best of our knowledge, this is the first attempt at developing a novel method of constructing tandem gene cluster for future research involving secondary metabolism of isoflavones and isoflavones engineering.Key words: Isoflavones biosynthesis, Novel method, Secondary metabolism, Tandem gene cluster     

The role of Mg2+-dependent phosphatidate phosphohydrolase in pulmonary glycerolipid biosynthesis

Rat lung microsomes washed with increasing concentrations of NaCl show a displacement of protein from microsomes to the wash supernatant. Among the proteins removed from the microsomal surface was the Mg2+-dependent phosphatidate phosphohydrolase, while the Mg2+-independent activity remained associated with the microsomes. The Mg2+-dependent activity could be quantitatively assayed in the wash supernatant. Microsomes washed with increasing concentrations of NaCl showed a progressive impairment in the synthesis of labelled neutral lipid and phosphatidylcholine from [14C]glycerol 3-phosphate with a concomitant increase in the labelling of phosphatidic acid. The impairment was sigmoidal and correlated highly with the decrease in Mg2+-dependent phosphatidate phosphohydrolase activity. When Mg2+-dependent phosphatidate phosphohydrolase from wash supernatant was incubated with microsomes previously washed with high salt concentrations, the labelling of neutral lipid and phosphatidylcholine was returned to control levels. Labelling of neutral lipids and phosphatidylcholine could be restored upon addition of a cytosolic Mg2+-dependent phosphatidate phosphohydrolase isolated by gel filtration. Mg2+-independent phosphatidate phosphohydrolase isolated from cytosol was incapable of restoring the labelling of neutral lipids and phosphatidylcholine. These findings confirm that the Mg2+-dependent phosphatidate phosphohydrolase of rat lung is involved in pulmonary glycerolipid biosynthesis. The role of the Mg2+-independent phosphatidate phosphohydrolase activity remains unknown.     

Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules

miRNAs are ～21 nt non-coding RNAs and play important roles in plant development and response to stress. Symbiotic nitrogen fixation (SNF) is agronomically important for reducing the need of nitrogen fertilizers. The soybean root nodule is the place where SNF takes place. To identify miRNAs that are possibly involved in nitrogen fixation in soybean functional nitrogen-fixing nodules, a small library of RNAs was constructed from the functional nodules harvested 28 days after inoculation with rhizobium. Thirty-two small RNA sequences were identified as belonging to 11 miRNA families. Eight miRNAs are conserved across plant species, twenty are specific to soybean, and the four remaining miRNAs are novel. Expression analysis revealed that miRNAs were differentially expressed in the different tissues. Combinatorial miRNA target prediction identified genes that are involved in multiple biological processes. The results suggest that miRNAs play critical and diverse roles in SNF, nutrient acquisition, and plant development.     

Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate

The effect of nitrate on N₂ fixation and the assimilation of fixed N₂ in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, ¹⁵N₂ fixation and relative abundance of ureides ([ureides/(ureides + nitrate + α-amino nitrogen)] × 100) in xylem exudate, gave similar results for the effect of nitrate on N₂ fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N₂ fixation precedes the loss of the potential of bacteroids to fix N₂. This in turn precedes the loss of the potential of nodules to assimilate fixed N₂.     

A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy

1. The accumulation of triglyceride in the liver remnant after subtotal hepatectomy (removal of 82% of the liver) exceeded that described for partial hepatectomy (removal of 70% of the liver). 2. Palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and diglyceride acyltransferase activities were measured in the microsomal fraction, and phosphatidate phosphohydrolase activity was measured in the particle-free supernatant fraction, prepared from the liver remnant at various times after subtotal hepatectomy. 3. The only enzyme showing a significant change in specific activity was phosphatidate phosphohydrolase. The specific activity was approximately fivefold that of the control value at 6h after operation and threefold that of the control at 10, 16 and 24h after operation. A smaller increase in the specific activity of the enzyme in sham-operated animals occurred only at 6h after operation. 4. However, at this time the total phosphohydrolase activity of the remaining liver in the sham-operated rats was approximately threefold that in hepatectomized rats. 5. Injection of actinomycin D prevented the increase in activity of phosphatidate phosphohydrolase but did not prevent the accumulation of triglyceride.     

大豆根茬木霉腐解产物的鉴定及其化感作用的研究

采用不同极性溶剂环己烷、乙酸乙酯、正丁醇连续提取及XAD 4树脂提取大豆根茬木霉腐解产物 ,并对各提取组分进行了化感作用研究 ,对化感作用明显的组分用GC MS进行了化学成分分析 .结果表明 ,大豆根茬木酶腐解产物的 2周、4周环己烷、乙酸乙酯、XAD 4树脂提取组分能显著抑制初期大豆种子萌发 (2 1h) ,随大豆种子萌发时间的延长 ,抑制作用减弱 .2周、4周根茬各提取组分对大豆种子萌发中的胚根生长 ,所表现出的化感抑制作用规律是 :0 .5 0、1.0 0gDW·ml-1乙酸乙酯、XAD 4树脂提取组分与 0 .10、0 .2 5 gDW·ml-1乙酸乙酯、XAD 4树脂组分及环己烷提取组分胚根长的RI值 (化感作用效应指数 )差异达显著或极显著水平 .乙酸乙酯、正丁醇、XAD 4树脂提取组分中的有机化合物种类有 :有机酸、酚、醛、酮、苯、胺、腈、酯等 ,其中大多为化感物质 .