Stable integration and expression of β-glucuronidase and NPT II genes in mango somatic embryos

Summary Somatic proembryos of mango (Mangifera indica L. cv. Hindi) were co-cultivated withAgrobacterium tumefaciens strain A208 harboring pTiT37-Se::pMON 9749 (9749 ASE). Transformed somatic proembryos capable of growing on selection medium containing 200 μg/ml kanamycin produced the characteristic indigo blue precipitate in the presence of 5-bromo-4-chloro-3-glucuronic acid. These proembryos were chimeral consisting of transformed (blue) and nontransformed (yellow/white) cells. A stepwise selection strategy was found necessary to eliminate chimeras. a) Initial screening at 200 μg/ml kanamycin to enable growth of transformed cells, b) further screening at 400 μg/ml kanamycin to reduce chimeras, and c) recovery of pure transformed clones of proembryos in liquid selection medium with 100 μg/ml kanamycin. The integration of the NPT II and GUS genes into mango genome was confirmed by Southern hybridization.     

A rapid method for qualitative assay of both neomycin phosphotransferase II and β-glucuronidase activities in transgenic plants

A rapid and efficient method for assaying both NPT II and GUS activities was developed. In this method, which is modified from that of McDonnell et al. (1987), and Jefferson (1987), no sample processing procedures such as grinding and centrifugation are necessary. Cut plant tissues (leaves) or intact calli or cells expressing the genes of interest are placed in wells of a microtiter plate containing reaction mixture, and after incubation the reaction mixture is directly used for both NPT II and GUS assays. For the NPT II assay, aliquots of the reaction mixture are blotted onto Whatman P81 paper through a manifold, and the product of the reaction is detected by autoradiography. For GUS activity, aliquots or the rest of the reaction mixture are observed for fluorescent emission under a hand-held UV light or read in a fluorimeter after adding stop buffer to the reaction mixture. This method is the simplest, cheapest, and quickest assays for NPT II and GUS reported to date, and is extremely efficient and suitable for assaying small amounts of samples (as little as 0.3 mg tissue), such as in transient expression assays, or for the quick screening of large numbers of samples, such as in studies of gene inheritance in transgenic plants. In our laboratory, it has been used successfully in assaying NPT II activities for transient and stable gene expression in transformed protoplasts, calli, and leaf tissues of various transgenic plants. It has also been used for detecting both NPT II and GUS activities in transgenic rice plants, in which more than 400 samples could be assayed per day per person.     

Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl^–1 kinetin, and 100 mgl^–1 kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl^–1 each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the -glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl^–1 myo-inositol, 1 mgl^–1 2,4-D, 0.5 mgl^–1 BA, and 0.5 mgl^–1 kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.Abbreviations BA 6-benzyladenine; 2,4-D,2,4-dichlorophenoxyacetic acid - DIG digoxigenine - GA₃ gibberellic acid - X-gluc X-glucuronide - GUS -glucuronidase - MS Murashige and Skoog (1962)     

Non-radioactive detection of β-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC

Summary The use of transient gene expression assays for the study of natural or engineered plant promoters is affected by a considerable degree of inter-experiment variability. As a means of obtaining interpretable data from a limited number of experiments, we worked out conditions for the simultaneous determi nation of the activity of two reporter genes, a sample and a reference, ona single extract of co-transformed protoplasts. ß-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) genes, both under the control of the CaMV 35S promoter, were transferred into tobacco (Nicotiana tabacum L.) protoplasts on two independent plasmids. The parallel expression of the two reporter genes in several independent co-transformation experiments was verified. Conditions for the use of a single protoplast extraction buffer and for the simultaneous assay of both reporter gene activities were set up. A HPLC method for the non-radioactive determination of both enzyme activities on a single aliquot of the reaction mixture was developed. The resulting procedure was tested using the GUS gene as reference and the CAT gene, under the control of either wild type or upstream-deleted (–90) CaMV 35S promoter, as sample. The protocol is simple and allows the fast analysis of plant promoters in the presence of a true internal standard under conditions in which assay manipulations are reduced to a minimum and both reporter gene activities are subjected to the same experimental treatments.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyl transferase - EDTA ethylenediaminetetraacetic acid - GUS ß-glucuronidase - HPLC high performance liquid chromatography - MES 2-morpholinoethanesulphonic acid - MS medium after Murashige and Skoog (1962) - MUG 4-methyl umbelliferyl glucuronide - MU methylumbelliferone - NOS nopaline synthase - PEG polyethylene glycol - TRIS tris-hydroxymethyl aminomethane - UV ultraviolet     

Genotype- and promoter-induced variability in transient β-glucuronidase expression in pea protoplasts

Summary Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of -glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 F; and using 125 g of calf thymus carrier DNA and 75 of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.NRCC No. 30910     

Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants

In order to construct transgenic rice plant with an introduced oryzacystatin (OC)--glucuronidase (GUS) fusion gene, we first introduced it into rice protoplasts by electroporation, together with a marker gene conferring hygromycinresistance (pUC-HPH). In a transient assay using the transfected protoplasts, both OC and GUS activities were detected. The GUS activity was higher when the OC-GUS fusion protein was expressed than when only a single GUS protein was expressed. Next, to isolate stable transformants, hygromycin-resistant calli were selected. Forty one out of 116 hygromycin-resistant calli expressed a 2.2 kb mRNA transcribed from the chimeric gene and their extracts exhibited the activities of both OC and GUS. Finally, the transgenic calli were regenerated into rice plants whose tissues (leaves, roots and seeds) exhibited GUS activity probably derived from the fusion protein.     

Biochemical Characterisation and Cloning of α-Kafirin Gene from Sorghum (Sorghum bicolor L Moench)

Seed storage proteins from naturally occurring lysine-rich cultivars namely IS 217O₂, CVS 365, G 1058, G 205 and CVS 549 were analyzed biochemically, immunologically and compared with a low-lysine cultivar (White Martin) and a chemically induced high-lysine mutant (P7210). Protein fractionation studies indicated that the high lysine cultivars contained 25% less kafirin and an increased alcohol insoluble reduced glutelin without affecting the total protein content. SDS-PAGE analysis of total kafirin showed the absence of 25.3 kD and 25.9 kD a-kafirin proteins in lysine-rich cultivars IS 217O₂, CVS 365 and G 1058, while in G 205 only the 25.9 kD protein was absent compared to low-lysine cultivar White Martin. A genomic clone λGK5 encoding an a-kafirin has been isolated from cv White Martin genomic library using pSKR3 as hybridizing probe and sequenced. Transient expression studies by particle bombardment of immature seeds of sorghum allowed to detect β-glucuronidase (GUS) activity only in endosperm cells confirming that the α-kafirin gene promoter is functional and tissue specific.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β?triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β-triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites

Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied. Incubation of 1 with C. elegans yielded five hydroxylated metabolites 2–6, while M. phaseolina transformed compound 1 into polar metabolites 7–11. These metabolites were identified as 6β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (3), 11α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6β,12β,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6β,15α,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (6), 17β-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7β,17β,-dihydroxy-17α-methylandrost-1,4-dien-3-one (8), 15β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (9), 17β-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12 and oximes 13, and 14. Compounds 6 and 12–14 were identified as new derivatives of dianabol (1). The structures of all transformed products were deduced on the basis of spectral analyses. Compounds 1–14 were evaluated for β-glucuronidase enzyme inhibitory activity. Compounds 7, 13, and 14 showed a strong inhibition of β-glucuronidase enzyme, with IC₅₀ values between 49.0 and 84.9 μM.     

Endophytic bacteria expressing β-glucuronidase cause false positives in transformation of Dioscorea species

Summary False positive transformants obtained during plant transformation experiments on species of the monocotyledonous genus Dioscorea (yam) are described. The false positive results were found to be due to endophytic bacteria which exist within aseptically micropropagated shoot cultures and which express -glucuronidase (GUS). The bacteria were isolated and identified as two species of Curtobacterium. The expression of GUS in these organisms was found to be induced by a variety of glucuronide substrates. The induction of GUS activity in the bacteria can be inhibited by chloramphenicol, tetracycline, ticarcillin and sodium azide. Implications of these results for use of the gus gene in plant transformation work are discussed.Abbreviations DTT Dithiothreitol - EDTA Ethylenediamine tetra-acetic acid - ELISA Enzyme-linked immuno-sorbent assay - GUS -glucuronidase - LB Luria Bertani - MUG 4-Methylumbelliferyl--D-glucuronide - PNPG p-nitrophenyl--D-glucuronide - PVP Polyvinylpyrrolidone - SDS Sodium dodecyl sulphate - TAE Tris-acetate-EDTA buffer - X-Gluc 5-Bromo-4-chloro-3-indolyl--D-glucuronide     

Allelic variation of the β-, γ- and δ-kafirin genes in diverse Sorghum genotypes

The β-, γ- and δ-kafirin genes were sequenced from 35 Sorghum genotypes to investigate the allelic diversity of seed storage proteins. A range of grain sorghums, including inbred parents from internationally diverse breeding programs and landraces, and three wild Sorghum relatives were selected to encompass an extensive array of improved and unimproved germplasm in the Eusorghum. A single locus exists for each of the expressed kafirin-encoding genes, unlike the multigenic α-kafirins. Significant diversity was found for each locus, with the cysteine-rich β-kafirin having four alleles, including the first natural null mutant reported for this prolamin subfamily. This allele contains a frame shift insertion at +206 resulting in a premature stop codon. SDS-PAGE revealed that lines with this allele do not produce β-kafirin. An analysis of flour viscosity reveals that these β-kafirin null lines have a difference in grain quality, with significantly lower viscosity observed over the entire Rapid ViscoAnalyser time course. There was less diversity at the protein level within the cysteine-rich γ-kafirin, with only two alleles in the cultivated sorghums. There were only two alleles for the δ-kafirin locus among the S. bicolor germplasm, with one allele encoding ten extra amino acids, of which five were methionine residues, with an additional methionine resulting from a nucleotide substitution. This longer allele encodes a protein with 19.1% methionine. The Asian species, S. propinquum, had distinct alleles for all three kafirin genes. We found no evidence for selection on the three kafirin genes during sorghum domestication even though the δ-kafirin locus displayed comparatively low genetic variation. This study has identified genetic diversity in all single copy seed storage protein genes, including a null mutant for β-kafirin in Sorghum.     

Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance

Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.     

Design and characterization of new β-glucuronidase active site variants with altered substrate specificity

Objective

To isolate and characterize the kinetics of variants of E. coli β-glucuronidase (GUS) having altered substrate specificity.

Results

Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-β-d-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved k_cat/K_M value towards pNP-gal compared to wild-type GUS, up to 9 × 10⁸-fold shift in specificity from p-nitrophenyl-β-d-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 10³-fold increase in specificity shift compared to a previously evolved GUS variant.

Conclusions

The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.

     

PCR Amplification，Cloning and Sequencing of RbcL Coding Region in Mesophyll Cell and Bundle Sheath Cell of Sorghum（Sorghum bicolor L．）

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Preparation of Water-soluble and Stable Chlorophylls and β-Carotene by Forming Complexes with Egg Albumin

Chlorophyll- and β-carotene-egg albumin complexes were prepared. As these pigment-protein complexes were soluble in water, the chlorophylls and β-carotene were endowed with a water-soluble character. The molecular numbers of pigments incorporated into the complexes were increased by using proteins treated with charcoal, and additionally by using chemically modified proteins, acetylated or carbamoylated egg albumin. When compared with the stabilities of these pigments in detergent or in an organic solvent, the chlorophyll in the complexes was stable against photobleaching and for a wide range of pH values (pH 5 ~ 10). β-Carotene in the egg albumin complexes was relatively stable under UV irradiation.