GUS Gene Activity Research Articles

Maize Autophagy-Related Protein ZmATG3 Confers Tolerance to Multiple Abiotic Stresses.

Abiotic stresses pose a major increasing problem for the cultivation of maize. Autophagy plays a vital role in recycling and re-utilizing nutrients and adapting to stress. However, the role of autophagy in the response to abiotic stress in maize has not yet been investigated. Here, ZmATG3, which is essential for ATG8-PE conjugation, was isolated from the maize inbred line B73. The ATG3 sequence was conserved, including the C-terminal domains with HPC and FLKF motifs and the catalytic domain in different species. The promoter of the ZmATG3 gene contained a number of elements involved in responses to environmental stresses or hormones. Heterologous expression of ZmATG3 in yeast promoted the growth of strain under salt, mannitol, and low-nitrogen stress. The expression of ZmATG3 could be altered by various types of abiotic stress (200 mM NaCl, 200 mM mannitol, low N) and exogenous hormones (500 µM ABA). GUS staining analysis of ZmATG3-GUS transgenic Arabidopsis revealed that GUS gene activity increased after abiotic treatment. ZmATG3-overexpressing Arabidopsis plants had higher osmotic and salinity stress tolerance than wild-type plants. Overexpression of ZmATG3 up-regulated the expression of other AtATGs (AtATG3, AtATG5, and AtATG8b) under NaCl, mannitol and LN stress. These findings demonstrate that overexpression of ZmATG3 can improve tolerance to multiple abiotic stresses.

Constitutive Expression of Arabidopsis Senescence Associated Gene 101 in Brachypodium distachyon Enhances Resistance to Puccinia brachypodii and Magnaporthe oryzae.

Brachypodium distachyon, as an effective model of cereal grains, is susceptible to most destructive cereal pathogens. Senescence associated gene 101 (SAG101) has been studied extensively in Arabidopsis. SAG101 is one of the important regulators of plant immunity. However, no homologous genes of AtSAG101 were found in B. distachyon. In this study, the AtSAG101 gene was transformed into B. distachyon. Three transgenic plant lines containing the AtSAG101 gene were confirmed by PCR and GUS gene activity. There were fewer Puccinia brachypodii urediospores in the AtSAG101-overexpressing plants compared to wild type plants. P. brachypodii biomass was obviously decreased in AtSAG101 transgenic plants. The length of infection hyphae and infection unit areas of P. brachypodii were significantly limited in transgenic plants. Moreover, there were small lesions in AtSAG101 transgenic plants challenged by Magnaporthe oryzae. Salicylic acid accumulation was significantly increased, which led to elevated pathogenesis-related gene expression in transgenic B. distachyon inoculated by P. brachypodii or M. oryzae compared to wild type plants. These results were consistent with infected phenotypes. Overexpression of AtSAG101 in B. distachyon caused resistance to M. oryzae and P. brachypodii. These results suggest that AtSAG101 could regulate plant resistance in B. distachyon.

“Floral-dip” transformation of Amaranthus caudatus L. and hybrids A. caudatus × A. paniculatus L.

After “floral-dip” transformation of Amaranth plants with Agrobacterium tumefaciens strain GV3101 carrying pCBV19 gene vector that contained bar and gus genes, transgenic seeds were obtained. The functioning of the tran+sferred genes in Amaranthus tissues was confirmed with herbicide selection (PPT herbicide – phospinotricin) and gus gene activity. Positive results were obtained for cultivars “Karmin” and “Kremoviy rannii”. The percentage of GUS positive samples was 1% (for “Karmin”), 2.2% (for “Kremoviy rannii”) from the total initial quantity of plants that was prior to selection with the herbicide. The seeds of six amaranth cultivars were received after treatment with A. tumefaciens by the method “floral dip”. The lowest lethal dose of herbicide PPT was established – 40 mg/l. After spraying with herbicide, resistant plants were obtained for cultivars: “Kremoviy rannii” (21%) and “Karmin” (20%). After conduction of PCR analysis, positive results were obtained for four cultivars. The percentage of bar positive plants was 0.3% (“Helios”); 0.26% (“Sterkch”); 0.06% (“Kremoviy rannii”); 0.3% (“Rushnichok”) from total initial quantity of plants.

English

The plasmids pULGU1 and pEmuGN were introduced by biolistics in embryogenic cell suspensions of pearl millet, Pennisetum glaucum . The plasmid pULGU1 contained the mutant acetolactate synthase (ALS) gene of Arabidopsis thaliana , responsible for resistance to the chlorsulfuron herbicide. The plasmid pEmuGN carried the reporter β-glucuronidase (GUS) gene of Escherichia coli . Two months after bombardment of the cells, transformed calli were selected on Murashige and Skoog medium containing 30 nM chlorsulfruron, a completely concentration inhibitory for non-transformed cells. A concentration of 200 nM chlorsufuron did not affect the growth of these selected resistant calli. Genomic DNA of resistant cells presented an electrophoretic pattern revealing the integration of the mutant ALS gene into the genome of Pennisetum glaucum . Expression of the GUS reporter gene was revealed by histochemical assay of the blue coloration of the calli in the presence of the substrate Xgluc (5-bromo-4-chloro-3-indolyl-β-D-Glucuronic acid). The presence of GUS gene was furthermore confirmed, by southern blot hybridization of non-radioactive probes, labelled with digoxigenin, on genomic DNA extracted from selected chlorsulfuron-resistant calli. GUS gene activity observed in all selected calli was high during the first 6 months after bombardment and then decreased. Pearl millet plants were regenerated from cell lines derived from chlorsulfuron resistant calli. Southern blot hybridization of non-radioactive probes with genomic DNA extracted from these regenerated plants showed the presence of the GUS and ALS transgenes, confirming the stable transformation of these plants. Key words : Pennisetum glaucum , stable transformation, chlorsulfuron (ALS), β-glucuronidase (GUS), CaMV35S and Emu promoters.

Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration

The present study describes the successful development of vacuum infiltration method in the oomycete-resistant wild Piper sp., Piper colubrinum, as a rapid transient method for expression of GUS (β-Glucuronidase) reporter gene and introduction of hairpin vector for endogenous gene silencing. The GUS reporter gene construct pCAMBIA 1305.2 was used as a positive control to test the efficiency of vacuum infiltration strategy. Agrobacteria (EHA 105) harbouring GUS binary vector were vacuum-infiltrated into young detached in vitro leaf explants, which showed detectable GUS gene activity within 4 days of infiltration. This paper also reports for the first time the application of transient gene silencing in P. colubrinum by the delivery of in vitro synthesized hairpin vector construct (pHELLSGATE) containing endogenous serine threonine protein kinase (STPK) gene homologue into in vitro shoots. Introduction of hairpin vectors for the STPK gene into in vitro plantlets by vacuum infiltration resulted in significant reduction in transcript accumulation of the endogenous gene. The results indicate that transient gene silencing could be used as a rapid, preliminary high-throughput tool for P. colubrinum functional genomics.

Evaluation of Parameters for High Efficiency Gene Transfer via Agrobacterium tumefaciens and Production of Transformants in Indian Mulberry, Morus indica cv. K2

Agrobacterium-mediated transformation of Indian mulberry, Morus indica cv. K2, employing the highly regenerative hypocotyl, cotyledon, leaf and leaf callus explants is reported. Preculture of explants on regeneration medium for 5 days and co-cultivation for 3 days was found optimal. With the GUS-histochemical assay up to 40% hypocotyl, 50% cotyledon, 50% leaf and 54% leaf callus explants tested positive. Agrobacterium strain LBA4404 was more infective than GV2260 and A281 and amongst the plasmids tested, pBI121 successfully transformed 100% of the explants followed by p35SGUSINT (90-100%). Approximately, 50% of the explants survived selection. Up to 35.7% hypocotyl, 38.5% cotyledon, 23% leaf and 40% leaf callus derived shoots tested positive for GUS gene activity by the spectrofluorometric analysis. Transgene integration in the regenerated transformants was confirmed by polymerase chain reaction (PCR) and Southern hybridization making this the first report of stable transformation of mulberry.

Agrobacterium tumefaciens-mediated transformation of Indian mulberry, Morus indica cv. K2: a time-phased screening strategy.

An efficient and reproducible protocol for the production of transgenic plants was developed for Morus indica cv. K2 by Agrobacterium tumefaciens-mediated transformation. The hypocotyls, cotyledon, leaf and leaf callus explants precultured for 5 days on regeneration medium were co-cultivated with a bacterial suspension at 10(9) cells/ml for 3 days in the dark. Infectivity of A. tumefaciens strain LBA4404 was more than that of strains GV2260 and A281, and among the various plasmids tried, pBI121 and pBI101:Act1 transformed nearly 100% of the explants followed closely by p35SGUSINT. About 90-100% of the explants tested positive in the beta-glucuronidase (GUS) histochemical assay performed after 3 days of co-cultivation. This high level of transient expression, however, decreased to 20-25% after 15 days. Gus activity was most stable in the callus explants, which emerged as the explant of choice for transformation. The transformed explants were selected on 50-75 mg/l kanamycin for 1 month, and 25-50% of the explants developed adventitious buds. On the basis of kanamycin-resistant shoots produced from the total number of explants inoculated, the transformation efficiency was 44%. After 1 month, 40% of these shoots displayed high gus activity as assessed by the GUS fluorometric assay. On a selection-free root induction medium, 80% of the shoots developed roots and 90% of the potted plantlets acclimatized to the growth room conditions. The 3-month-old regenerates showed gus and nptII(neomycin phosphotransferase II) gene activity as assayed by the GUS fluorometric assay and nptII enzyme assay, followed by PCR polymerase chain reaction (54.5%) analysis after 6-months. Transgene integration into the nuclear genome of 1-year-old regenerates was confirmed in 10 of the 18 transformants tested by Southern analysis. The transformation efficiency as defined by the number of transgenic plants produced from the total number of explants co-cultivated was 6%.

Evaluation of diallel analysis using β-glucuronidase activity from transgenes in Nicotiana tabacum

A full diallel analysis is a tool for selection in plant breeding that has been subject to many discussions and controversies regarding its interpretation and merits. The analysis of well-defined transgenes by such an approach permits assessment of the value of diallel analyses. The performance of the Eberhart/Gardner diallel approach is analysed for the β-glucuronidase (GUS) activity of six well-defined, homozygous one-locus tobacco (Nicotiana tabacum L.) transgenic lines, each carrying differently located alleles of the GUS gene, and the nulliplex wild type. Tobacco is an inbreeding plant species, therefore all these lines are fully isogenic apart from the T-DNA insertion. The analysis shows that additivity of GUS gene activity as well as epistatic gene silencing translate well in the diallel parameters of general combining ability (GCA) and specific combining ability (SCA) or more detailed versions thereof, when compared to a parsimonious model based on the precise genetic constitution of the transgenic plants lines used as parents. The tobacco line with the highest GUS activity also has the highest GCA, demonstrating that an evaluation of parental phenotype would be sufficient for determining breeding potential. In case of the epistatic gene silencing, however, there is no positive correlation between GCA and parental performance, the reduction in GUS activity is more severe than is to be expected on the basis of parental performance.

Effect of six promoter-intron combinations on transient reporter gene expression in einkorn, emmer and common wheat cells by particle bombardment

The effect of six promoter-intron combinations on transient expression of β-glucuronidase (GUS) as the reporter gene was estimated in cultured einkorn ( Triticum monococcum), emmer ( Triticum durum) and common ( Triticum aestivum) wheat cell lines. Four promoters; cauliflower mosaic virus (CaMV) 35S, tandem CaMV35S, maize alcohol dehydrogenase gene ( Adh1) and rice actin gene ( Act1) promoters, and two introns; Adh1 intron1 and castor bean catalase intron, were tested. Six vectors having different promoter-intron combinations were introduced into wheat cells by using particle bombardment. Different levels of GUS gene activity in three wheat calli were detected by an in situ enzyme assay. The CaMV35S promoter gave the lowest level of transient expression in wheat cells. On the other hand, the rice Act1 promoter showed the highest level of transient expression in all three wheat cells and was also the most efficient promoter of all, suggesting that the rice Act1 promoter is efficient for use in wheat transformation.

Transformation of Elite Cotton Cultivars via Particle Bombardment of Meristems

A variety-independent protocol was developed to allow the production of transgenic cotton. High velocity gold beads coated with DNA were used to deliver foreign genes directly into the meristematic tissue of excised embryonic axes. Bombarded explants were allowed to develop into plants which were subsequently screened for gus gene activity. Buds in the axils of transformed leaves were forced to develop into plants by pruning away non-transformed primary shoot tips. Plants derived from this process carried the foreign gene in one or more of their tissue layers. Transformation frequencies varied with the genotype used, but all cultivars attempted to date have yielded transgenic progeny. Molecular and genetic characterization of primary transformants and their progeny established that foreign genes were stably integrated and transmitted to progeny in a Mendelian fashion.

Transient expression of the ?-glucuronidase gene in embryogenic callus of Picea mariana following microprojection

A microprojection protocol using the DuPont Biolistic(™) particle delivery system and the β-glucuronidase (GUS) reporter gene fused with the 35S promoter of Cauliflower mosaic virus (CaMV) was developed for Picea mariana callus. Comparison of four tungsten microprojectile sizes showed the highest transient gene expression with 1.11μm diameter particles. Adsorption of DNA on the microcarriers using calcium chloride led to higher GUS gene activity than using polyethylene glycol. GUS gene activity in P. mariana was the highest when cells were treated 5 and 6 days after subculturing to fresh media. The wheat ABA-inducible Em gene promoter yielded 4.5 times higher GUS gene activity than the 35S CaMV promoter. Comparison of transient GUS gene expression among 10 P. mariana embryogenic cell lines from six different open-pollinated families showed comparable gene activity, with the exception of one family showing no GUS gene activity.

In situ detection of transposition of the maize controlling element (Ac) in transgenic soybean tissues

The development of a transposon mutagenesis system in soybean would aid in the isolation of unknown genes. The maize controlling element (Ac) has, therefore, been introduced into the soybean (Glycine max (L.) Merr.) genome byAgrobacterium-mediated transformation.Ac was inserted into the untranslated leader region of the bacterial ß-glucuronidase gene (GUS) such that the excision ofAc resulted in restoration of the GUS gene activity. Excision events of theAc element were monitored by detecting blue cells or sectors in transgenic soybean tissues. Using the GUS gene assay and with hybridization data, we have demonstrated that theAc element transposes in transgenic soybean calli, leaves, stems, and roots.