Copper induction and differential expression of laccase in Aspergillus flavus.

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/µg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus . Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.

Copper induction and differential expression of laccase in Aspergillus flavus

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.

An efficient and reproducible protocol for the production of salt tolerant transgenic wheat plants expressing the Arabidopsis AtNHX1 gene.

We present an efficient method for the production of transgenic salt tolerant hexaploid wheat plants expressing the Arabidopsis AtNHX1 gene. Wheat mature zygotic embryos were isolated from two hexaploid bread wheat (Triticum aestivum) cultivars (namely: Gemmeiza 9 and Gemmeiza 10) and were transformed with the A. tumefaciens LBA4404 harboring the pBI-121 vector containing the AtNHX1 gene. Transgenic wheat lines that express the gus intron was obtained and used as control. The results confirmed that npt-II gene could be transmitted and expressed in the T2 following 3:1 Mendelian segregation while the control plant couldn't. The data indicate that, the AtNHX1 gene was integrated in a stable manner into the wheat genome and the corresponding transcripts were expressed. The transformation efficiency was 5.7 and 7.5% for cultivars Gemmeiza 10 and Gemmeiza 9, respectively. A greenhouse experiment was conducted to investigate the effect of AtNHX1 gene in wheat salt tolerance. The transgenic wheat lines could maintain high growth rate under salt stress condition (350 mM NaCl) while the control plant couldn't. The results confirmed that Na(+)/H(+) antiporter gene AtNHX1 increased salt tolerance by increasing Na(+) accumulation and keeping K+/Na(+) balance. Thus, transgenic plants showed high tolerance to salt stress and can be considered as a new genetic resource in breeding programs.

Expression of S-adenosyl methionine decarboxylase gene for polyamine accumulation in Egyptian cotton Giza 88 and Giza 90.

Developing drought tolerance in Egyptian cotton varieties is a strategic goal considering the need to expand cotton cultivated area and water scarcity in the Nile valley. In the present study, increasing levels of polyamine accumulation via expressing S-adenosyl methionine decarboxylase (SAMDC) gene was the main goal. SAMDC cDNA isolated from Saccharomyces cerevisiae isolate was isolated and genetically engineered into Egyptian cotton varieties Giza 88 as an extra long staple and Giza 90 as a long staple by means of particle bombardment through meristem transformation.T(0) transgenic plants were screened using basta herbicide (200 mg/l). RT- PCR analysis was used to confirm gene expression while gene integration was confirmed by Southern blot analysis. Control plants from Giza 88 and Giza 90 were subjected to drought regime using different concentrations of PEG 6000 (2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17%, and 20%) for 9 hrs to record drought stress symptoms and determine the potential concentration level for inducing polyamine accumulation. 17% of PEG 6000 was considered the sublethal concentration showing drought stress symptoms and therefore was used as potential stress concentration for estimating the level for spermine accumulation in both control and transgenic Giza varieties. T(1) transgenic plants grown under induced drought stress regime were tested positive for gene integration and expression and subjected to HPLC analysis to determine levels of spermine as polyamine accumulated compound in response to drought stress regime. Elevated spermine accumulation in Egyptian cotton varieties Giza 88 and Giza 90, were compared as non transgenic plants grown under same induced drought conditions with T(1) transgenic plants using reverse-phase HPLC analysis. Elevated spermine accumulation expressing SAMDC gene reflect main cause for increasing drought tolerance in both transgenic varieties.

Genetic transformation of mature embryos of bread (T. aestivum) and pasta (T. durum) wheat genotypes.

The objective of the present study is to develop an efficient protocol for regeneration of transgenic wheat plants using Agrobacterium- mediated transformation of mature embryos of hexaploid bread wheat (Triticum aestivum) and tetraploid pasta wheat (Triticum durum). The data indicated that embryogenic calli were formed within 7 days in the presence of 2 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic calli in the presence of 2 mgl-1 BA. Shoot regeneration frequency varied between wheat cultivars according to their genetic background differences. Regeneration frequency was higher in the cultivar Gemmiza 10 (95 %) compared with the other cultivars tested. Mature embryos derived callus of the cultivars Gemmiza 10 and Gemmiza 9 were co-cultivated with A. tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (npt-II). The resulted putative transgenic plantlets were able to grow on kanamycin containing medium. A successful integration of the transgene was confirmed by analyzing the T0 plantlets using Southern hybridization and PCR amplification. The gus gene expression can be detected only in the transgenic plants. The reported protocol is reproducible and can be used to regenerate transgenic wheat plants expressing the genes present in A. tumifaciens binary vectors.