Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

Is a non-synonymous SNP in the HvAACT1 coding region associated with acidic soil tolerance in barley?

Abstract The barley HvAACT1 gene codes for a citrate transporter associated with tolerance to acidic soil. In this report, we describe a single nucleotide polymorphism (SNP) in the HvAACT1 coding region that was detected as T-1,198 (in genotypes with lower root growth on acidic soil) or G-1,198 (greater root growth) and resulted in a single amino acid change (L/V-172). Molecular dynamic analysis predicted that HvAACT1 proteins with L or V-172 were stable, although the substitution led to structural changes within the protein. To evaluate the effect of the SNP on tolerance to acidic soil, barley accessions were separated into haplotypes based on the presence of a 1 kb insertion in the HvAACT1 promoter and a 21 bp insertion/deletion. These markers and the SNP-1,198 allowed the identification of five haplotypes. Short-term soil experiments showed no difference in root growth for most of the accessions containing the 21 bp insertion and T or G-1,198. In contrast, genotypes showing both the 21 bp deletion and G-1,198, with one of them having the 1 kb insertion, showed greater root growth. These results indicate that the SNP was not advantageous or deleterious when genotypes from the same haplotype were compared. The occurrence of the SNP was highly correlated with the 21 bp insertion/deletion that, together with the 1 kb insertion, explained most of the barley tolerance to acidic soil.

Resistance to Anticarsia gemmatalis Hübner (Lepidoptera, Noctuidae) in transgenic soybean (Glycine max (L) Merrill Fabales, Fabaceae) cultivar IAS5 expressing a modified Cry1Ac endotoxin

Somatic embryos of the commercial soybean (Glycine max) cultivar IAS5 were co-transformed using particle bombardment with a synthetic form of the Bacillus thuringiensis delta-endotoxin crystal protein gene cry1Ac, the beta-glucuronidase reporter gene gusA and the hygromycin resistance gene hpt. Hygromycin-resistant tissues were proliferated individually to give rise to nine sets of clones corresponding to independent transformation events. The co-bombardment resulted in a co-transformation efficiency of 44 percent. Many histodifferentiated embryos and 30 well-developed plants were obtained. Twenty of these plants flowered and fourteen set seeds. The integration and expression of the cry1Ac, gusA and hpt transgenes into the genomes of a sample of transformed embryos and all T0, T1, T2 and T3 plants were confirmed by Gus activity, PCR, Southern and western blot, and ELISA techniques. Two T0 plants out of the seven co-transformed plants produced seeds and were analyzed for patterns of integration and inheritance until the T3 generation. Bioassays indicated that the transgenic plants were highly toxic to the velvetbean caterpillar Anticarsia gemmatalis, thus offering a potential for effective insect resistance in soybean.

Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene

Penicillium brevicompactum é um fungo filamentoso que apresenta um potencial para a aplicacão industrial devido a sua eficiente producão de enzimas do complexo pectinolítico. Neste trabalho foi desenvolvido um sistema de transformacão heterólogo para P. brevicompactum baseado na complementacão de um mutante nitrato redutase. Mutantes nitrato redutase foram obtidos pela resistência ao clorato de sódio em uma taxa de 23,24per center. O mutante denominado 4457-18X foi escolhido para os experimentos de transformacão com o vetor pNH24, que contém o gene da nitrato redutase de Fusarium oxysporum. Uma freqüência de cerca de 3 transformantes/mg de DNA foi obtida utilizando-se o vetor pNH24 na forma circular e um aumento de cerca de 10 vezes nessa freqüência foi alcancado com a utilizacão desse vetor linearizado com a enzima de restricão Xba I. A análise dos transformantes pela técnica de hibridizacão revelou uma tendência do vetor linearizado diminuir o número de integracões em relacão ao vetor circular. A integracão foi aleatória e estável nos transformantes analisados. O estabelecimento de um sistema de transformacão para P. brevicompactum é essencial para a manipulacão genética desse microrganismo.