Identification of genes associated with Egyptian cotton fiber development using combination of SSH, microarrays and real time RT-PCR

One of the major limitations in the application of genetic engineering or conventional breeding methods in improving cotton fiber is the paucity of information about fiber related genes. Availability of Gossypium barbedense Giza 88 extra-long staple cotton fiber traits provides a unique opportunity to study fiber-associated genes because of its high-quality fiber compared to Giza 90 long staple fiber. To understand the molecular basis of cotton fiber development, we used the combination of suppression subtractive hybridization [SSH], microarrays and real-time reverse transcription-polymerase chain reaction [RT-PCR] technologies to identify the potential genes related to cotton fiber development. Utilizing mRNAs from 15 days post anthesis [dpa] fibers, we constructed a SSH cDNA library from Giza 88 extra long staple fiber as the tester and Giza 90 long staple fiber as the driver. The SSH cDNA library was then screened using microarrays. Microarrays analysis showed that 20 genes were differentially expressed in Giza 88 15-dpa fiber compared to Giza 90 as confirmed by real time RT-PCR. These genes include two beta-tubulins, an actin, a putative kinesin light chain, a cellulose synthase, glycosyl hydrolase family protein, pyruvate decarboxylase, glycoside hydrolase family, GDP-mannose pyrophosphorylase, dynaminlike protein, annexin and a number of genes involved in signal transduction, and protein, nucleic acid metabolism and lipid metabolisms

16S rDNA analysis for characterization of a bacillus isolate and its tolerance profile after subsequent subculturing

The isolation of microorganisms from different highly contaminated environments offers novel bacteria of unique functionality and potential applications in different biotechnological processes. It also entails a variety of genes responsible for microbial tolerance or defense against extreme conditions or xenobiotics present in the media. In a previous work, Bacillus spp. isolated from textile wastewater was exploited in terms of characterization, tolerance to pH, salinity, cold temperature and hydrogen peroxide, and the mechanism of resistance against hydrogen peroxide [Gomaa and Momtaz, 2006]. This isolate was phenotypically identified as Bacillus maroccanus/Bacillus simplex. In this study, the primary aim was to characterize this strain using 16S rRNA partial gene amplification and sequencing. It was assigned as Bacillus simplex TWW-04. DNA-DNA hybridization showed 96% relatedness to Acinetobacter. The tolerance profile of this strain was examined in regard to the four traits previously mentioned. A 36-month subculturing of this strain showed no change in tolerance to pH, salinity and cold temperature, whereas the tolerance to hydrogen peroxide was lost. A sample, frozen for the same duration, showed no change in any of the tested traits. The tolerance alleviation was evident after 24 months of subculturing. Testing the KatA gene responsible for hydrogen peroxide tolerance in the two samples showed higher band intensity in the frozen sample when compared to the subcultured sample. This indicates that the hydrogen peroxide tolerance trait is extrachromosomal and has been lost during successive cell division, while the other traits are chromosomal

Identification of actin-related gene family from G.barbadense Giza 88 cultivar using PCR-based positional cloning

Plant actins contribute strongly in cell cytoskeleton, microtubule filaments regulation and cellulose deposition orientation during cotton fiber cell development, which directly affects fiber quality. Identification of actin-related gene family from the Egyptian cotton is considered a corner stone in future engineering of fiber cell traits. P1-derived artificial chromosome [PAC] library has been constructed for the Egyptian extra long stable variety Giza88. The Giza88-PAC library comprised 8900 PAC clones with 70 Kb average size; representing 0.3 equivalents to the haploid genome [2118 Mb] of Gossypium barbadense. Randomly selected PAC clones were subjected to actin PCR-based screening using GhACT2 degenerate primers, which resulted in 14 actin positive clones. MPAC94 as one of these positives was purified and subjected to physical mapping and PCR-based positional cloning. The results indicated the recovery of a positive MPAC94/EcoRI actin fragment [16.26 Kb], which was confirmed by RT-PCR and sequence alignment at upland cotton database. This study is the first from its kind to identify one gene fragment of the actin-related gene family in Egyptian cotton Giza88 using PCR-based positional cloning technology