Mervat Ragab Mansour Diab
Date : 2015
Molecular Genetic Studies on a Midgut Related
Gene in Pink Bollworm (Pectinophora gossypiella).
Vacuolar -H+ ATPase (V-ATPase) is a multi subunit enzyme. It consists of two domains (V1 and VO). V1 domain has eight subunits (A-H) while VO domain has six subunits (a, c, c', c'', d and e). The V-ATPase is an ATP-driven proton pump and is essential to the function of eukaryotic cells. In insects, V-ATPase is located in apical membrane of goblet cells in the midgut. It acts as an energizer of the plasma membrane, driving nutrient uptake, fluid secretion and in some cases alkalizing the gut lumen. The aim of the present study was knocking down the V-ATPase gene(s) using RNAi in an attempt to disturb the food process within the pink bollworm (PBW) midgut and eventually causing the insect death. Based on the conserved regions of the V-ATPase subunits A and D genes in different insects available in the GenBank, degenerate primers were synthesized and used to amplify the two genes. The amplified genes encoding the V-ATPase subunits A and D transcripts were sequenced. The sequencing results confirmed the isolation of the full length genes encoding the V-ATPase subunit A was 2602 bp in length, encoding 618 amino acids. While, the gene encoding V-ATPase subunit D was 1467 bp, encoding 249 amino acids. Three dsRNA fragments were designed, two of them (VATPA756-1155 bp and VATPA347-753 bp) were targeting subunit A and the third (VATPD221-703 bp) to knockdown subunit D. Three bioassay methods were assayed on the larvae of the pink bollworm (PBW), i.e., droplet feeding, mixing the dsRNA with the diet and dsRNA injection. Injection of 200 ng of the three dsRNA fragments (VATPA756-1155 bp, VATPA347-753 bp and VATPD221-703 bp) into the thorax of the third instar caused larval mortality of 46.3, 43.9 and 25%, respectively. Furthermore, treating the larvae with the two dsRNA fragments targeting the V-ATPase subunit A caused shrinkage of the bodies and slower development indicating the starvation effect induced by the treatment with the dsRNA.
Key words: Pink bollworm, Pectinophora gossypiella, RNA interference, Subunit A, Subunit D and Vacuolar ATPase.
Mai M. Labib
Date : 26-4-2015
GENE ISOLATION FROM HALOPHILIC
In Egypt, the degradation of soil by salinity and alkalinity has been classified as a major agricultural problem especially in the northern part of the Nile Delta. This increase in the external salt concentration causes a serious problem to any living cells due to the loss of water. However, it leads to cell death if no countermeasures are taken. Some prokaryotes have developed different strategies to cope with increasing salinities by producing some amino acids such as proline, glycerol, glycine betaine, L-α- glutamate and ectoine. The present study was conducted on five isolates of salt tolerant bacteria that isolated from Egyptian solis. These isolates are producing proline to cope with the external salt stress. Three genes coding for: ornithine cyclodeaminase, proline iminopeptidase (pip) and pyrroline-5-carboxylate reductase (proC) were studied. The five isolates showed good growth in salt media up to (7.7 M NaCl). The microscopic examination in free media and T3 media showed thin and long vegetative cells, sporulated cells were thick and short and some crystal proteins began to appear. In salt media cells appeared with highly super-coiled shape. The existences of the three proline genes were 684 bp from pyrroline-5-carboxylate reductase gene, 757 bp from proline iminopeptidasegene, 674 bp from ornithine cyclodeaminase gene. Total protein pattern for all the isolates showed differences such as appearance of new bands between 72 kDa and 95 kDa in salt media. Proline was measured in all the isolates showing gradually increasing in proline concentration accompanied with regular increase in salinity treatment. The maximum proline concentrations was recorded at 1 M for isolate 2, 2.5 M for isolate 3, 2 M for isolate 4 and 5, and 1.5 M for isolate 6 of salt. The PCR products of the detected genes were sequenced and analyzed with NCBI BLAST program and they hit 19 of different Bacillus thuringiensis. One of them was Bacillus thuringiensis str. Al Hakam (CP000485.1). The sequences of isolate 2 produced significant alignments reached 99% for pyrroline-5-carboxylate reductase gene, 92% for proline iminopeptidasegene, and 91% for ornithine cyclodeaminase gene. Isolate 4 reached 99% for pyrroline-5-carboxylate reductase gene, 93 % for proline iminopeptidasegene, and 92% for ornithine cyclodeaminase gene. Isolate 5 reached 99 % for pyrroline-5-carboxylate reductase gene, 90% for proline iminopeptidasegene, and 91% for ornithine cyclodeaminase gene. Isolate 6 reached 94% pyrroline-5-carboxylate reductase gene, 94% for proline iminopeptidase gene, and 96% for ornithine cyclodeaminase gene.