Molecular and Genetic Study on Pseudomonas fluorescens Siderophore Genes and Trials for Transfer to Soybean (Glycine max L. Merr)

number: 
2456
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Mohammed Abbas Jasim
Supervisor: 
Dr. Nabeel K. Al-Ani
Dr. Kadhim M. Ibrahim
year: 
2010
Abstract:

This study was all done in Southern Illinois University/Carbondale, Illinois, USA in the period 1/4-1/10/2009. Thirty bacterial isolates from Southern Illinois University soybean fields were isolated and grown on King B medium which gave green-yellowish colonies and were fluorescens under UV light. These isolates re-cultured on King B media and then subjected to common identification procedures (Cultural, microscopic, biochemical characteristics, 16S rRNA hybridization with species-specific primers). Result showed that 8 isolates referred as Ps. fluorescens, belong to different biovars, Biovar I (1), Biovar II (1), Biovar III (1), and Biovar V (5) isolate. These isolates were screened for their abilities to produce an iron chelating agents known as siderophores and they all showed capability to produce this important compound, then checked in vitro for its ability to inhibit certain fungi Mucor (ATCC 7933), Aspergillus fumigatus (ATCC 14110), As.niger (ATCC 1004), As. flavus (ATCC 11655), As. oryzae (ATCC 1003), As. Ochroceus, (ATCC 12066), Fusarium oxysporum (ATCC 12581), Rhizopus (ATCC 52751). All isolates exhibited 100% activity against Mucor and Aspergillus fumigates, different activities against As.niger, As. flavus, As. oryzae, As. ochroceus , Fusarium oxysporum , Rhizopus compared with the control. After figuring out these results, one of the isolates (Ps. fluorescens MA4) which displayed the best activity against studied fungi was selected for later experiments and transformation. Later, the type of these siderophore was compared with 4 different known siderophore types (Pyoverdine; Pyochelin; Enterobactin; Pseudobactin) using a new innovative High Resolution Melting –PCR. The results showed that the studied siderophore from Ps. fluorescens is related to Pyoverdine. This siderophore was borne on plasmid; this plasmid was eliminated using acridine orange under a concentration of 5μg/ml. that was proved by running on gel. Then this isolate (MA4) screened for its activity against used fungi, in this case fungi were able to grow in a rate equal to control, and one of the important observations is that this isolate had lost its ability to produce the fluorescent, green yellowish pigment on King B plates. This result was the same in the case of using iron source (FeCl This compound (siderophore) was extracted from bacterial culture and analyzed using High performance liquid chromatography, the result gained indicates that it was consisted of tryptophan, histidine, lysine, and methionine. Transformation of desirable gene was successfully done from Ps. fluorescens to soybean and trials of transgentic plant production was conducted. Many steps were followed to gain these results starting with constructing of vectors pBR322, XAL10, pUC19 and a binary vector pCLD04541, succesful tranformants was just obtained from pCLD04541 while others exhibited no succesful transformation, transformed to A. tumafaciens competent cells, and was selected using AmpR and KanR selection procedures to ensure successful transformants. Soybean plants (variant Forrest) were subjected to the transformants A.tumafaciens using floral-dip procedure and was selected on the base of antibiotic selection and β-glucuronidase (GUS) activity. Successful plant transformants then transferred into 5 cm tubes with a soil containing 25% organic matter and 15% vermiculite in suitable pots and incubated in greenhouse at 24°C. After two weeks of incubation period, plants were checked for growth promotion. Results showed that transgenic plants were averaged 26.3cm in height with weight 80g for transformed while wild type (control) showed an average 22cm long and 68g per plant weight.3 ).