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Mushroom growth promoting gene profiling from Bacteria. By Mr. Aradhya Molecular genetics lab. Introduction. Mushrooms are being used as food and medicine since time immemorial.
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Mushroom growth promoting gene profiling from Bacteria By Mr. Aradhya Molecular genetics lab
Introduction • Mushrooms are being used as food and medicine since time immemorial. • Their cultivation on extensive scale can help solve many problems of global importance such as protein shortage, resource recovery and reuse as well as part of environmental management. • Presents the latest cultivation and biotechnological advances that will help ensure mushroom cultivation without pollution and provide mushroom products with health enhancers • Edible mushrooms are a valuable nutritional supplement for us, and how they and their derivative products can serve as important pharmaceutical/medicinal agents
Brief Introduction about Pleurotus erynigii • The Pleurotus sp. is one of the most widely cultivated mushroom in the world. Wild Pleurotus erynigii collected in southern Europe, North Africa and central Asia. • It has many subspecies and similar taxa such as P.fuscus var. from china. • Since 1997, Pleurotus erynigii has become a popular crop due to its unique flavor, shape and texture as a reason its been cultivated in many parts of Korea. • Mushroom growth Stimulation limited by several factors like media , abiotic parameters –temperature, humidity , pH and carbon di-oxide concentration. • In the current study among Agaricus sp. and Pleurotus sp. showed that many micro-organisms promote vegetative and reproductive growth.
Materials and Methods Bacterial isolation by Serial dilution Soil sample Centrifuge the culture Bacterial culturing Growth promotion checking Control Supernatant added to plate
Becterial characterization 16srDNA gene amplification For bacterial characterization Phylogeny tree construction 1.B.cereus 2.Comamonas 3.Pseudomnas 4.Bacillus sps 5.Ochrobactrum Isolation of Growth promoting compound Culture supernatant Butanol extract Ethyl acetate extract
TLC of ethyl acetate fraction IAA STD IAA Sample 14
Bioassay by plate Method Ethyl acetate Extract TLC Fraction control Sample
IAA SYNTHESIS Tryptophan Tryptamine N-Acetyltryptophan Indolepyruvic acid Indolyl-lactic acid N-Acetyltryptamine Indolylacetaldehyde acid Tryptophol Acetyltryptophols Indole acetic acid Indolecarboxaldehyde Anthranilic acid
Profiling of IAA gene Bacillus lincheniformis Specific Primers for IAA gene Lamda/P 1 2 3 4 5 6 7 Positive Control 2.2Kb Colony-PCR result- Clone in pGEM-T-vector
Quantification of IAA Clone containing IAA gene was propagated overnight in 5ml of M9 salts minimal media Supplemented with tryptophan ; 0 , 50 , 100 , 200 , 500 ug/ml After 42hr , the density was measured at 600nm, Bacterial cells were removed from culture by centrifugation, 1ml aliquot of the supernatant was mixed vigorously with 4ml of Salkowski’s reagent. Allow to stand at room temperature for 20min before the absorbance at 535 nm. The concentration of IAA in each culture medium was determined by comparison with a standard curve.
Result : • As the tryptophan concentration increases the cell density and the production of IAA also increases. • After adding Salkowski’s reagent the supernatant color turned in to reddish pink. • The maximum production of IAA was observed test tube containing 500ug of tryptophan, beyond that there was a inhibition of bacterial growth.
Result : Culture supernatant Ethyl acetate extract Further confirmation was done with TLC and HPLC. Bioassay
Future work Agrobacterium mediated gene transformation Agro bacterium containing our gene Transformation of mushroom Bottle culture of transformed mushroom
Conclusion : • Till these days we studied more about plant and microbial interactions but there are no reports on Bacterial and Fungi interactions. • More over the very interesting thing in this is how the Bacteria and Mushroom will interact • Mushroom growers in all countries will learn how to produce greater and more reliable yields. • Finally edible mushrooms are a valuable nutritional supplement for us, and how they and their derivative products can serve as important pharmaceutical/medicinal agents. • Further work is needed to analyze final derivative structure. • Gene should be transformed in many other edible mushroom to commercialize.
