Mushroom growth promoting gene profiling from Bacteria
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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

Mushroom growth promoting gene profiling from Bacteria

By Mr. Aradhya

Molecular genetics lab


Mushroom growth promoting gene profiling from bacteria

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

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.


Mushroom growth promoting gene profiling from bacteria

Materials and Methods

Bacterial isolation by Serial dilution

Soil sample

Centrifuge the culture

Bacterial culturing

Growth promotion checking

Control Supernatant added to plate


Mushroom growth promoting gene profiling from bacteria

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


Mushroom growth promoting gene profiling from bacteria

TLC of ethyl acetate fraction

IAA STD

IAA Sample 14


Mushroom growth promoting gene profiling from bacteria

Bioassay by plate Method

Ethyl acetate Extract

TLC Fraction

control

Sample


Mushroom growth promoting gene profiling from bacteria

IAA SYNTHESIS

Tryptophan

Tryptamine

N-Acetyltryptophan

Indolepyruvic acid

Indolyl-lactic acid

N-Acetyltryptamine

Indolylacetaldehyde acid

Tryptophol

Acetyltryptophols

Indole acetic acid

Indolecarboxaldehyde

Anthranilic acid


Mushroom growth promoting gene profiling from bacteria

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


Mushroom growth promoting gene profiling from bacteria

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.


Mushroom growth promoting gene profiling from bacteria

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.


Mushroom growth promoting gene profiling from bacteria

Result :

Culture supernatant

Ethyl acetate extract

Further confirmation was done with TLC and HPLC.

Bioassay


Mushroom growth promoting gene profiling from bacteria

Future work

Agrobacterium mediated gene transformation

Agro bacterium containing our gene

Transformation of mushroom

Bottle culture of transformed mushroom


Mushroom growth promoting gene profiling from bacteria

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.


Mushroom growth promoting gene profiling from bacteria

Thank you


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