Evaluating Resistance for Hygromycin B

Transforming of  p ostaligo were obtained and planted on the myg plates in which the hygromycin b was absent. Its cultivation was then performed at 28 degree c. after cultivation it was then incubated on the plates that contain myg. Vigorous growth of tranformants were occurred and the transformation that were obtained was so stable.

Amplification of hph gene by PCR

For the purpose of transformation DNA,s of Transformants were obtain and used as a template. Then

·         After this plasmid was then transfused into p. ostaligo

·         The band that was amplified it were appear as PCR product on agrose gel.

·         But the band was absent when the p. ostaligo was not transformed.

·         The appearance of this band was used as confirmation test for the transforming of plasmid.

Analysis by Southern Blotting

In order to confirm that the plasmid is present in the p.ostaligo Sothern blotting was performed.

·         In southern blotting hph gene was used as probe

·         The data that remain un transformed it can not contain any hybridization signal.

·         While the data that was transformed contain hybridization signal

·         Dna region was also present in it.

·         The DNA that was introduce its integration was occurred in chromosomal Dna.

·         The mycelia that was cultivated in liquid medium in which hygromycin b was absent from this mycelium dna sample was formed

·         The transformation that were that were obtain were stable

 

Evaluation of expression of GFP Gene

The expression of gfp gene in the mycelium was very difficult and it should that changes must be performed in gfp gene. Experiments were performed that gfp gene were ready to enter or transformed in the p .ostaligo a gfp gene was obtained from a vector and a permotor. And both of these were fused to obtain new expression of vector which was then classified into the p, ostaligo. After this transformation the analysis of the protoplast was performed by the use of florescent microscopy in which different rays of different wave length were used. The purpose of this use of florescent microscopy was to evaluate the transformation efficiency. At that time the transformation was 150 transform ants and in control no green light was detected.

After the time period of 4-6 hours the green light that was observed were transformed into the protoplast. After the time period of 19 hours the green light that was observed was weak and after the few time of 19 hours the green light that was observed contain maximum intensity. After reaching to its maximum intensity the green light started to fade . after the time duration of thirty ours the gfp protein might be degraded.

Because of methaylation gfp protein might be inactivated. This modifiable gfp protein is suitable for expression in the plants and it is not suitable for expressing in fungi. There is a need to modify the gfp protein so that it can be expressed in the p. ostaligo that was of high efficiency. In our transformation method the prominent markers were gfp and hph gene. Our method must show resistance to hygromycin b and after the evaluation reputation by five times our transformation then will be resistant to hygromycin b.

Above process for pfg of the transformation p. ostaligo. After this procedure another type of mushroom which we can discus by the pfg protein transformation. The mushroom is popular would be and its popularity is because of its successful cultivation.

In the bioremediation process p. ostaligo is also used in order to remove the contamination and also to remove the heavy metals. Despite of its demands and uses only few biological tools are available for its modification.

·         There is a need for the development of highly efficient method for the transformation of gene.

·         Different types of plasmids and strains were prepared.

·         These strains were obtain from the  denature agrose

·         After this these strains were cultivated at 28 degree c.

·         Its shaking was performed for 10 days.

·         Speed of shaking was 130 rpm.

·         After this isolation of genomic Dna was performed.

·         Two pairs of primers were design for terminator and permotor.

·         The target bands were inserted.

·         After this sequencing of permotor was performed and it was then analysed.

Preparation of Protoplast

·         For this purpose incubation of mycelia was done for one week

·         Then its filtration was performed.

·         Enzyme was prepared and it was then purified.

·         It can also be washed with the buffer.

·         After washing the protoplast were suspended.

·         A mixture was prepared by mixing it with plasmid.

·         This mixture is also add in the ptc buffer.

·         This mixture was placed for the 20 min.

·         Then again Ptc buffer was added in the mixture and its incubation was performed.

·         The duration of the incubation period was 25 minutes.

·         Then mixture was done with the STC buffer.

·         The stc buffer was spread on the medium and in our medium hygromycin b was present.

·         Again incubation of protoplast were performed for the purpose of regeneration.

 

Molecular Analysis

The procedure of molecular analysis was given below

·         Bullet blander was used for the mixing of mycelia pellet.

·         So it can be used for the PCR analysis.

·         Mini kit was used to extract the our DNA.

·         RNA mini kit were used to extract the total amount of RNA for the purpose of rt-pcr analysis.

·         Then a primer pair was generated.

·         Then after this the transcription of the transformats were performed.

·         Positive transformants acted as the template.

·         A hyphe of p. syringe was fixed on the glass slide.

·         After this hygromycin was added and its growth were observed.

·         After the  five days the samples that were obtained they were analyzed.

·         Technique using florescence microscopy that uses many types of different wavelength of rays.

·         After analysis images were obtain and we get final results.

Discussion

A fragment of 1065 base pairs were subjected to amplification by using pcr. For the transformation of the method of the e syringe the desirable thin is antibiotic resistance. For determining the maximum concentration of hygromycin that is used in the screening analysis. For this purpose spreading of protoplast is done on the rm or gym plates. In Rm plates different concentration of the hygromycin b was present. The protoplast regeneration was inhabited due to the presence of the hygromycin b.

Protoplast then transformed with the help of plasmid in the presence of the cacl2.it can be spreads until the transformants were obtain that were hygromycin resistant the points are given below to explain this procedure

·         After the time period of three weeks the non hygromycin resistant transformants were obtain.

·         But after the period of 30 days the transformants that were obtain on the RM plates were hygromycin resistant.

·         This transformants method was applied in many basidimycytes.

·         The results that were obtained were of the different efficiencies.

·         In starting experiments the peg transforming method efficiency were low.

·         But after the gradual time period its efficiency was increased.

·         After obtaining five generations the transformants were analyzed by PCR.

·         The transcription of the transformants was then confirmed by using the RT_PCR.

·         Transcription level was varied among different transformants.

·         Highest and lowest levels were shown by Pe B11 and Pe-B12.

·         Green light is shown by all transformants of p. syringe.

·         For the study of gene expression different fungs were visualized.

·         Green light and hygromycin resistance become the fusion gene.

·         It is very beneficial mushroom and it has importance in many asian and Chinese countries.

·         This species can be better visualized in biotechnological and agricultural aspects.

·         An antibiotic marker was used for the successful development of the anti resistance transformants expression system.

·         Then after this successful expression of hph gene was obtained.

·         Transcriptional levels were visualized by using the RT- PCR technique.

·         This method can be successful in proceeding to the different fields.

Now we will discus the polyetyleneglycol mediated transformation in the fungal pathogens.

·         In this process genomic dna was transformed into an organism.

·         As DNA is transformed into genome of organism so the changes take place in the genome.

·         There are three chances that these changes might be transformed in to the new generations.

·         This transformation method was successfully transformed in to many fungal species.

·         Dna is exposed in the presence of the calcium ions and greater concentration of peg.

·         This process was letter on applied on the fungus and plant species.

·         This process in pathogens is very successful in greater extent.

·         In order to over come the deficiencies associated with the peg method.

·         Some other methods like as the electroporation method was used.