Effect Of Vitreoscilla Hemoglobin Gene Expression On The Physiological Characteristics Of Lentinus Edodes

Lentinus edodes is one of the most popular edible fungi.It has wide cultivation area,high nutritional value,rich flavor and delicious taste.Oxygen is one of the important environmental factors in the production of edible fungi,especially for Lentinus edodes.In this study,Agrobacterium tumefaciens-mediated transformation was used to transfer the Vitreoscilla hemoglobin gene vgb into Lentinus edodes.The oxygen-intaking capability and the other physiological characteristics of the Lentinus edodes strain expressing vgb gene were investigated.(1)The CDS regin of Vitreoscilla hemoglobin gene sequence was found from Gene Bank and be synthesized.The recombinant plasmid p BHg-vgb-gpd was constructed using plasmid p BHg-BCA1-gpd as original plasmid.By Agrobacterium tumefaciens-mediated transformation of edible fungi,the recombinant plasmid was transferred into Agrobacterium tumefaciens EHA105 first,then the Agrobacterium tumefaciens EHA105 containing plasmid p BHg-vgb-gpd was induced toxicity and co-cultured with mycelium of Lentinus edodes L26.The co-cultured mycelium was transferred to the screening medium containing hygromycin and cefotaxime.Then extract the genome of the growing hyphae to make PCR detection,the results of PCR showed that the target gene had been successfully integrated into the recipient strain L26.(2)After a series of genetic stability and PCR validation,four transformants(two of which were purified protoplasts)were obtained.RT-PCR analysis q PCR analysis of the transformants confirmed the successful transcription of vgb gene in Lentinus edodes and calculated the relativeexpression of the target gene.(3)By the CO differential spectroscopy analysis,we found that there was a characteristic absorption peak appeared at about 419 nm,but there was no peak value in the samples without CO treatment and wild type control.The results showed that the bioactive hemoglobin was successfully expressed by the transformant strain.(4)By plate culture,plastic bag planting and tube culture,we get the results that the expression of vgb gene in the four transformants increased the growth rate by 17.2% ~61.9%.The growth rate of the transformed strain was faster than that of the wild type,especially under the condition of relative anoxic environment,and the mycelium was thick and stronger.(5)The polysaccharide production of liquid strain cultured in shaking flask was detected.The results showed that the polysaccharide production ability of transformed strain was higher than that of wild type at the same time.The growth rate of four transformants increased by 19.0%,16.9%,24.0% and 12.9% respectively.The activity of Carboxymethyl cellulase,laccase and amylase in the culture material filled with mycelium was determined,and it was found that the activity of carboxymethyl cellulase didn't change obviously.Laccase activity and amylase activity were increased by 17.7% ~ 40.3% and 16.7%~37.9% respectively,which indicated that the ability of mycelium to decompose lignin and polysaccharides in culture material was enhanced.The results of above showed that the expression of vgb gene in Lentinus edodes could increase the growth rate of mycelium,the yield of polysaccharides and the ability of substrate utilization enzyme,which could shorten the cultivation period of Lentinus edodes and improve the yield and quality of Lentinus edodes.