Study On The Expression Of Cold Induced Gene In Volvariella Volvacea During Low Temperature Treatment And The Analysis Of Its Correlation To Antolyzation Phenomenon

Volvariella volvacea is an important edible fungus of the tropics and subtropics area. With its delicious taste and nutritious value,V.volvacea has a long cultivation history in China.Like some tropical and subtropical plants,V.volvacea can not be stored at low temperature.When it is stored at 4℃,its mycelia of V.volvacea will autolyze quickly and the fruit bodies will become waterlogged and perish more easily than other artificially cultivated mushrooms,which seriously restricts the production,transport,shelf life and export of V.volvacea,even the development in china and around the world.By far,we still do not understand the mechanism why the mushroom autolyzes at low temperature yet.Real-time quantitative PCR developed in 90s last century has become one of the most important methods to detect the expression of gene for its high accurate,sensitivity, specificness and repetitiveness.Because it is performed in a containing system,and can be read the result directly after PCR without any manipulate,so the possibility of contaminant is greatly reduced.Real-time quantitative PCR technology detects the entire PCR process by measuring the accumulation of fluorescent signals,and quantifies the input DNA target according to a standard curve.This allows the quantitative detection of the target DNA in a straightforward,rapid,sensitive,specific and accurate manner.Contamination is greatly reduced because the sample is amplified and quantified in a closed-tube system without post-PCR manipulations.Then,a cold full-length cDNA library of V.volvacea was constructed using SMART^TM cDNA Library Construction kit.The cold induced gene fragments isolated by DDRT-PCR labeled with DIG,were used to screen the cDNA library.Cold induced fragments of genes were obtained.As part of our previous research on the mechanisms underlying cold temperature autolysis in V.volvacea,Cor1 gene and Cor4 gene were selected from cold induced cDNA bank from V.volvacean by mRNA differential display technology followed with library screen,which showed high homology with according to the results of DNA sequencing and blast in the GenBank. Volvariella volvacea strain V23 and VH3 were tested in this experiment,cDNA were synthesized by reverse transcription reaction with total RNA extracted from mycelia which were treated at 4℃for 0,2,4,6,8,10h respectively.Plasmid containing part of cor1,cor4 and gpd gene were constructed as standard for real-time quantitative PCR to investigate the expression of cor1 and cor4 in strain V23 and VH3 treated under cold stress.In V.volvacea V23,relative expression of cor1 gene exhibited a continuous decrease after stored at 4℃.It reached the lowest,only 15 percent of control,when stored at 4℃for 6h.Then it increased slightly.In V.volvacea VH3,relative expression of cor1 gene showed an increase at the treatment of 2h,6h and 8h,which were 1.62,1.11 and 1.42 folds higher than those of the control.Its lowest expression took place at the treatment of 10h,40 percent of control.The results showed that the expression of cor1 at 4℃reached the maximum at 0h,decreased gradually at 2h,4h and 6h,then increased gradually at 8h and 10h in strain V23; expression of cor1 at 2h was higher than that at 0h,and decreased at 4h,then increased gradually at 6h and 8h,but decreased at 10h in VH3 during low temperature treatment at 4℃.In V.volvacea V23,relative expression of cor4 gene decreased gradually after stored at 4℃.Its expression also reached the lowest,only 25 percent of control,when stored at 4℃for 6h.In V.volvacea VH3,relative expression of cor4 gene showed an increase at the treatment of 2h and 4h,which were 1.06 and 1.1 folds higher than those of the control.Its lowest expression also took place at the treatment of 8h,more than 40 percent of control.The expression of cor4 at 4℃reached the maximum at 0h,decreased gradually at 2h,4h and 6h, then increased gradually at 8h and 10h in strain V23;expression of cor4 at 2h was higher than that at 0h,and reached the maximum at 4h,then decreased gradually at 6h and 8h,but decreased at 10h in VH3 during low temperature treatment at 4℃.The results indicated that cor1and cor4 gene might play a role in cold endurance of V. volvace,which which would lay good foundation for assigning a role for the gene in the low temperature autolyzation phenomenon.