Regulation Of Fungi Immunomodulatory Protein Expression

Fungal immunomodulatory proteins (FIPs) is a small molecule protein with immune regulating activity. Till now, six kinds of FIPs gene have been isolated from G. lucidum, G. tsugae, Flammulina velutipes, G. sinensis, G. japoncium, and G. microsporum, respectively. FIPs have widely immune regulating activity, such as mitogenicity, regulating cell cycle and inducing apoptosis, inducing expression of cytokine and anti-allergic effects, etc.In this dissertation, both the expression and regulation of fungal immunomodulatary protein are explored and discussed as following:1. Cloning, forecasting and identifying the expression regulatory elements of the fungal immunomodulatory protein gene promoter.In this study, the fungal immunomodulatory protein gene of Ganoderma astum, FIP-gas, was cloned for the first time, and then using bioinformatic analysis predicted its amino acid composition, physical and chemical properties, hydrophobicity, secondary and tertiary structure. The results showed FIP-gas contained336bp, and encoded111amino acid residues which named FIP-gas. The molecular weight of FIP-gas is12.4kDa and isoelectric point is4.80. It is a new member of the FIPs family. The amino acid sequence of the FIP-gas was similar with FIP-gsi and FIP-gja. Using genomic walking technology, the promoters of FIP-gas and FIP-glu were cloned from genomic DNA of Ganoderma astum and Ganoderma lucidum and analysed of the expression regulatory elements. In order to verify their function, the promoter were cloned into plant expression vector pCAMBIA1304, and transferred into Arabidopsis thaliana by agrobacterium-mediated transformation.2. Analysis of the fungal immunomodulatory protein gene induced expressionIn this dissertation, FIP-gas and FIP-glu gene expression changes by regulating the ambient pH(4.0,5.5,7.0,8.5,10.0), joining inducer like SA(0mg/L,10mg/L,100mg/L,500mg/L,1000mg/L), MeJA(0μmol/L,10μmol/L,100μmol/L,500μmol/L,1000μmol/L). The expression of this gene was confirmed in transcription level by RT-qPCR technique. The results showed that:under MeJA500μmol/L condition, the expression of FIP-gas and FIP-glu gene were higher respectively. The expressions of FIP-gas gene under SA10mg/L, and FIP-glu gene under SA1000mg/L condition were the highest. And in different ambient pH condition, the expression of two genes were the highest under ambient pH=10.0condition. This research lay a foundation for the study of fungal immunomodulatory protein expression and regulation mechanisms.