Cloning And Analysis Of Glyceraldehyde-3-phosphate Dehydrogenase Gene And Promoter From Inonotus Obliquus

Inonotus obliquus belongs to Hymenochaeetaceae,Inonotus,which contains polysaccharides,inooidiol,fuscoporine,and oxygenated triterpenes.It has many medicinal functions such as anti-cancer,hypoglycemic,hypolipidemic,anti-oxidation and so on.GPD is one of the most basic enzymes in life-sustaining activities and participates in the process of glycolysis in tissues.GPD is commonly used in the stress of fungi and the identification of strains.The GPD promoter contains TATA box,CART box and a multi CT sequences,and has a strong function of regulating the transcription of heterologous genes.At present,the GPD promoter has been used as an exogenous gene promoter to transform Lentinus edodes,Pleurotus ostreaus,Volvariella volvacea,Agaricus bisporus,Ganoderma lucidum.The results are as follows:1.The full length sequences of gene were successfully cloned by PCR and RACE technology.The results show that the full length of cDNA sequences of the GPD gene were 1120 bp,encoding 339 amino acids.2.The amino acid sequence analysis showed that the IoGPD protein has no signal peptide sequence and is located in the cytoplasm,belonging to type I in the glyceraldehyde-3-phosphate dehydrogenase family.3.Encoding the amino acid sequences of IoGPD gene were carried out on similarity search.The results show that the IoGPD have high similarity with other fungal GPD.IoGPD protein has the highest similarity with Sanghuangporus baumii and Fomitiporia mediterranea which is 88%.4.The GPD promoter sequence of 446 bp was cloned from Inonotus obliquus strain JL01 using chromosome walking technique.5.The analysis showed that the GPD promoter sequence has two transcription initiation sites at 152 bp and 160 bp,containing TATA box,CAAT box,CCAAT box,and important elements such as LTR,02-site,circadian,Sp1,and TCCC-motif.