cDNA library construction and isolation of genes expressed during the presymbiotic stage of fungus: glomus intraradices
Student name: Ms Aditi Jain
Guide: Dr Deepti Gupta
Year of completion: 2010
Host Organisation: Centre for Mycorrhizal Research, Biotechnology and Management of Bioresources Division, The Energy a
Supervisor (Host Organisation): Dr Alok Adholeya
Abstract: Mycorrhizae are symbiotic associations between plant roots and fungi of phylum Glomeromycota.AM fungi can contribute to the increasing survival and fitness of plants to limiting environments due to their ability in improving nutrient uptake and abiotic and biotic stress tolerance. Despite their ecological significance, the molecular and genetic mechanisms controlling the AM development and functioning are largely unknown. The present study involved the construction of cDNA library from the presymbiotic stage of model AM fungi Glomus intraradices to get a snapshot of the transcriptome apparatus induced during this stage. Total of 300 clones from cDNA library were screened. Nine putative full length ORF sequences were obtained, which included Copper transport protein, DNA binding protein, Trm112p like protein, DUF962 domain containing protein, phosphotransferase protein, dTDP glucose 4,6 hydratase and three novel proteins. Blastx results assigned functions to other partial ORF sequences also like cytokine-induced apoptosis inhibitor, small subunit protein of Acetolactate synthase, MAT2, Zygote specific protein, Oxysterol binding protein, transposase and autophagy related protein. Out of total 25 sequences, 12 translated sequences showed the presence of conserved domains. One of the interesting EST sequence encoding phosphotransferase protein with function in osmotic stress signalling was found important and primers were designed based on this sequence to conduct 5' and 3' RACE to isolate the full length cDNA.The total length of cDNA obtained was 643bp which included a coding sequence of about 324 bp and 3'UTR of 312 bp. The cDNA is partial as it is incomplete from 5'end.Coding sequence of this protein was translated into a protein to do primary,secondary and tertiary structure analysis. Finally a phylogenetic tree was also constructed using this protein sequence from other fungal members which displayed that Glomus intraradices is closest to Schizosaccharomyces pombe as they were present in same cluster.The protein is important as it suggests a signalling mechanism activated in AM fungus as a result of osmotic stress which helps the both fungus and plant fight water imbalance.
Keywords – Arbuscular mycorrhizal fungus, Glomus intraradices, cDNA library construction, RACE, Expressed sequence tags, Presymbiotic stage, phosphotransferase gene