Cloning And Function Analysis Of Phosphate Transpoter Genes From A Dark Septate Endophyte(DSE)

Phosphorus is one of the most important nutrient elements for life. However, the amount of soluble phosphorus is very small in terrestrial ecosystem, particularly in farmland ecosystem. A large number of phosphatic fertilizer is applied to make up for the immobilization and loss of soluble phosphorus in farmland. Microorganisms play a crucial role in contributing to the adaptation of plants to soil environment with low soluble phosphorus. Dark septate endophytes (DSE) are ubiquitous fungi that colonized in different plant roots, worldwide distribution and especially in stressed environments. Although their classification and ecological function is not very clear, DSEs have been reported to stimulate plant growth under specific environmental conditions and enhance plant tolerance to variety of environmental stresses by improving the supply of organic and inorganic nutrients. However, reports on the mechanism of absorption and transportation of phosphorus by DSEs are rare. Phosphate transporters are a family of proteins widely distributing from plants to microorganisms, and reported to be divided into two groups:low and high affinity phosphate transporters. Cloning and function verification of fungal phosphate transporter genes have been reported only in a few species, but not yet in DSEs. To understand the phosphate transporter gene function of DSEs would contribute to revealing the molecular mechanism of DSEs phosphorus nutrient, and the ecological function of DSEs at gene level, and subsequently providing fundamental basis for further study on how DSEs promote phosphorus uptake of plants. Based on transcriptome sequencing analysis of the DSE Exophiala pinsciphila (H93), cDNA coding sequences of four phosphate transporter genes EpPTl, EpPT2, EpPT3and EpPT4were obtained in this thesis. Function verification of two genes were further studied. Results are as below:1. Cloning of phosphate transporter genesBased on the transcriptome sequence of DSE strain H93, bioinformatics of genes labeled as phosphate transporter were analyzed. EpPTl, EpPT2, EpPT3and EpPT4were selected as target genes, subsequently cloned from H93. cDNA coding sequences of the four genes were confirmed and obtained.2. Function verification of EpPTl and EpPT2The expression of proteins encoded by cDNA-ORF region of EpPT1and EpPT2in yeast heterologous expression systems was tested, and the results indicated that EpPTl and EpPT2were able to complement phosphorus uptake mutant yeast lacking of PHO84. The growth of yeasts transferred by EpPTl was better than that of EpPT2.These results suggested that EpPT1and EpPT2could help the defective yeast to transport phosphorus.And under the conditions of low phosphate concentration,the phosphate transport capacity of EpPT1was significantly stonger than EpPT2.