Cloning And Functional Analysis Of PsICL1and PsG6PDH1from Puccinia Striiformis F. Sp Tritici

Puccinia striiformis f. sp. Tritici (Pst.), the causal fungus of wheat stripe rust, is anobligate biotrophic basidiomycete. Urediniospores are the most common spore involved in theepidemiology of the disease. Basic nutrient metabolism plays a very important role in thegermination and infection of wheat stripe rust.But few studies have investigated to date aboutthe glyoxylate cycle and the pentose phosphate pathway of wheat stripe rust.In this paper, wedid research about the PsICL1gene and the PsG6PDH1.The main results are as follows:The sequences of PsICL1and PsG6PDH1were checked out from a previously describedcDNA library. The open reading frame (ORF) of PsICL1, consisting of1611nucleotides,encodes a polypeptide of536amino acids; PsG6PDH1is1497bp, and encodes498aminoacids.The expression pattern showed that the PsICL1was continuously down-regulated, theexpression level of PsICL1was extremely low after24hpi. PsG6PDH1showed highexpression level during6-168hpi compared to urediniospores, but down-regulated after168hpi.The function of the two genes were identified through complementation of the S.cerevisiae mutant, the result showed that the PsICL1and the PsG6PDH1partially rescued themutant in growth.The Pst. urediniospores and germinated tubes were stained with Nile red and observedvia confocal microscopy, we observed that translocation of lipid bodies along the developingtubes. The carbohydrate synthesis analysis showed that there was a significant increase in thecontent of anthrone-positive material in the germ tubes. When isocitrate lyase enzyme activitywas measured, highly enzyme activity in geme tubes was observed. When treated theurediniospores with3-NP, nearly100%of the urediniospores loss of germination, these allindicated that ICL plays a key role during Pst. germ tube formation. And the prokaryoticexpression technology was used to obtained the PsICL1protein.When expressed the gene PsG6PDH1in S. cerevisiae, SDS-PAGE, westernblot andenzyme activity analysis all showed that the PsG6PDH1was successfully expressed in S.cerevisiae. This work analysed the function of PsICL1and PsG6PDH1, and initially determined theaffect on growth of P. striiformis f. sp. Tritici. The results provides essential information thatsheds light on P. striiformis f. sp. Tritici nutrient metabolism, may helps us to understand theinfection mechanism of P. striiformis f. sp. Tritici, and provide theoretical basis for furtherresearch.