Analysis Of Influencing Factors In Dimorphism And Preliminary Functional Study Of Glyoxylate Cycle Key Genes In Ustilago Esculenta

Abstract:Ustilago esculenta is an endophytic fungus of Zizania latifolia. It might be the interaction of U.esculenta and Z. latifolia that formed the swollen gall, becoming an edible aquatic vegetable-Jiaobai. There are three phenotypes in the field:normal jiaobai, grey jiao, male jiao. They are all closely related to growth situation of U.esculenta in the jiaobai. The previous studies found U.esculenta isolated from the normal jiaobai existed in the form of hyphae, showing M-T (mycelia-teliospore) type, while U.esculenta isolated from grey jiaobai existed in the form of spores, showing T (teliospore) type. The current researches on U.esculenta mainly focus on its morphology and proteomic differences. In this study, influences of medium composition, pH, temperature on the morphology of U.esculenta in vitro culture were analyzed systematically; full-length sequence of two key genes in the glyoxylate cycle were cloned; expression differences of the two genes during morphological changes of U.esculenta were analyzed; we constructed genetic transformation system of U.esculenta, and made a preliminary study on the function of the gene.(1) We compared effects of seven carbon sources, eight nitrogen sources, ten different carbon/nitrogen ratio, four different pH, and three incubating temperature on M-T type and T type U.esculenta isolated from Z. latifolia Trues cultivar’ Longjiao2’. The results showed that M-T type U.esculenta changed to yeast-like form in the mediums of galactose or sucrose as the sole carbon source and in the medium that pH was5. While the T type U.esculenta changed to hypha form when incubating for8-10days in the following conditions:when the mediums with sucrose, glucose, galactose, lactose, mannitol, sorbitol, or fructose as the sole carbon source; or urea, tartaric acid amines, peptone, glycine, ammonium sulfate, potassium nitrate, ammonium nitrate, sodium glutamate as the sole nitrogen source, or the C/N ratio being10:1,20:1,40:1,50:1; or pH value equaling to3,5, or6. Ustilago esculenta grew slowly at20℃, more quickly at28℃,and they both did not change form. While at37℃, the two types of U.esculenta both could not grow.(2)According to the transcriptome data, the genes UeICL(logFC=4.935) and UeMCP(logFC=1.986) which showing significantly differential expression between M-T type U.esculenta and T type U.esculenta were chosen for cloning and Real-time quantitative PCR (qRT-PCR). The results showed total length of UeICL and UeMCP were2059bp and1317bp. They both expressed differently in U.esculenta cultivated in different carbon sources medium at different times. Take gene expression levels of UeICL and UeMCP in fourth day’s culture in PDA medium as a control, the relative expression of UeICL in maltose medium, sucrose medium, glucose medium were17.106,2.929,1.996, respectively; the relative expression of UeMCP in maltose medium, sucrose medium, glucose medium were3.107,2.154,0.881, respectively. The highest expression levels of two genes were both detected in maltose medium.(3) U.esculenta genetic transformation system was constructed through protoplast transformation methods. T-type U.esculenta was cultivated in PDA medium until OD600reached0.8, treated with7mg novozyme/mL in SCS buffer for40min. The protoplast had a good regenerative effect in the PDA medium containing0.6mol/L sucrose. Under the STC-PEG mediation, using linearized pUMa207plasmid to transform into U.esculenta protoplast can gain stable genetic transformant.(4) Construction of UeICL knockout strains. On the basis of pUMa1467plasmid, golden gate cloning technology was used to build the UeICL gene knockout vector. The vector was verified by PCR, sequencing and restriction enzyme digestion. Then the knockout vector was transformed into T type U.esculenta protoplast using the established transformation system.23transformants were gained by four generation of transgenerational screening. PCR identification results showed that among the23transformants,3 transformants possessed both specific sequence of UeICL gene and upstream/downstream boundary sequences of insertion gene, speculating that the original strain had multiple copies of UeICL. Compared with wild-type strains,1positive strain can form hyphae. We concluded that UeICL played an important role in morphological changes of U.esculenta when culticated in vitro.