Clone And Expression Analysis Of Key Genes Of CAMP Pathway And MAPK Pathway In Ustilago Esculenta

Zizania latifolia is the second largely cultivated aquatic vegetable in China.It is widely cultivated in the drainage area and southern of the Yangtze River and hasimportant economic benefits. The enlargement of Z.latifolia is closely related toinfection and interaction of its endophytic fungus, Ustilago esculenta. The hyphamorphology of U.esculenta is the key factor in determining the commodity andedibleness of Z.latifolia. Proteomics and expression profiling analysis showed thatdifferentially expressed UePka and MAPK existed between the yeast-form andhypha-form. Both genes actively participated in the process of hyphae formation andthe transformation from yeast-form to hypha-form in fungi.In this study, the yeast-form and hypha-form U. esculenta isolated fromZ.latifolia were used as the research material. Using molecular techniques andbioinformatics, the gene structure of cAMP pathway key gene (UePka) and MAPKpathway key genes (UeErk, UeMkk, UeSsk), expression differences in differentcarbon sources and genes interaction were studied. The results were as following:1. Cloning and sequence analysis of UePka, UeErk, UeMkk and UeSsk of U.esculentaAccording to the transcriptome database of U. esculenta, the primers of UePka,UeErk, UeMkk and UeSsk were designed. cDNA full-length sequence of UePka,UeErk, UeMkk and UeSsk were obtained by RACE. Sequence homology analysisshowed that, UePka, UeErk, UeMkk and UeSsk were highly homologous with theother species (especially other species of the Ustilago).The UePka gene of U.esculenta has a cDNA full length of1949bp, containing aopen reading frame of1230bp encoding410amino acids. Predicted molecularweight was approximate45.78kDa. Theoretical isoelectric point was8.29. The genecontains a5‘untranslated region (5’UTR) of555bp and a3‘UTR of161bp.The UeErk gene of U.esculenta has a cDNA full length of1828bp, containing aopen reading frame of1659bp encoding553amino acids. Predicted molecularweight was approximate62.05kDa. Theoretical isoelectric point was6.60. The gene contains a5‘untranslated region (5’UTR) of7bp and a3‘UTR of10bp.The UeMkk gene of U.esculenta has a cDNA full length of2204bp, containinga open reading frame of2040bp encoding680amino acids. Predicted molecularweight was approximate72.68kDa. Theoretical isoelectric point was8.09. The genecontains a5‘untranslated region (5’UTR) of54bp and a3‘UTR of107bp.The UeSsk gene of U.esculenta has a cDNA full length of7625bp, containing aopen reading frame of5871bp encoding1957amino acids. Predicted molecularweight was approximate214.54kDa. Theoretical isoelectric point was5.19. Thegene contains a5‘untranslated region (5’UTR) of1669bp and a3‘UTR of82bp.According to the cDNA full-length sequences of UePka, UeErk, UeMkk andUeSsk, we designed primers for genome sequence amplification and obtainedgenome sequences of four genes. By compared with the cDNA sequences, we foundthat only UePka contains a intron of113bp.2. Different expressions of UePka, UeErk, UeMkk and UeSsk of U.esculenta indifferent carbon sourcesUsing real-time PCR, expression pattern of four genes were detected inmediums with different carbon sources. The results showed that compared to maltoseas solely carbon source, the expression of UePka, UeErk, UeMkk and UeSsk hadsignificantly risen when incubating in medium with sucrose as solely carbon sourcefor four days. When incubating time elonged to eighth, twelfth, sixteenth days, nosignificantly expression variation could be found for UePka, UeErk, UeMkk andUeSsk between mediums with sucrose or maltose as the solely carbon source.Microscopic observation showed that the fourth day in culture using sucrose ascarbon source, the yeast-form U.esculenta began to branch and became longer, onthe eighth day the elongation and branching were more obvious, like the hypha-form.It illustrated that different carbon source can induce the morphologicaltransformation of U.esculenta. We deduced on the varied expression levels andrelation to morphological changes that four genes might be related to themorphological transformation of U.esculenta in varying degrees.3. Interaction analysis of UePka, UeErk, UeMkk and UeSsk of U.esculentaProkaryotic expression plasmid of UePka, UeErk, UeMkk and UeSsk wereconstructed, they are pPorf-pET-28a, pEorf-pET-28a, pMorf-pET-28a and pSorf-pET-28a. Expression inducing conditions were realized. In this study,appropriate conditions to inducing expression were selected:37℃,180rpm, shakeculture1.5-2h; while OD600was0.4-0.6, added IPTG to a final concentration of0.2mM, and continue culturing5-7h. Prokaryotic expression products were analyzed bySDS-PAGE, specific bands were consistent with the predicted size.Ni-NTA affinity resin was used to purify the protein. SDS-PAGEelectrophoresis analysis showed that, UEPKA, UEERK, UEMKK and UESSK had arelatively purified single band. UEPKA had a high affinity with Ni-NTA and itspurification effect was the best.Protein interaction analysis showed that, interaction could be detected betweenUEPKA and total protein, UEERK and total protein. No interaction detected betweenUEMKK and total protein, UEPKA and UEMKK. Detailed interaction study betweenpairs of proteins remains to be revealed.