Preliminary Functional Analysis Of Several Genes Related To Mycorrhizal Formation From Cymbidium Hybridum

Orchidaceae is a kind of rare and endangered plants with high ornamental value and medicinal value.Because of the growth shorten,Orchids need to beassociated with the orchid mycorrhizal fungi to supply nutrition under natural conditions.In order to understand the essence of orchid mycorrhizal(OM)formation mechanism,In this experiment,we dig into a few induced expression genes by the orchid mycorrhizal fungi through the analysis of the transcriptome data from Cymbidium hybridum,and the molecular characteristics of these genes were studied by means of the existing molecular techniques.The aim of this study was to find the key genes for the formation of orchid mycorrhiza,and to pave the way for the subsequent molecular studies in orchid.The main results of this paper are as follows:(1)Based on theCymbidium transcriptome data,an PUP-like gene,CyPUP3 and three SWEET-like genes,CySWEETs,were selected for this experiment.These genes were cloned and the PC R products were Sanger sequenced and used for blastn with the assembled sequences,which revealed these ESTs were reliably assembledand could be used for further study.(2)The transmembrane prediction and phylogenetic analysis identified that the amino acid sequences of these genes have the typical transmembrane domain,among them,CyPUP3 has 10 transmembrane domains,and is highly homologous with OsPUP3;CySWEETs has 7 transmembrane domains,similar to OsSWEET4 and OsSWEET14 from Oryza sativa.(3)The result ofSemi quantitative and q RT-PCR revealed that the transcripts of CyENT3,CyPUP3 and CySWEETsare not only induced by Orchidaceous Rhizoctonias,but also by a new type of orchid fungus,Piriformospora indica.(4)Transient CyPUP3 expression vector was constructed through Gateway system and used for transformation of benthamiana.LCM observation showed that the expressedprotein of CyPUP3 located in the plasma membrane.Based on the results abovementioned,it is infered that the protein CyPUP3 is a kind of trans-membrane transporter.(5)In the previous research,CyENT3 is presumed to be a balance nucleoside transporter.To verify this function,CyENT3 was transformed into Saccharomyces cerevisiae mutant BYL042 c,a lcking endogenous transport system strain.Cultured on synthetic dropout medium containing nucleoside,compared with the control group,C yENT3 can grow on the medium containing adenosine instead of medium toxic 5-fluorouracil,the experimental results shows that CyENT3 can effectively transport nucleoside.(6)CySWEET4a and CySWEET4 b are predicted to transport monosaccharides,In this test,these two genes was transformed into Saccharomyces cerevisiae mutant EBY.VW4000 lacking transmembrane monosaccharide transporterscultured on synthetic dropout medium containing glucose,the yeast containing two genes can grow on the medium containing glucose and grew well,while the control group is very weak,the results shows that C ySWEET4 a and CySWEET4 b can transport monosaccharides.