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

Orchid mycorrhizal(OM)is a typical endophytic fungus,has long been considered to be parasitic on fungi.In resent years,Cameron and his workmates revealed that it exists a mutualistic symbiotic relationship between green Orchidaceae and mycorrhizal fungi(OMF)by carrying out the radioactive isotopes labeling experiment.Mycorrhizal fungi assist plants to colonize in the terrestrial environment and provide mineral nutrition to plants,while the host feedback carbohydrates to fungi.However,how the carbohydrates are transported to fungi? Which factors regulate the process? By studying the regulation mechanism of cloning,localization and expression of sugar transporter gene,it well be provided substantial evidence for constructing OM mechanism of mutualistic symbiosis of green Orchidaceae plants.In addition,it is significant for the protection and cultivation of precious Orchid varieties.The experimental group have proved that Cymbidium hybrids can form mycorrhizal symbiosis structure with Piriformospora indica in the culture medium DE,and the fact is that the sugar transporter of CySWEET14 CySWEET4 a and CySWEET4 b gene which are up-regulated,which induced by mycorrhizal fungi.in the process of the mycorrhizal formation.The heterologous expression of CySWEETs in the Arabidopsis thaliana was Conducted in this research,which verified the sugar transport activity and the possible functions of SWEETs family in the establishment process of symbiotic relation with plants.The main research results in this paper as follows:(1)By regulating the Nitrogen forms,carbon source and the concentration of phosphorus,we screened a beneficial symbiotic culture medium which is suitable for the symbiosis between P.indica and C.hybridum,A.thaliana.The experimental results show that in LW01 c culture medium(The ratio of nitrate nitrogen and ammonium nitrogen is 1:2;Total inorganic nitrogen 9 mM,the concentration of phosphorus 5 mM,with 0.25 g/L yeast extract and 5 mM soluble starch),P.indica can form symbiotic structures with those hosts and promote their growth.(2)On the basis of LW01,the effect of carbon source addition on the symbiosis medium between C.hybridum and P.indica was compared.The results showed that medium adding sucrose and soluble starch are conducive to the fungal colonization,but P.indica have inhibitory effect on the growth of the cymbidium in the LW01 medium adding sucrose,and roots were grown malformed and negative geotropism,the browning of roots is serious,the spore was hardly colonized in the cortex of C.hybridum in the LW01 medium without carbon source.(3)The abduction expression pattern of CySWEET4 a,CySWEET4b,CySWEET14 gene which relate to formation of mycorrhiza was quantitated by using qRT-PCR.(4)In the low concentration glucose(5 mM)treatment,the growth performance about the overexpression plant of CySWEET4 a and the revertants plant of CySWEET4 b is better than the wild type,but the root growth was inhibited in the 50 mM glucose treatment,with leaves chlorosis,indicating that CySWEET4 a has high sugar transport activity.The revival effect of the revertants of CySWEET14 of the atsweet11;12 was obvious in the 5 mM sugar treatment,which could improve the physical defect,indicating that CySWEET14 had sugar transport function.(5)In the symbiotic medium LW01,the treatment(7dpi,14dpi)caused the downregulation of AtSWEET11 and AtSWEET12 in the root of A.thaliana,while the mycorrhizal growth responses of atsweet11;12 of root fresh weight is promoted by P.indica,that is significantly higher than the wild type,which indicated that AtSWEET11 and AtSWEET12 negatively regulated the symbiotic process of A.thaliana and P.indica Therefore,the homologous genes AtSWEET11 and AtSWEET12 of CySWEET14 in Arabidopsis have different functions in the symbiotic process between plants and P.indica.(6)The revertants of CySWEET14?CySWEET4a of the atsweet11;12 at the third generation lines were obtained,which mycorrhizal growth responses of root fresh weight is better than that of the wild type,but it is weaker than the double mutant,which shows the intermediate type of functional recovery.