Identification And Preliminary Functional Characterization Of MicroRNA827 Ang Its Target Genes In Tobacco

Phosphorus(P)is a structural component of many biologically important macro-molecules,such as nucleic acids,phospholipids and P-containing enzymes.It plays important roles in energy transfer,signal transduction,and photosynthesis processes.Therefore,phosphorus is one of the essential macro-nutrients for plant growth and development.However,the predominant form of P accessible to plant roots is inorganic orthophosphate(Pi,H2PO4-).It is easily fixed by cations and precipitated in the soil,resulting in their low mobility and availability.Therefore Pi is one of the major limiting factors for plant growth and development.During the long evolutionary process,plants have evolved a series of adaptive mechanisms for the P-deprived environment,including modification of root architecture,release of acid phosphatase,formation of mutualistic symbiotic associations with arbuscular mycorrhizal fungi,and activation of phosphate transporter.In the past twenty years,an increasing number of genes have been found to be involved in maintaining Pi homeostasis in plant,among which microRNAs(miRs)and SPX(SYG1/PH081/XPR1)family members play a key role.MiRs are a class of noncoding RNAs with a length of?21 nucleotides.MiRs are processed from long stem-loop structions by a Dicer-like enzyme.MiRs are incorporated into silencing complexes that contain Argonaute proteins,wherein they can guide repression of their target genes through mRNA cleavage or translational inhibition.MiR399 and miR827 are two known Pi starvation-induced miRs families conserved in plants.In Arabidopsis thaliana(Arabidopsis),the target gene of miR827 is AtNLA which belongs to the SPX-RING subfamily;whereas in rice,miR827's target genes are OsSPX-MFS1/2 which belong to SPX-MFS subfamily.In this work,the expression pattern of miR827 and the target genes,as well as the subcellular localization and the function of miR827 target genes were studied in tobacco.The main results obtained are as follows:1.Based on bioinformatics prediction,we successfully identified and cloned miR827 in tobacco.Bioinformatics analysis showed that the miR827 precursor in tobacco could be folded into characteritic stem-loop structure.RT-qPCR of analysis showed that,the transcriptions of NsmiR827s were dramatically enhanced by Pi starvation,this is consistent with that found in Arabidopsis and rice,suggesting that the expression of miR827 in response to Pi starvation among different species is highly conserved.2.The online miR target prediction server psRNATarget(http://plantgm.noble.org/psRNATarget)was utilized.The partial sequences of two miR827 target genes were obtained in Nicotiana benthamiana(N.benthamiana).Based on these two partial sequences from N.benthamiana,the full length cDNA sequnences were cloned in N.sylvestris by RNAlegase-mediated rapid amplification of cDNA ends technology.The target genes of NsmiR827 contain two conserved domains,namely SPX and MFS domain,and were designated as NsSPX-MFS1 and NsSPX-MFS2.RT-qPCR analysis showed NsSPX-MFS1 and NsSPX-MFS2 were responsive to Pi starvation in the opposite trends.Specifically,NsSPX-MFS1 was downregulated by Pi starvation,while NsSPX-MFS2 was upregulated by the same stimulus.3.We fused the full length ORF of NsSPX-MFS1/2 as well as their fragments lacking the SPX domains to the reporter gene,enhanced GFP.The subcellular localization analysis by infiltration of N.benthamiana leaf epidermal cells showed that they were localized to the tonoplast(vacuolar membrane)and the nucleus,which suggesting that the MFS domain might play an important role in protein location.4.The functions of NsSPX-MFS1/2 were analyzed by their complementation of a yeast mutant strain defective in five major Pi transporters,EY917.The results showed that under high-Pi condition(10 mM)the growth of the EY917 mutant strain was restored by the introduction of either of NsSPX-MFS1/2;in addition,the fragments of NsSPX-MFS1/2 lacking the SPX domains could partially complement the mutant strain.These indicate that the MFS domains of NsSPX-MFS1/2 play a major role in Pi transport,whereas the SPX domains are also required.Nevertheless,the specific roles of NsSPX-MFS1/2 in plants and the underlying molecular mechanism remain to be studied.5.The transgenic miR827a/d overexpression lines were generated through Agrobacteriu,m tumefaciens-mediated transformation.Under four treatments in which different levels of Pi were supplied(100 ?M,1 mM,5 mM,10 mM),no significant difference in growth performance,root-to-shoot ratio,and soluble Pi concentration was observed in the overexpression lines as compared with the wild-type plants.