| Sweet potato[Ipomoea batatas（L.）Lam.]is a tuberous root crop with strong resistance and barrenness tolerence.It is important to explore and study the molecules playing function in both storage root development and stress resistance of sweet potato for breeding high quality sweet potato varieties and improving agricultural economic value.We obtained a number of miRNAs that may be closely related to storage root development by small RNA sequencing,and further screened the candidate miRNAs by abiotic stress response analysis.The results showed that miR398 was not only differentially expressed between storage roots and fibrous roots,but also responded strongly to abiotic stresses.We analyzed the function of miR398 in sweet potato by transcriptome sequencing,expression pattern analysis,agrobacterium tumefaciens mediated in vivo transgenic technology and non-invasive micro assay technology.The main results are as follows:（1）Small RNA libraries were constructed from sweet potato fibrous roots,1 cm storage roots,3 cm storage roots,5 cm storage roots and 10 cm storage roots for high-throughput sequencing.The results showed that 145 miRNAs were differentially expressed in fibrous roots and storage roots.There are 30 known miRNAs and 115 new miRNAs.Eight candidate miRNAs including four known miRNAs（miR398,miR156g,miR394a,miR156g-5p）and four new miRNAs（novel＿272,novel＿246,novel＿96,novel＿9）were selected for real-time quantitative PCR analysis,and the results were consistent with the sequencing data,which further confirmed the sequencing results.（2）The responses of candidate miRNAs to abiotic stresses（Cold,Heat,Salinity and Drought）were analyzed.The results showed that novel＿9,miR156g,miR394a and miR398 all responded to abiotic stresses,and miR398 responded to salt stress strongly.（3）We cloned pre-miR398 and performed hormone treatment analysis and the results showed that the expression of miR398 can impact by storage root development related hormones（IAA,ZT and GAs）and stress related hormones（ABA and GAs）.Compared with the control plants,the expression of miR398 increased under IAA and ZT treatment.However,after ABA and GAs treatment,the relative expression of miR398 decreased significantly compared with control.This further indicated that miR398 may play a role in storage root development and stress response.（4）The overexpression vector of pre-miR398 was constructed,and the hairy roots of transgenic sweet potato were obtained.The transcriptome sequencing of transgenic and non transgenic adventitious roots revealed 593 up-regulated and 482 down regulated transcripts.The results of GO and KEGG analysis showed that these transcripts were mainly involved in abiotic stress and photosynthesis of sweet potato.（5）Fluorescence quantitative PCR analysis showed that the expression of stress resistant genes IbABF4,IbSOS2-like and IbLEA14 in transgenic hairy roots increased significantly,while the expression of IbNAC1-like decreased significantly.The ion flow detection of transgenic roots showed that the discharge velocity of sodium ions in the meristem and elongation area of the transgenic roots was significantly higher than that of wild type.Coro Na TMGreen AM staining results showed that the accumulation of sodium ions in transgenic adventitious roots was significantly lower than that of wild type,which indicated that miR398 could promote sodium ion efflux and improve salt tolerance of sweet potato.In addition,Fluorescence quantitative PCR analysis showed that storage root development regulator IbSRD1 was significantly induced in the transgenic adventitious roots,and the expression of IbAGPase was inhibited.These results suggested that miR398 may play a certain role in the regulation of sweet potato storage root development.（6）The quantitative PCR showed that the expression of IbCCS1 was significantly decreased in the over expressed roots,which was negatively related to miR398 expression pattern.Moreover,the cleavage position of miR398 in IbCCS1 transcript was predicted by ps RNATarget online software.The activities of SOD and Cu Zn SOD in transgenic adventitious roots were significantly decreased,which indicated that IbCCS1 may be the potential target gene of miR398 in sweet potato.In conclusion,the expression pattern,salt tolerance and target genes of miR398 were studied.The results showed that miR398 may play an important role in storage root development and salt tolerance of sweet potato. |
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