| MicroRNAs(miRNAs)are a class of non-coding RNAs that play important roles in leaf development and stress tolerance.Short Tandem Target Mimic(STTM)technology can efficiently and specifically silence the target miRNA family,while the expression and function of other miRNAs are not affected,which act a crucial role to investigate miRNA functions and miRNAs interaction.In this study,we used the STTM technology to analyze the interaction of mi R160 and mi R165/166 in Arabidopsis and the functions of mi R166 in maize.In Arabidopsis,the double mutant,STTM160×165/166,was generated by crossing two single mutants,STTM160 and STTM165/166.We identified the leaf and drought phenotypes,and analyzed the expressions of mi R160 and mi R165/166 in the double mutant.Using RNA-seq and Small RNA-seq technologies,we compared the expression level of multiple miRNAs and genes between single and double mutants.Using High Performance Liquid Chromatography(HPLC)approaches,we also compared the IAA and ABA content between single and double mutants.Thus,we dissected the interaction of mi R160 and mi R165/166 in controlling leaf development and drought tolerance in Arabidopsis.In maize,the transgenic line,STTM166,was generated by vector construction and genetic transformation.We identified the phenotype of plant and ear,and analyzed the exprrssion of mi R166 in the transgenic line.Using RNA-seq technology,we compared the expression level of multiple genes between the transgenic line STTM166 and wild-type C01.Therefore,we expected to uncover the functions of mi R166 in regulting leaf development and flowering time in maize.The main results are as follows:1.The single mutant,STTM160,showed upward leaves with serrated edges and advanced flowering time.The single mutant,STTM165/166,showed increased leave number,spoon or trumpt leave morphology and predominant drought tolenrence.The double mutant,STTM160×165/166,exhibited a series of compromised phenotypes,such as increased leaves numbers,rough surface,jagged edges,upward growth leaves morphology,enhanced drought tolerance,reduced leaf dehydration and slightly promoted flowering time.Compared with the wild type,the STTM160×165/166 displayed remarkable decreased in the expressions of mi R160 and mi R165/166,whereas notable increased in the expressions of ARF10,ARF16,PHB and PHV.Compared with STTM160 and STTM165/166,mi R160 was decreased,but mi R165/166 was increased,ARF10 and ARF16 were increased but PHB and PHV was decreased in the STTM160×165/166.These alterations in leaf/drought phenotype and miRNA/target expression indicate that there is a novelty link bettween mi R160 and mi R165/166.2.Using RNA-Seq and small RNA-seq,we analyzed the differentially expressed miRNAs and genes in STTM160,STTM165/166 and STTM160×165/166.Compared with STTM160,seven miRNAs and 728 genes were differentially expressed in STTM160×165/166.Compared with STTM165/166,22 miRNAs and 4732 genes were differentially expressed in STTM160×165/166.These differentially expressed miRNAs and genes are involved in photosynthesis,circadian rhythms,plant hormonal signal transduction and biotic/abiotic stress responses.The interaction between mi R165/166 and mi R160 may be achieved through the miRNAs and genes associated with these pathways.3.The double mutant displayed notable changes in leaf development and drought tolerance.Also,leaf morphological defects may be associated with important miRNAs alterations in leaf developmental proceses.Drought stress response may be associated with important genes alterations in plant hormone metabolism.Thus,we analyzed some critical miRNAs and genes associated with these regulatory roles among WT,STTM160,STTM165/166 and STTM160×165/166.Compared with STTM160 and STTM165/166,the expression levels of mi R159 and mi R319 were highest in STTM160×165/166,and the expression levels of mi R156,mi R164,mi R390 and mi R396 were higher than STTM160 but lower than STTM165/166.Compared with STTM160 and STTM165/166,TAA1,YUC1,PYR1 and BG1 showed significantly differences in STTM160×165/166.It is remarkable that changes in the IAA and ABA metabolism related genes may lead to changes in the endogenous contents of IAA and ABA.Using the High-Performance Liquid Chromatography(HPLC)approach,we analyzed the IAA and ABA content among WT,STTM160,STTM165/166 and STTM160×165/166.The content of IAA and ABA in the STTM160×165/166 was higher than STTM160 and wild-type but lower than STTM165/166,and the difference reached a significant level.4.The maize STTM166 plants showed curled leaves,delayed flowering,reduced plant height,decreased in tassel length as well as tassel branch number.The maize STTM166 ears showed shorter ear length,while almost unchanged in ear diameter,row numbers per year and kernel numbers per row.Compared with C01,the kernel length of STTM166 was 6.50 mm,a decrease of 1.98 mm,and the difference reached a very significant level.The 100-grain weight of STTM166 was 13.20 g,a decrease of 5.5 g,also a very significant level.The 100-grain weight of STTM166 was notably decreased by 29.41%,which may due to the remarkable reduction in grain length.These results suggest that the fuctions of mi R166 is conserved between the dicots(Arabidopsis)and the monocots(maize).5.Using RNA-seq technology,we analyzed the differentially expressed genes in the transgenic line STTM166 and wild-type C01.A total of 178 differentially expressed genes were identified,including 118 up-regulated genes and 60 down-regulated genes.Of these,we found the key gene ZCN8 in maize flowering time and phase change regulation,the important gene MADS-Box in floral development,and the critical genes RLD2,KAN1 and KAN3 in leaf polartity regulation.The alterations in these genes may be a possible reason of rolled leaves and delayed flowering in maize by silencing of mi R166. |
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