Application Of Short Tandem Target Mimic In The Model Plant Lotus Japonicus

The model legume Lotus japonicus is very important as agriculture forage sources.It is a very important source of plant protein in livestock feed,as well as key components of natural and agricultural resources.It also has a variety of functions such as improving soil structure,enhancing soil fertility and moisturizing water and soil.Study of its growth development and environmental-adaptability is of great significance.micro RNAs(miRNAs)are a class of endogenous non-coding small RNAs(20-24 nucleotides in length)that bind to target genes by sequence complementation and negatively regulates their expression at the post-transcription or translation level.In plants,miRNAs are involved in many biological processes,such as growth and development,as well as responses to stresses.Lotus japonicus harbors huge amounts of miRNAs,but the study of miRNAs is still lacking.Short Tandem Target Mimic(STTM)is a powerful and effective approach to silence miRNAs.Although it has been widely used in the model plants and crops such as Arabidopsis thaliana and Oryza sativa,its feasibility in the model legume is still unknown.Therefore,this work mainly focuses on the analysis of miRNAs in Lotus japonicus and explores whether STTM is effective in Lotus japonicus to pave the way for the future study of miRNAs function.In this study,we first analyzed miRNAs in shoot apical meristem(SAM)and compound leaves by high-throughput sequencing of small RNAs.Both conserved miRNAs,such as miR166,miR167,miR168 and miR396,and specific miRNAs such as miR7516 and miR11115 were also highly expressed in SAM and compound leaves.By comparing the miRNAs that were highly expressed,we found miR11148 was specifically expressed in compound leaves,while miR11097 b,miR11122 and miR11137 were specifically expressed in SAM.Although Lotus japonicus has a good environmental adaptability,its cold tolerance is very poor.Therefore,we searched for miRNAs in response to low temperature stress through high-throughput sequencing of small RNAs.Results showed that the expression of miR398,miR11173 and miR11078 c were induced under low temperature stress,while some miRNAs,such as miR11093 and miR11131 were downregulated.To further investigate the function of miRNAs,miR166,miR396 and miR398 were selected and knocked down using by STTM.We optimized the tissue culture transformation system of the Lotus japonicus to obtain positive transformants,the transgenic plants of Lj STTM166,Lj STTM396 and Lj STTM398 were generated.Further analysis showed that,the expression of miR166,miR396 and miR398 in transgenic lines were significantly decreased compared to the wild type,while their target genes expression was dramatically upregulated.These indicate that STTM was effective in Lotus japonicus.In summary,miRNAs involved in growth and development,as well as responses to low temperature stress in Lotus japonicus were analyzed by high-throughput sequencing of small RNAs in this work.Some miRNAs were selected and knocked down by STTM.The tissue culture transformation system of Lotus japonicus was improved and the high efficiency of STTM in silencing miRNAs in Lotus japonicus were proved.This study laid a foundation for further study of miRNAs function in compound leaves development and cold resistance in Lotus japonicus.