Analysis Of Salt Tolerance Related MiRNAs And Prediction Of Target Genes In Medicago Sativa ’Millenium’

With the growth of China’s population,development of social and economic and the change of climate,water shortages,soil salinity and desertification have become a prominent problem.The most ecological and economic ways to develop the coastal zone are cultivate salt-tolerant plants and using biological technology to improve the soil.Medicago sativa,is a perennial herbs,It is one of the important pastures with rich nutrients,high grass yield and nitrogen fixation.In order to understand the molecular mechanism of Medicago sativa‘Millenium’under salt stress,the salt-tolerant miRNAs and related target genes were explored.Illumina HiSeq^TM2000 was performed on the Medicago sativa‘Millenium’and its mutant seedlings roots to explore the salt-tolerant miRNAs.Using psRNATarget,TargetFinder and Tapirhybrid software to forecast the target genes related to the differentially expressed miRNAs,and GO enrichment analysis of predicted target genes is carried out to analyze the main functional categories of target genes,explore the main biological functions and metabolic pathways involved in salt-tolerant target genes,in order to lay the foundation for the further study of salt-resistance of Medicago Sativa and contribute to the cultivation of new salt-tolerant varieties The results of this study are as follows.（1）Determination of salt tolerance physiological indexes of Medicago sativa roots under salt stressTreating the wild type and salt-tolerant mutants of the Medicago sativa‘millenium’with250mMol·L^-11 NaCl,changes of 5 physiological indexes at 0h and 72h after salt stress were measured.The results showed that after 72h salt stress,the plant roots’s length is shortened,the number of fibrous roots is reduced.We also found that under salt stress,inside the wild type and mutant type plants,the proline content,malondialdehyde content,superoxide dismutase activity and ascorbate peroxidase activity in the roots of plants were up-regulated.The soluble protein content was down-regulated,and all physiological indexes in the body showed obvious changes,it can be inferred that 72h is a good salt stress response time.At the same time,it was found that,in the mutant type,the up-regulation of proline content,superoxide dismutase activity and ascorbate peroxidase activity were significantly higher than that in the wild type after 72h of salt stress.The changes of malondialdehyde content and soluble protein content in mutant type were all lower than the wild type,the results of physiological indicators showed that the salt tolerance of the mutant was stronger than that of the wild type.（2）Analysis of salt related miRNAs in Medicago sativaBuild six southern type Medicago sativa mutant root sRNA library and six wild type alfalfa roots sRNA before and after salt stressed.In the mutant type,discovered 447 known miRNAs and 146 new miRNAs.46 differentially expressed miRNAs are predicted from the known miRNAs（12 up-regulated,34 down-regulated）;19 differentially expressed miRNAs（7 up-regulated,12 down-regulated）are predicted from the new miRNAs.In the wild type alfalfa,Discovered 437 known miRNAs and 142 new miRNAs.36 differentially expressed miRNAs are predicted from the known miRNAs（10 up-regulated,26 down-regulated）;16 differentially expressed miRNAs（8 up-regulated,8 down-regulated）are predicted from the new miRNAs.（3）Analysis of target genes related to differential expressed miRNAs in Medicago sativaUsing psRNATarget,TargetFinder and Tapirhybrid softwares to predict the target genes,and an amount of 1190 target genes were predicted in mutant type,and 1143 target genes were predicted in wild type.The Functional analysis of gene ontology revealed two main functional categories of these target genes in mutant type,predicted target genes function mainly occurred in molecular biology function,combining,purine nucleoside triphosphate combined and ribonucleic acid.In the wild type,it was predicted that the main biological processes of its target genes were enriched in the replication process and the metabolic process.In the molecular function category,the gene function and the mutant function were roughly the same,mainly based on molecular function,binding and RNA binding.（4）qRT-PCRIn order to ensure the credibility of high-throughput sequencing data,Among the miRNAs with common differential expression of wild-type and mutants,9 miRNAs and their target genes were selected for qrt-pRT-PCR.The selected miRNAs are miR5272b、miR167a、miR2604、miR156a、miR5208d、miR169j、N-miR54、N-miR10、N-miR73.The results showed that those miRNAs were detected and most of the expression results were consistent with the sequencing results.The experimental data is nuine and believable.among the 9 miRNAs selected from the mutant type,except for miR156a,the other 8 miRNAs were negatively regulated by their target genes,among the wild type,except for miR5272b and N-miR10,the other 7 miRNAs were negatively regulated by their target genes,which proves that miRNA regulates plant growth,development and environment by negatively regulating or silencing target genes.Through the prediction and relative expression level analysis of the predicted miRNAs target genes,the salt tolerance of the mutants was verified to be stronger than the wild type.