Expression Analysis And Function Research Of Micrornas In Apple Under Drought Stress

MiRNAs are a class of endogenous small RNAs whose length is about 21-24 nt.MiRNAs interacted with the target and involved in regulating the growth and development,morphogenesis,fruit development,response to biological and abiotic stresses.In recent years,with the development of high-throughput sequencing and bioinformatics,more and more miRNAs and their targets have been identified.Malus domestica is an important economic crop.Drought stress affects the yield and quality of apple,so the research of drought resistance of apple is important.MiRBase-2.1 has record 206 apple-specific pre-miRNAs,207 mature miRNAs,but the drought-related miRNAs were rarely reported.In this study,miRNAs associated with drought resistance were analyzed by high-throughput sequencing.The differentially expressed miRNAs were identified by qRT-PCR.Osa-miR171h_L-1R + 1₁ss2TC and mdm-MIR169f-p5 were overexpressed and transgenic researched.The results are as follows:According to the sequencing results,14 known miRNAs and 12 new miRNAs were screened for qRT-PCR,and quantitave results of 10 known miRNAs and 10 novel miRNAs were consistent with sequencing results.The dissolution curve of mdm-MIR7121d-p3 and PC-5p-101486₁30 had no peaks,and the quantitative results of mdm-miR477 b,mdm-miR7122 a,mdm-miR393 a and mdm-miR156 t were not consistent with sequencing.Ssecondary structure of 20 miRNAs whose quantitative results consistented with the sequencing were analyzed,results showed that the precursors of 20 miRNAs had stem-loop structures.Using the online analysis software,we analyzed the target of 10 new miRNAs,the results demonstrated that the target may be related to serine/threonine protein kinase,triangular pentapeptide repeat compatible protein.According to the target analysis and quantitative results,overexpression vector and artifical miRNAs of osa-miR171h_L-1R + 1₁ss2TC and mdm-MIR169f-p5 were constructed.The content of Proline,tissue relative water,malondialdehyde and chlorophyll were examined for transgenic Arabidopsis that successfully overexpress osa-miR171h_L-1R + 1₁ss2TC.The results showed that the content of proline,tissue relative water and chlorophyll in transgenic Arabidopsis thalianas were lower than wild type under drought stress,while the content of malondialdehyde was lower.Osa-miR171h_L-1R + 1₁ss2TC can enhance the drought resistance of Arabidopsis thaliana.