Screening And Identification Of Rice Stress Response MicroRNA

Plant microRNAs(miRNAs)are a class of non-coding single-stranded small RNAs that are spontaneously transcribed from plant genomic DNA and that can silence the target genes after processing.In order to screen rice miRNAs in response to abiotic stress,miRNAs related to abiotic stress in rice were obtained through literature query,database screening and bioinformatics analysis.The wild-type Nipponbare(Oryza sativa L.ssp.japonica cv.Nipponbare)at tillering stage was subjected to high temperature(50?),drought(10% PEG6000 simulation),low temperature(6?)and salt(100 mM)stress treatment,and qRT-PCR analysis confirmed the stress responses of these rice miRNAs.The overexpression vector pOsa-mi RNAs-OE and the interference vector pOsa-miRNAs-STTM of Osa-microRNAs were successfully constructed for subsequent Osa-miRNAs gene function research.(1)Database screening and bioinformatics analysis: A rice miRNA library related to abiotic stresses was constructed by miRNA transcriptome database screening and literature review.The upstream promoter cis-elements of the miRNAs in the library were analyzed.The predicted results showed that the upstream promoter regions of the pre-selected Osa-miRNAs had cis-acting elements related to abiotic stresses.(2)qRT-PCR verification: Rice miRNAs were reverse transcribed by stem-loop method,and rice U6 was used as an internal reference gene,and qRT-PCR was performed by SYBR Green I dye method.The results showed that rice miRNAs were differentially expressed under different abiotic stress conditions and miRNAs responsive to abiotic stresses were screened.The expression levels of these miRNAs in roots and leaves of rice were different,indicating that there were spatial expression differences of miRNAs in rice plants.(3)Construction of Osa-miRNAs overexpression vector and STTM interference vector: Using Osa-miRNA1876 as an example,the gene sequence of Osa-pre-miRNA1876 was obtained by primer synthesis and genomic amplification,and integrated into plant expression vector pCAMBIA1300-ubi,Osa-pre-miRNA1876 overexpression vector pOsa-miRNA1876-OE was obtained;two target gene targetmimics were designed based on the mature sequence of Osa-mi RNA1876,which were ligated to the 5' and 3' ends of the 48 nt hairpin structure,respectively.The Osa-mi RNA1876 STTM functional fragment was obtained and integrated into the plant expression vector pCAMBIA1300-ubi to obtain the Osa-miRNA1876 interference vector pOsa-miRNA1876-STTM.