Identification And Biological Function Analysis Of The MiR393-AFB2-tasiRNA Cascade Pathway In Mulberry

The miRNA-TAS-tasiRNA-target gene cascade pathway which links miRNA and siRNA into an gene regulatory network play an important role in modulating the expression of gene at transcriptional and post-transcriptional levels and has been identified as an important elements in gene regulation networks involved in the plant development, metabolism and response to biotic and abiotic stresses. Based on the attained information of sRNA,transcriptome, and degradome of mulberry, the miR393 of mulberry was found target the transport inhibitor response 1/auxin signaling F-box 2(AFB2) transcript and most likely initiate the biogenesis of secondary siRNAs from the transcript. The siRNAs produced were in a 21 nt phasing form and likely as tasiRNAs and were detected by Northern blot and qRT-PCR. On this basis, the expression patterns and biological functions of MulMIR393 A,MulMIR393B and their target MulAFB2 were analysised with PCR and transgene technology.The results obtained in this study would be provide further insights into the regulatory mechanism of miR393-AFB2-tasiRNA-target gene cascade pathway, and promote the research on the functional genomic analysis in mulberry. The main results of this study can be summarized as follows.(1) Identification of the miR393-AFB2-tasiRNA cascade way in mulberryThe target gene of miR393 of mulberry was predicted the plant small RNA target analysis server, psRNATarget(http://plantgrn.noble.org/psRNATarget/) and validated by the analysis of degradome database. The results showed that mul-miR393 may target the TRANSPORT INHIBITOR RESPONSE1(TIR1), AUXIN SIGNALING F-BOX2 gene and most likely initiate the biogenesis of siRNAs from the transcript. The siRNAs produced were in a 21 nt phasing form and likely as tasiRNAs, and the existence of these siRNAs was proved by Northern blot and qRT-PCR analysis. We provide the evidence for the existence of miR393-AFB2-tasiRNA cascade pathway in the mulberry.(2) Cloning and activity analysis of the promoters of MulMIR393 A and MulMIR393 B genesThe promoter sequences of MulMIR393 A and MulMIR393 B genes were successfully isolated from mulberry by PCR technology and were named as pMulMIR393 A and pMulMIR393 B, respectively. Sequence analysis of pMulMIR393 A and pMulMIR393 B were performed using the Plant CARE algorithm, and the results showed that the two sequencescontained some core elements of promoter, multiple transcription factor binding sites, and a variety of environmental factors responsive elements. The plant expression vectors containing pMulMIR393 A and pMulMIR393 B, respectively, fused with GUS were constructed and transformed into Arabidopsis, and the transgenic Arabidopsis plants obtained were used to analysis the activity of pMulMIR393 A and pMulMIR393 B with GUS histochemical staining and fluorescence quantitative detection method. The results showed that the expression activity of the two promoters were different though they both were tissue-specific expressed and induced by pathogenic bacteria and hormones.(3) The biological functions of MulMIR393 A and MulMIR393BThe MulMIR393 A and MulMIR393 B genes were transformed into Arabidopsis thaliana plants, respectively, and the transgenic Arabidopsis plants of the genes were obtained successfully. Northern hybridization and qRT-PCR analysis showed that all mi R393 a and miR393 b precursors were processed in the transgenic Arabidopsis plants, but the accumulation of mature miRNA 3P and 5P of MulMIR393 A and MulMIR393 B were different.In MulMIR393 A transgenic plants, the accumulation of miRNA-5P was higher than miRNA-3P, while the accumulation of miRNA-3P was higher than miRNA-5P in MulMIR393 B transgenic plants. Northern blot and qRT-PCR analysis showed that mul-miR393 can silence the AFB2 transcript in the transgenic plants. Both the MulMIR393 A and MulMIR393 bB transgenic plants have the phenotypes different from that of the wild type plant, and the phenotypes of the MulMIR393 A and MulMIR393 bB transgenic plants were also different. This indicated that both the MulMIR393 A and MulMIR393 B genes were associated with plant development, but the roles of them were different. When the transgenic Arabidopsis were subjected to Pseudomonas syringae pv. tomato DC3000(Pst DC3000), the transgenic Arabidopsis plants of MulMIR393 A showed more resistance to Pst DC3000 infection compared with wild type plants, while the transgenic Arabidopsis plants of MulMIR393 B showed sensitive to Pst DC3000 infection. Therefore, both the MulMIR393 A and MulMIR393 B genes were involved in the response to the Pst DC3000 infection, but the MulMIR393 A may be as a positive regulator in the response while the MulMIR393 A may be as a negative regulator to Pst DC3000 infection.(4) The biological functions of MulAFB2 targeted by miR393Based on the mulberry transcriptome information, the MulAFB2 gene was cloned using PCR technology. The gene has an open reading frame of 1772 bp encoding a protein composed of 573 amino acids which were highly homology to Arabidopsis thaliana AthAFB2.The plant expression vector of MulAFB2 was constructed and then the gene was transformedinto wide type Arabidopsis successfully. The transgenic Arabidopsis plants of MulAFB2 had a shorter root and less numbers of leaves compared with wild type plants. When the transgenic plants were subjected to Pst DC3000, the transgenic plants of MulAFB2 showed more sensitive to Pst DC3000 infection compared with wild type plants. Therefore, MulAFB2 gene was not only involved in plant growth and development, but also as a negative regulator to Pst DC3000 infection.