Iditififation And Biological Functions Of The MiR3954-MuLnc1-tasiRNA Cascade In Mulberry

The miRNA--TAS--ta-siRNA--target gene cascade has been identified as an important element in gene regulation networks involved in the plant development, metabolism and response to biotic and abiotic stresses. In this study, a new ta-siRNA regulatory cascade: Mul-miR3954--MuLnc1--ta-siRNA--Mul-CML27 was identified, and the expression patterns and biological functions of the elements in the cascade were analysized. Furthermore, the mul-miR3954--MuLnc1--ta-siRNA was established in the Arabidopsis using transgene and hybridization technique, and the function and regulatory mechanism of cascade was discussed. The results obtained in this study would be will provide further insights into the function and regulatory mechanism of mul-miR3954--MuLnc1--ta-siRNA--target gene cascade passway, and enrich the ta-siRNA regulatory cascades, 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 mul-miR3954--MuLnc1--ta-siRNA--target gene cascade in mulberryThe MuLnc1 transcript, which was a new long noncoding RNA in mulberry, was predicted as the target gene of mul-miR3954 based on the analysis of the transcriptome, sRNA, and degradome databases. Our results showed that mul-miR3954 may target t MuLnc1 transcript and most likely initiate the biogenesis of siRNAs, which were produced in a 21 nt phasing form and likely as ta-siRNAs. Moreover, the existence of these siRNAs was proved by Northern blot analysis, and this provided the evidence for the existence of mul-miR3954--MuLnc1--ta-siRNA--target gen cascade in the mulberry. Therefore, MuLnc1 was a new potential TAS gene.(2) Cloning and biological functions of MulMIR3954The MulMIR3954 gene was cloned using PCR, and its expression patterns in mulberry was analysized. The results showed that MulMIR3954 had no specific expression in different tissues. However, its expression abundance was diverse in different tissues and organs, and the expression level was highest in roots and lowest in fruits. The MulMIR3954 gene was transformed into Arabidopsis thaliana plants, and the transgenic Arabidopsis plants of the gene were obtained successfully. Northern hybridization and qRT-PCR analysis showed that the mul-miR3954 precursor was processed and there was the accumulation of miR3954 in the transgenic Arabidopsis plants. The phenotype of the transgenic plants was found different with the wild type plants, and the expression of the mul-miR3954 in the transgenic Arabidopsis may hasten the growth of the roots. In addition, the transgenic Arabidopsis plants of MulMIR3954 showed lee resistance to Pseudomonas syringae pv. tomato DC3000(Pst DC3000) infection, drought and salt stresses compared with wild type plants. Therefore, mul-miR3954 may be involved in the regulation of plants growth and development, and it may be as a negative regulator in the response to Pst DC3000 infection, drought and salt stresses.(3) Cloning and biological functions of MuLnc1The MuLnc1 gene was cloned using PCR and showed little conservatism among different plants. The expression abundance of MuLnc1 was diverse in different tissues and organs, and its expression level was highest in fruits but lowest in barks. The MuLnc1 gene was transformed into Arabidopsis thaliana plants, and the transgenic Arabidopsis plants of the gene were obtained successfully. Northern hybridization and qRT-PCR analysis showed that there was the accumulation of MuLnc1 transcript in the transgenic Arabidopsis plants. The phenotype of the transgenic plants was found different with the wild type plants, and the expression of the MuLnc1 in the transgenic Arabidopsis may hasten the growth of the plants. In addition, the transgenic Arabidopsis plants of MuLnc1 showed more resistance to Pst DC3000 infection, drought and salt stresses. Therefore, MuLnc1 may hasten the growth of the plants and be as a positive regulator in the response to Pst DC3000 infection, drought and salt stresses.(4) Cloning and biological functions of the calmodulin-like protein gene CML27 in mulberryThe calmodulin-like protein gene CML27 was cloned using PCR and designated as Mul-CML27. The gene has an open reading frame of 507 bp encoding a protein composed of 168 amino acids with a predicted molecular mass of 18.5 kDa and an isoelectric point of 4.48. It was predicted that the 3D structure of the Mul-CML27 was consist of ? helixs and random coils, and the protein possesses no typical signal peptide sequence and significant across membrane structure zone, but has some phosphorylation sites and two conserved Efh domain. The expression abundance of MuLnc1 was diverse in different tissues and organs, and its expression level was higher in fruits and foots but lower in barks and leaves. The phenotype of the transgenic plants was found different with the wild type plants, and the expression of the Mul-CML27 in the transgenic Arabidopsis may hasten the growth of the roots. In addition, the transgenic Arabidopsis plants of Mul-CML27 showed more resistance to Pst DC3000 infection, drought and salt stresses indicting Mul-CML27 may be as a positive regulator in the response to Pst DC3000 infection, drought and salt stresses.(5) Biological functions of Mul-miR3954-Mu Lnc1-ta-siRNA cascadeThe transgenic Arabidopsis plants of Mul-MIR3954 were hybridized as male parent with the transgenic Arabidopsis plants of MuLnc1 as female donor, and the hybrid F1 seedlings were obtained. The transcripts of Mul-MIR3954 and MuLnc1 were detected by Northern hybridization and qRT-PCR analysis. Moreover, one of the secondary siRNAs, si161579, from the MuLnc1 transcript was also detected by Northern blot and qRT-PCR. This indicates that the Mul-miR3954-Mu Lnc1-ta-siRNA was established in the hybrid F1 Arabidopsis successfully. The hybrid F1 Arabidopsis plants has no difference with the wild type plants in the resistance to Pst DC3000 infection, drought and salt stresses. This provide the further evidence for that the mul-miR3954 may target MuLnc1 transcript and the Mul-miR3954-Mu Lnc1-ta-siRNA-target gene cascade in the mulberry may be as a negative regulator in the response to Pst DC3000 infection, drought and salt stresses.