Biological Function Analysis Of Mul-miR482 And Mul-miR58 From Mulberry Phloem Sap

The development of potent systems for long-distance transportation for the distribution of nutrients and signals throughout the plant was one of the most important achievements in the evolution of higher plants. The phloem is not only the major route for the translocation and distribution of organic metabolites but also is an important mediator of whole-plant communication and involved in whole plant events including stress responses and long-distance signaling. Recently a large number of mi RNAs have been identified in the phloem, and some mi RNAs in the phloem have been demonstrated acting as long-distance signaling and have a role in mediating gene expression for developmental patterning and stress responses. In this study, based on the existing mulberry transcriptome information, the mul-mi R482 and mul-mi R58 genes were cloned from mulberry phloem sap using PCR technology, and their tissue expression pattern were investigated using quantitative PCR. Meanwhile, the biological functions of mul-mi R482 and mul-mi R58 were analyzed using transgenic technology. Furthermore, Tail-PCR technique was used to amplify the promoter of mul-mi R482 gene, and the expressive activity of the promoter obtained was investigated using GUS Histochemical staining. In addition, the transmission property of mul-mi R482 in Arabidopsis was examined using micro-grafting technique. The research results obtained in this study will be useful for revealing the the biological function of mulberry mul-mi R482 and mul-mi R58 and providing further insights into the regulatory mechanism of mi RNAs long-distance transported in the phloem and lay a foundation for functional genomic analysis in mulberry. The main findings are as follows:(1) Analysis of the biological function of mul-mi R482 from mulberry phloem sapBased on the mulberry transcriptome sequence information, the mul-mi R482 gene was cloned using PCR technology and named as Mul MIR482. The plant expression vector of Mul MIR482 was constructed, and the transgenic Arabidopsis plants of the genes were obtained successfully. Northern hybridization and Real-time PCR analysis showed that the Mul MIR482 could be processed into mature mi RNA in the transgenic Arabidopsis plants. The phenotypeof the transgenic plants was found different with the wild type plants, and the transgenic Arabidopsis show less resistant to salt stress and more sensitivity to disease compared with the wild type Arabidopsis plants. Therefore, mul-mi R482 may be involved in the regulation of plants growth and development, and it may be as a negative regulator in the response to salt stress and disease resistance.(2) Tissue expression characteristic analysis of mul-mi R482 from mulberry phloemThe regulatory sequence 1937 bp upstream of Mul MIR482 was amplified using Tail-PCR and named as pmi R482. Sequence analysis showed that it contained many elements, such as CAAT-BOX, TATA-BOX, which are necessary for the normal transcription initiation, and many other transcription factor binding sites and transcriptional regulatory elements. The expression vector containing this sequence fused with GUS was constructed for transformation into Arabidopsis by using agro bacterium-mediated method. Histochemical staining of transgenic Arabidopsis showed that GUS reporter gene was predominantly expressed in stems and flowers. However, its expression in roots was very low and no expression was detected in the leaves and siliques. This displayed a strong tissue-specific expression pattern in Arabidopsis transformed with pmi R482 fused with gene GUS. The expression pattern of mul-mi R482 in the mulberry was analyzed using quantitative PCR, and the results showed that it 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 leaves.(3) Analysis of the transmission property of mul-mi R482 from mulberry phloem sapThe transgenic Arabidopsis of mul-mi R482 and wild-type Arabidopsis were used as rootstock and scion(or scion and rootstock), respectively, and grafted together using micro-grafting technique. Then the expression abundance of mul-mi R482 in the rootstocks and scions were detected quantitative PCR technique. The results showed that the transmission of mul-mi R482 in the transgenic Arabidopsis was transported from top to bottom predominantly, though it can be transported in two-way directions.(4) The biological function of mul-mi R58 from mulberry phloem sapThe mul-mi R58 gene was cloned using PCR technology and named as Mul MIR58. Real-time PCR analysis showed that the expression of mul-mi R58 was significantly higher in roots,fruits and barks than that in the leaves. The Mul MIR58 gene was transformed into Arabidopsis plant, and Northern hybridization and Real-time PCR analysis showed that the Mul MIR58 could be processed into mul-mi R58 in the transgenic Arabidopsis plants. The phenotype of the transgenic plants was found different with the wild type plants, and the transgenic Arabidopsis showed less resistance to salt stress and more sensitivity to disease compared with the wild type Arabidopsis plants. Therefore, mul-mi R58 may also be involved in the regulation of plants growth and development, and plays a negative role on the resistance to salt stress and Pst DC3000 infection.