Biological Function Analysis Of The Pathogenesis-related Protein Gene MuPR1c And Its Promoter In Mulberry

Mulberry varieties are the material basis for sericultural production and the important ecological species for soil and water conservation, wind-prevention, sand-fixation and improving the quality of environment. Mulberry diseases and pests are important factors affecting the sericulture development and realization the ecological value of mulberry trees. Research on the molecular mechanisms of disease resistance in mulberry and breeding resistance cultivars are beneficial for mulberry varieties, being the economic and ecological tree species, to play their important roles in forestry production. As a class of defense response genes, the pathogenesis-related protein gene PR1 s are closely related to the systemic acquired resistance of plants, but their biological activities and resistance mechanisms are not clear yet. At the same time, many studies on PR1 s focus on herbs, and little information about PR1 s of woody plants has been reported. In this study, the c DNA encoding PR1 c was isolated from mulberry and was designated as Mu PR1 c, and its biological functions were analysised. In addition, the promoter of Mu RP1 c was cloned and its activity was investigated, and the expressional regulation patterns of Mu RP1 c gene were discussed. The results obtained in this study would provid a candidate gene for the molecular breeding for mulberry disease resistance, and laid the foundation for the research on the functions and mechanisms of PR1 proteins. The main results of this study can be summarized as follows:(1) Biological function analysis of the pathogenesis-related protein gene Mu PR1cBased on the mulberry transcriptome sequence information, the mulberry pathogenesis-related protein gene PR1 c was cloned using PCR and designated as Mu PR1c(Gen Bank accession No. KC453994). The gene has an open reading frame of 609 bp encoding a protein composed of 202 amino acids with a predicted molecular mass of 22.97 k Da and an isoelectric point of 6.30. It was predicted that the secondary structure of Mu PR1 c was rich in alpha helix, followed by beta folded and extended fragments, and the corner in the structure only accounted for 4.46%, and the Mu PR1 c was folded into a hexahedron shape with α-β-α sandwich structure. The protein possesses a typical signal peptide sequence, a significant across membrane structure zone and a nuclear export signal sequence. The coding region of the Mu PR1 c was inserted into the expression vector p BI121 and transformed into Arabidopsis thaliana plants, and the transgenic Arabidopsis plants of the gene were obtainedsuccessfully. The transgenic plants show strong resistance to Pst DC3000 and Botrytis cinerea, indicating that Mu PR1 c plays an important role in the resistance to the bacteria and fungi.(2) Subcellular localization of the mulberry pathogenesis-related protein MuPR1c The coding region of the Mu PR1 c was inserted into an expression vector PROKⅡ containing GFP, and the expression vector containing this sequence fused with GFP was constructed for transformation into Arabidopsis by using agrobacterium-mediated method. Then the transgenic Arabidopsis thaliana plant roots were observed through fluorescence microscopy and the results showed the Mu PR1 c localizated in cell wall or secreted into the intercellular space.(3) Cloning and functional analysis of the promoter of Mu PR1 c geneThe promoter sequence of Mu PR1 c gene(p Mu PR1c) was successfully isolated from mulberry by Tail-PCR technology. Sequence analysis of p Mu PR1 c was performed using the Plant Care algorithm, and the results showed that it contained some core elements of promoter, multiple transcription factor binding sites, and a variety of environmental factors responsive elements.This indicated that p Mu PR1 c might be involved in the diverse response processes to environment stresses though different signaling pathways in plants. The activity of p Mu PR1 c was investigated with the method of transient expression of tobacco leaves, and the result indicated that p Mu PR1 c can initiate the transcription of the GUS gene which was used as a reporter gene and be induced upon pathogen attack. At the same time, the plant expression vector containing p Mu PR1 c fused with GUS was constructed for transformation into Arabidopsis by using agrobacterium-mediated method. The transgenic Arabidopsis thaliana plants were obtained and were used to detect the activity of p Mu PR1 c using GUS histochemical staining method. The results showed that the promoter was tissue-specific expression and induced by pathogenic fungi and bacteria.