Functional Analysis Of Pathogenesis-Related NP24 Protein In Tomato

It is a major problem to overcome plant disease problem that affecting the yield and quality of crops. Tomato is one of the most widely cultivated fruit vegetables in the world, and the affection of the disease situation is also quite grim. Traditional prevention measure such as improving the cultivation is limited to avoid of disease prevention. Chemical control brings the environmental pollution, and drug residues also creates a potential threat to human health. We can obtain new tomato varieties that have high disease-resistant ability by means of transgenic method. They overcome the long period of traditional breeding, species isolation and other issues, which undoubtedly opens up a new road for the selection and optimization of tomato germplasm.NP24 gene is a member of PR5 pathogenesis-related protein family. Many commentators have noted that its biological functions, the molecular mechanisms in plant disease resistance and the regulation of defense gene study increasely day by day. Particularly their relationship with disease resistance in plants has attacted scholars' attention. The cloning and validation of gene function is necessary and have great value on tomato breeding. In this study, we cloned NP24 gene and analyzed tissue expression patterns from the wild-type tomato material. Aslo we use software to analyze and predict its biological function and evolutionary relationships. Meanwhile, over-expression and interfering vector of the gene were constructed. Therefore, we obtained overexpression transgenic lines and interference lines by genetic transformation respectively. For the first time we purified actived NP24 protein in vivo by eukaryotic expression, and done in vivo vaccination experiments which verifies the relationship between NP24 protein and resistance of the plant pathogenesis. This study obtained following results:(1) We cloned NP24 gene which contains 718 nucleotides of the open reading frame, and encods 247 amino acids. It belongs to TLPS of PR5 pathogenesis-related protein family.(2) Tissue-specific and temporal expression patterns: we detected NP24 gene expression of wild-type tomato in different tissues and organs such as root(R), stems(S), leaves(L), flowers(FL), fruit 0(IM0), fruit 10(IM10), fruit 20(IM20), fruit 30(IM30), mature green(MG), broken color(Br), orange fruit(YF), red fruit(RF) by RT-qPCR. The results show that the high expression in roots and fruits, especially in breaking fruit stage.(3) Construction of transgene vector: We successfully constructed over-expression vector and interference vector of the NP24 gene and obtained transgenic plants by genetic transformation. Drought stress experiment found that the overexpression transgenic plants have more tolerance than the wild type. The interference transgenic plants appear dwarf phenotype. The gene plays an important role in growth and development of the plant.(4) Eukaryotic expression: For the first time we purified actived NP24 protein in vivo by eukaryotic expression, and enzyme activity show it has ?-1,3-glucanase. NP24 can degrade the ?-1,3-glucan on fungal spore surface, which may affect the permeability of the cell wall and thus inhibit its growth.