Antifungal Protein PscAFP Expressed By Yeast And The Analysis Disease Resistance Of Transgenic Tobacco

All crops are subject to disease both in the field and post-harvest, the major groups of pathogens being viruses, bacteria, fungi, and nematodes. These biotic constraints can, at times, severely restrict agricultural development and seriously compromise food security. Moreover, plant diseases caused a great of loss of the production and lower the quality of production. A variety of pesticides is available for plant disease control, however, applying too little pesticide will result in lack of disease control, and applying excessive amounts may lead to damage plants and residue problems. At the same time, it may result chemical resistance followed by the appling of pesticide and lower the efficiency of pesticide. It is timely and more effort that selecting the varieties of resistant by the traditional methods (hybridization breeding), and the traditional breeding methods can not meet the need for agriculture. With the development of molecular biology, we can use the antifungal protein genes and the disease resistance genes to enhance the resistant of crop by genetic modified approach.In this paper, we isolated a protein with trypsin inhibitor activity from a herb, Psoraleae corylifolia seed. The protein, designated PscAFP, decreased the growth speed of bacterial and fungi, in vitro. Then we cloned the sequence of PscAFP, expressed it using Pichia yeast, and tested the activity of the recombined protein (PscAFP). Moreover, the gene was transformed into tobacco, and the disease resistance of the transgenic tobacco was detected. The result is that the resistance of transgenic tobacco was improved, compared with the wild type.The main results are as follow:1. We obtained the sequence of PscAFP.2. The expression vector pPIC9K-PscAFP was constructed, then it was transformated into Pichia yeast, and 1% methanol was used to induce the expression of PscAFP in yeast. A protein was expressed by yeast with the molecular mass about 20KDa.3. The recombined PscAFP with the ability of lower the capability of trypsin to hydrolysis subtrate(Phe-Val-Arg-PNA, Boc-Gly-Pro-PNA, BNPNA, BANA). 4. The recombinate protein, PscAFP, can apparently inhibit the spore germination of A. alterant.5. The plant expression vector P5-PscAFP was constructed, then it was transformed into tobacco by Agrobacterium tumefaciens, and the transgenic tobaccos with strong resistance against fungal pathogen, A. alterant, bacterial pathogen Ralstonia solanacearum infection were obtained, compered with the wild type.