Study On Isolation And Antifungal Activity Of Polygalacturonase-inhibiting Protein (pgip) Gene

The plant diseases are very troublesome problems for agricultural production, and more than 80% plant diseases are caused by fungus. There are a few of methods to prevent and control plant fungus disease. Firstly, the resistant cultivars are gained through traditional breeding method, and this way is very difficult to achieve breeders' purpose. Secondly, the chemical pesticide is applied to control diseases, and this method could make serious environmental contamination. Lastly, crop rotation is applied, however, this method is not enough effective. Application of the above three methods are limited to some extent, and can not resolve the problem of fungus disease thoroughly. In recent years, with the prompt development of molecular biology and genetic engineering, antifungal genes were transferred into plants, and eventually the new antifungal plant varieties were obtained. Transgenic approach is able to fundamentally resolve the problems caused by plant fungus diseases. The genes encoding polygalacturonase-inhibiting proteins (PGIPs) are a kind of antifungal genes which had acquired considerable attentions. PGIPs are multifunctional proteins related to plant autoimmunity and belong to plant extracellular leucine-rich repeat (eLRR) protein superfamily. Polygalacturonases (PGs) are the first hydrolases when fungus invades plant, which hydrolyze plant cell wall so that fungus can enter to plant cells. PGIPs can specifically recognize and combine PGs secreted by fungus, then reduce the hydrolytic activity of PGs and favor the accumulation of long-chain oligogalacturonides (OGs) which are elicitors of a variety of defense responses. Finally the purpose of inhibiting fungal is achieved through above responses. Torch pear (Pyrus pyrifolia Nakai) is a kind of local sand pear in Yunnan, which possesses strong adaptability to soil and resist black spot, rot, and Psylla Chinensis. At the same time, torch pear acclimatizes late frost and low temperature. In a word, the resistant genes of torch pear have certain value to be studied.In this study, PpPGIP was isolated from torch pear, and the full-length cDNA of PpPGIP was analysed by a series of bioinformatics methods. The nucleotide sequence of PpPGIP was highly homologous with PGIPs from other species, The protein encoded by PpPGIP has several conserved LRR domains. The putative 3D structure of PpPGIP is highly consistent with PvPGIP2 from bean (Phaseolus vulgar is). Therefor the gene cloned in present study is PGIPs of torch pear. Transcriptional expression of PpPGIP in torch pear leaves and peels was analyzed by RT-PCR, and the result showed the expression of PpPGIP in leaves was very low, but its expression in peels was relatively high. Then the constitutive plant expresssion vector pCAMBIA2300S-PpPGIP was constructed by DNA recombination technique, and transferred into Agrobacterium (LBA4404 and EHA105) by freeze-thawed method. Before the genetic transformation experiments, we first optimized the genetic transformation system of three plants, Nicotiana tabacum L. cv Xanthi, Dianthus caryophyllus, and Eustoma grandiflorum by GUS transient expression assays. The concentration of kanamycin about D. caryophyllus and E. grandiflorum was confirmed respectively for screening out transformed explants. PpPGIP is transferred into three kind of plants via Agrobacterium-mediated method. Through the preliminary selection with kanamycin, we got lots of anti-kanamycin regenerated plantlets, in which the tobacco transformants was 52, carnation transformants was 13, and lisianthus transformants was 65. The positive transgenic plantlets with 24 tobacco transformants and 8 lisianthus transformants were obtained by PCR of genomic DNA. Out of the 24 positive tobacco plantelts,10 plantelts were screened out because PpPGIP was expressed in transcriptional level.In order to further study the antifungal activity of PpPGIP, the above 10 transgenic tobacco plantlets were used to evaluate vitro antifungal activity of PpPGIP by plate antifungal test. The results indicated that all the 10 transgenic tobacco plantlets inhibited the growth of Phomopsis sp., Peyroneuaea, Alternaria sp., Aspergillus niger, and Penicillium sp. in different degrees.