The Phytopathgon Resistance Of An Endochitinase Gene (Bbchit1) From The Entomopathogenic Fungi, Beauveria Bassiana

Chitinase is a group of enzymes which catalyze the hydrolysis ofβ-1,4-glycosidic bonds of chitin, an important component of many fungal cell wall. With the antifungal activity, chitinase has i attracted a lot of efforts to enhance plant resistance to pathogens by overexpressing this kind of genes in plants. In this study, we prepared a Beauveria bassiana chitinase Bbchitl using Pichia pastoris expression system, and determined the inhibition effects of Bbchitl on spore germination and mycelial growth of several plant pathogenic fungi. Then, the Bbchitl gene was transferred into tobaccos (Nicotiana tabacum), and 5 homozygous T₂ lines were selected to study the pathogen resistance in vivo. The main results are as followings :1. Inhibition Effects of chitinase Bbchitl on spore germination and mycelial growth of several phytopathogenic fungi were tested. Bbchitl can inhibit the mycelial growth of Botrytis cinerea, Sclerotinia sclerotiorum, Bipolaris maydis, Altenaria brassicae and Penicillium digitatum, and the spore germination of Alternaria alternata, Altenaria brassicae, Aspergillus niger, Verticillium dahliae and Penicillium digitatum.2. GUS histochemical staining and Southern blot analysis showed that Bbchitl gene has been integrated into the tobacco genome. Chitinase activity measurement indicated that the representative transgenic lines had higher chitinase activity than wild type. Western blot analysis showed that Bbchitl gene expressed stably in transgenic tobacco.3. Five homozygous lines were selected to assay disease resistance. when challenged by a bacterial pathogen Ralstonia solanacearum, the disease index of 5 T₂ homozygous lines (Bb35, Bb49, Bb57, Bb63, and Bb71) decreased by 8.75%, 53.75% 3.75%, 33.75%, and 23.75%, respectively. When inoculated with fungal pathogen Alternaria alternata, the lesion areas of the 5 transgenic lines reduced by 39.11%, 35.63%, 43.62%, 57.22%, and 61.36%. respectively. These observation suggested that the Bbchitl might be potential for improvement of plants resistance against a wide range of phytopathogenic fungi and bacteria by gene engineering.