AiiA Gene Transformed Amorphophallus Konjac Resistant To Soft Rot And Component Analysis Of An Antibiotic Protein Produced By An A. Konjac Endophyte

The research includs two parts:one is identification of resistance to soft rot of aiiA gene transformed Amorphophallus konjac;the other is two-dimensional electrophoresis and MALDI-TOF-MS analysis of a novel antibiotic protein Apn5 produced by Bacillus subtilis BSn5.In order to search for the encoding gene of Apn5,some job for gene knock-out has been done.1.Amorphophallus konjac is one of the important economic crops in our country, which is widely used in food,medical,chemical and construction industry.But its production is threatened seriously by soft rot caused by Erwina carotovora subsp. carotovora.This pathogen is a gram-negative bacterial with wide host.The expression of its pathogenic gene is regulated through Quorum sensing system(QS system).AiiA gene is widely distributed among Bacillus,encoding an enzyme which degrades Acyl-homoserine lactone(AHL) and attenuates disease by distributing QS system of the pathogen.In this study,34 aiiA gene transformed A.konjac were planted in greenhouse,and disease resistance detection for the transgenic plants was also carried out.The results revealed that A.konjac grew well under greenhouse conditions and there were no disease or insect damage happened.Leaves of transgenic and control A.konjac were incubated with 2×10~5,2×10~7,2×10~9 cfu of Erwina carotovora SCG1,morbidity was observed 96 h later.The results showed that all of the tested transgenic A.konjac lines exhibited high resistance to soft rot bacteria SCG1,the disease symptom was significantly alleviated and the rot area was also significantly decreased.The resistance to soft rot detection was carried out continously for two years,the same results were obtained.These results proved that aiiA gene conferred soft rot disease resistance to A.konjac and the exogenous gene was integrated stably into the genome of A.konjac and was heritable.2.Bacillus subtilis BSn5,an endophyte isolated from A.konjac calli,produces extracellular protein Apn5 which could inhibit Erwinia carotovora subsp,carotovora strain SCG1.To study the characteristics of Apn5 and to find its encoding gene,Apn5 was isolated from the BSn5 culture by ammonium sulfate precipitation with 30%relative saturation and further purified by ultrafiltration,then subjected to two-dimensional electrophoresis and MALDI-TOF-MS analysis.The peptide mass fingerprinting for each protein spot was searched in the NCBInr database with Mascot software.300μg Apn5 was prepared for two-demensional electrophoresis,and separated into 13 protein spots, which mainly centralized within molecular weight 10-30 kDa and pI 4-5.5.11 of these spots were identified with MALDI-TOF-MS,while only 4 were believable with mascot score greater than 51,and they were all flagellum proteins.Others were unbelievable with mascot score less than 51.To prove the relationship between Apn5 and flagellum protein,we attempted to knock out the flagellum gene in BSn5 in order to detect its influence on the production, characteristics,antibacterial activation etc.of Apn5.A double-exchange vector pUC19-hag::Erm was constructed in this study,and knock-out of the hag gene needs to be continued.