Expression Of Antimicrobial Peptides And Chitinase Gene In Transgenic Tobacco Plants Confers Enhanced Resistance To Black Shank And Brown Leaf Spot

Fungal diseases are one of the major factors limiting crops production worldwide.The important guarantee of agriculture foodstuff safety is to breed new disease resistance variety by using new antifungal resource and genes.It is offered a feasible approach with gene engineering technique basing on molecular genetics and molecular biology.At present, one of the important strategies to breed disease resistance variety is transfered disease resistence genes into plant to enhance resistance.In this paper, we selected the leaf dish of Nicotiana benthamiana as the explants and introduced six different genes (SPCEMA, AFP, CHI,SPCEMA-CHI, AFP-CH1, AFP-SPCEMA)into tobacco by Agrobacterium mediated transformation, we compared the resistant effect of different foreign genes in transgenic plants. The results are as following:1. Development of examination method of disease resistanceFrom experiment,it was shown that sesame agar can be used to culture Phytophthora nicatianae which grew well on it.At the same time, a new culture method which can produce a lot of zoosporangia was obstained.The outputs of zoosporangia were increased from 5×104conidia/ml to 1.5×105conidia/ml.The inoculative concentration of Phytophthora nicatianae and Alternaria alternate were 1 ×104conidia/ml and 5×105 个/ml.respectively.2. Acquirement of transgenic tobacco of different genesSterile tobacco leaves sliced into leaf dishes (0.5cm2/tablet) were immerged into theAgrobacterium suspension of OD600 0.1-0.2 for 5min, and the leaf dishes were co-cultured on MSB1(MSB0+ 2.0mg/L BA +0.1mg/L NAA) medium covered with a sterile filter paper disc for 3d indarkness at 24℃. After co-culture was over, the exolants were directlv transferred to the selectionMedium MSB2 (MSB1+Cef500mg/L+Km100mg/L) for three weeks. The explants were selected twice. After green buds appeared, they were sliced and transferred to radication medium MSB3 (MSB|+ Cef500mg/L+Kml50mg/L). Finally, green seedlings were selected for further assays.3. Molecular assays of transgenic plantExplants from the Kanamycin-resistant regenerated plants were stained for assaying GUS activity using a protocol derived from Jefferson (1987). Expression of the GUS gene confirmed that the T-DNA had been transferred and integrated into the regenerated plants. At the same time, the introduction of the target genes were confirmed by PCR analysis of the DNA samples isolated from the GUS positive regenerated plants.4. Resistance analysis4.1 Inoculation with Phytophthora nicatianae of TO- transgenic plantsTo-transgenic plants of different genes were inoculated with Phytophthora nicatianae in greenhouse. The results showed that the resistance of transgenic plants containing different g enes was strengthened distinctively, compared with control. The disease index of the six gen es were AFP-CHI41.67%, AFP-SPCEMA58.33%, SPCEMA-CHI60.87%, AFP63.71%,SPCEM A63.89%,CHI70.54%.4.2 Inoculation with Alternaria alternata of T1-transgenic plantsT1-transgenic plants of different genes were inoculated with Alternaria alternat in green house.The results manifested that the resistance of transgenic plants containing different gene s was strengthened distinctively, compared with control.The disease index of the six genes w ere AFP-CHI37.81%, AFP-SPCEMA39.69%, SPCEMA-CHI40.00%, AFP51.25%,SPCEMA55.6 3%,CHI56.25%.From the above,we can see that all of the six genes had antifungal effect an d can be transmitted and expressed in offspring.5. Assaying the stability of descendiblity in transgenic plantswe randomly selected two plants from transgenic plants of every gene. After germinated, they were stained for assaying GUS activity. The x c2 aptness workout indicated that the heritability of foreign gene accorded with the law of segregation.