Pest-resistance Of Pinellia Ternata Agglutinin And Establishment Of Gene Co-transformation System In Rice

Rice (Oryza sativa L) is an important worldwide cereal crop. It accounts for about fifty percent of cereal consumption in China. During growing period of rice,there are many stress factors restricting rice's yield enhancement,among which pests damage being a critical one. In this study. Pinellia Temata agglutinin (PTA) gene was introduced into mature seed-derived calli ofyaponica rice cultivar Eyi105. Zhonghua12 and indica rice line E-you532 through Agrobacterium tumefaciens-medtaied transformation,in which the "high toxic" strain EHA105 and the eukaryotic expression vector pCAMBIA1305 were adopted. Totally,36 PCR-positive transformants were obtained. This laid the foundation for further identification and selection for Brown leafhopper resistant rice germplasm and breeding materials. Furthermore,gus and npfll genes were constructed separately with their respective vectors and co-transformed into commercial rice cultivars Eyi105 and Zhonghua12 with a strain dosage ratio of 1:1 and 1:3 respectively. By using gus gene and hpt gene as selection markers,17 PCR-positive co-transformants were obtained. Several factors influencing Agrobacfe/ium-mediated co-transformation were discussed. This will shed light on the improvement of co-transformation efficiency in rice. The detailed results are as follows:1. Agrobacferium-mediated transformation of pta gene resulted in 36 PCR-positive rice TO transformants,of which 19, 12 and 10 transformants were gained in EY1105,Zhonghua12 and E-you532 respectively.2. Among the 8 PCR-positive transformants resulted from co-transformation of gus and npfll with the strain dosage ratio of 1:1,3 plants contained single gus gene and 5 contained both gus and npfll gene. Among the 9 PCR-positive transformants resulting from co-transformation with the ratio of 1:3. 2 plants contained single gus gene and 7 plants contained both gus and npfll co-transformed.3. In single gene transformation system,the transformation efficiency of pta gene was between 1.3% and 1.8%. In co-transformation system,two-gene co-transformation efficiency with the strain dosage ratio of 1:1 was higher than the resute with the ratio of 1:3. Southern blot analysis of the p/a-PCR positive transformants revealed different transgenic plants had different hybridization bands,indicating there were independent transformation events. Thus,Southern blot further confirmed that the pta gene had been integrated into rice genome.