| Rice is the staple food for a large number of people. However, rice blast disease, caused by the fungus Magnaporthe grisea, is one of the most devastating catastrophe in rice production. Now the main method to prevent such disease is to combine the high-yield resistant cultivars with fungistatic agent. Nevertheless, due to its complicated strain diversity, the resistance of most rice cultivars is usually disappeared after cultivated3to5years.Given to the situation above, on one hand, we catch the idea to introduce and express multiple broad-spectrum resistance genes in a single TAC vector and transform to rice to change its susceptibility. On the other hand, we attempt to accumulate resveratrol in rice through introducing the resveratrol synthase (RS) gene by transgenic methods, to make use of its natural antimicrobial effect, and broaden the way of rice disease resistance.Through multiple rounds of gene assembly with the Cre/loxP recombination system, multiple genes were sequentially delivered into the single TAC vector. Based on the method, we got the results as follows:1. We created constructs containing3blast resistant genes(Pid2,Pi37and Pi2) with two independent T regions and8valuable medial vectors.2. The construct with2resistant genes(Pid2and Pi37)were transferred into rice genome by Agrobacterium mediated transformation. Through the PCR test, GUS coloration and Real-time PCR test, we screen out the transgenetic rice including2resistant genes with high transcription levels, which guarantee two independent T regions integrated respectively. They will be used for subsequent disease resistance testing.For another part, a resveratrol synthase gene from two different Vitis spp. was obtained and placed under the control of Ubiquitin promoter respectively. The vectors were then introduced into japonica rice cultivar zhonghua11through Agrobacterium-mediated gene transformation, aimed at producing the phytoalexin resveratrol, a natural component of resistance to fungal diseases, synthesized by the key enzyme resveratrol synthase, and improving the rice resistance to the rice blast (caused by Magnaporthe grisea). Several results were obtained as below:1. We got8homozygous single-copy gene transformants through PCR test, Southern blot test and seed germination test.2. Most of the introduced genes were stable expressed in different single-copy gene transformants3. It is piceid (resveratrol-glucoside) not resveratrol detected in the leaves of the single-copy gene transformants, with the highest content10.26μg/g fresh weight, which is1000-fold to the wild type control. Resveratrol produced by the exogenous integrated gene should be metabolized into piceid by an endogenous glycosyltransferase.4. Although these transformants did not show apparent resistance towards Magnaporthe grisea, they still may have some beneficial effects on human health with the high content of piceid. |
