Tomato Root-knot Nematode Resistant Gene SlMi Transferring Into Lettuce (Lactuca Sativa.)

Plant root knot nematodes (Meloidogyne spp.), one of the four major plant infection pathogens, are parasitic roundworms that damage more than 3000 different crops. Root knot nematodes cause major economic damage to horticulture and agriculture in China and around the world. This research was to clone the root knot nematode resistance gene SlMi from tomato by the method of homologous sequence and established high-frequency regeneration and transformation protocols for lettuce (Lactuca sativa). Then SlMi gene was transferred into lettuce in which no genetic resources of root knot nematode resistance had been identified by Agrobacterium mediated transformation and we obtained the transgenic lettuce which laid a promising foundation for root knot nematodes resistance breeding, it also provided good perspective for the successful introduction of foreign genes. The main results were as follows:1. The protocols of lettuce regeneration and transformation were established. Through the comparison of 16 different medium suggested, the regeneration capabilities of different genotypes were varied on different medium. Among 3 cultivars tested, NaireErbaipi (Er) was the best one and the regeneration shoot ratio was more than 90ï¼…on seven different mediums. Its optimal medium is MS+6-BA 0.5mg/L+NAA 0.20mg/L with the regeneration shoot ratio of 99.21ï¼…. Huaxuan No.1 (HX) is the second and the regeneration shoot ratio is more than 90ï¼…on four mediums. The best one reached 97.58ï¼…on MS+6-BA 1.0mg/L+NAA 0.25mg/L; The regeneration shoot ratio of Naichoutaishengcai (Nai) is more than 90ï¼…on three mediums and the highest one reached 94.94ï¼…on MS+6-BA 0.5mg/L+NAA 0.25mg/L. 100 mg/L kanamycin can completely inhibited.2. Tomato root knot nematode resistance gene SlMi was cloned based on the published sequence (AF039682) of Mi by homologous sequence method. And the sense vector of SlMi gene was constructed.3. The SlMi gene was successfully transferred into the genome of lettuce cultivar Huaxuan No.1 (HX) by the Agrobacterium mediated transformation method. A total of 24 kanamycin resistance plants were obtained. Four of them were identified by PCR and southern blotting and three plants were confirmed to be transgenic plants with 1 to 3 copies of T-DNA integration.4. Semi-quantitative RT-PCR analysis indicated that the expression level of target gene was higher in the three transgenic plants than that in the control. 5. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in the three transgenic plants compared to untransformed susceptible control.6. Germination ratio and morphological comparison between T₁ transgenic plant seeds and the control indicated that the single copy plants L1, L4 weren't different with control, but the three copies plant L3 had great variation from the control.7. Paraffin section analysis indicated that the root ultrastructure was remarkably changed after root-knot nematode invading. In the root of untransformed susceptible plants, there were lots of nematodes and mutinucleotide giant cells. But in the root of sense SlMi transgenic plants, hypersensitive response (HR) was elicited and cell death was observed around the nematode after nematodes penetrated the root.