Studies Of Transformation Of TLPD34 Gene Into Maize By Agrobacterium-Mediated And Detection

Excellent maize inbred line Zheng 58 and ye 478 as receptor material, the shoot apical point and young ears ( zygote ) of maize as transformation receptors, using the Agrobacterium-mediated transformation technology. The rice thaumatin-like protein (TLPD34) gene that antifungal diseases was transferred into corn, after selecting with glufosinate, molecule detection and Fungi inoculation test, we have conclusions as followed:1. Through the plasmid pUbiTLPD34 complete physical map, choose enzymes cut plasmid sites, BamHI and HindIII, single and double digestion, get each segment with the same size as expected, the purpose clip is about 1100bp, the similarity is reached 97% after plasmid sequencing result compare with the sequencethat had submitted in NCBI.2. Bar Gene as screening marker gene, choose glufosinate herbicide, configurate different mass concentration herbicides solution (glufosinate), smear non-Transformed plant of corn in 3 leaf stage, 200 mg/L glufosinate for genetically modified corn is best screening mass concentration.3. Through Agrobacterium-mediated transformation, the gene was transferred into the shoot apical point of maize inbred line zheng 58, we have obtained 451 transgenic plants in all. After selecting with 200 mg/L effective mass concentration glufosinate, 57 transgenic plants were obtained, after T0 generation test, 13 positive transgenic plants were confirmed by PCR., the transformation frequency is about 2.9 %. 7 plants obtained after confirmed by RT-PCR amplification. The study showed that with this method the foreign gene TLPD34 has integrated into maize inbred line zheng 58 genome and transcription normally.4. Cell suspension of Agrobacterium tumefaciens (OD=0.6), carrying TLPD34 genes, was applied onto the young ears of Zheng 58 and Ye 478, which had been previously pollinated for 8-12h with the pollen(Zygote Transformation). We obtained 261 and 322 seeds respectively, percentage of germination all reached more than 70%, after the same screening concentration with glufosinate, zheng 58 got 7.42 % resistant plantlets,ye 478 got 10.24 % resistant plantlets, after confirmed by PCR,the transformation frequency is about 0.99 % and 1.18 %, thus the effect is better for ye 478 compare to zheng 58 with this method. 5. Use the shoot apical point and young ears(zygote) of zheng 58 as transformation receptors, use the same cell suspension of Agrobacterium tumefaciens(OD=0.6), the same concentration of herbicides screening, get 12.64 % and 7.42% resistant seedlings respectively, after confirmed by PCR,the transformation frequency is about 2.9 % and 0.99 %. The study showed that the method with shoot apical point as transformation receptor is better for zheng 58.6. The fungi inoculation test For transgenic and non-modified corn, corn started feeling sick at jointing stage. The transgenic seedlings have high resistance to Fusarium graminearum compared to non-modified seedlings, but transgenic seedlings resistance to Fusarium maniliforme increased significantly, non-modified seedlings leaf beginning to yellow, plant growing weaker, roots became corruption, and eventually death.7. At jointing stage, several wheat seeds co-cultivate with Rhizoctonia solani were placed into the third sheath of the maize, photographic observation after 12h,24h,36h,48h,72h,84h and 96h , compared with transgenic seedlings, non-modified seedlings acquired susceptibility later, and after fungal infection, transgenic seedlings can inhibit the spread of the mycelium further expansion,the resistance to disease is improved.