Establishment Of Safety Selection Marker System For Soybean By Lyr Gene

As the worldwide major oil crops, soybean is playing critical important role in social and economical benefits for human beings. Therefore, scientists are eager to solution for bigger output of soybean. The researchers use transgenic technology to produce insect-resistant, drought resistance, high yield and other new lines of genetically modified soybeans to meet the demand of soybean production. Genetically modified foods bring better benefits, however the transgenic plants based on the use of antibiotic-based and herbicides selectable marker genes in transgenic plants is questioned due to the ecological concerns and food safety issues raised mainly by environmentalists and consumers at the same time. So, transgenic plants without selectable marker has significant importantance.L-Lysine racemase( Lyr) exist in soil microorganism, it catalyze the mutual conversion between L-Lysine(L-Lys) and D- Lysine(D-Lys). wild plant cell is sensitive to L-Lys, but D-Lys can promote growth of plant cell, plant which is transformed with Lyr gene can grow naturally in medium which contain L-Lys and agar, but growth of plant which is not transformed with Lyr gene is inhibited. The experiment use Lyr gene to create security screening marker system of soybean, construct plant expression vector pBasta-Lyr with Lyr and Basta gene double gene marker adopt, use L-Lysine and Basta as the selection agent to determine regeneration selection pressure of the bud medium L-Lysine of cotyledon node, so that to compare the selection efficiency, growth state and conversion rate of genetically modified soybeans between two markers of transgenic soybean in the appropriate screening pressure, so that to provide reliable theoretical basis by using the Lyr gene as a security screening marker. The result of the experiment is as following:1. Lyr gene was synthetized and constructed to pBasta expression vector which resulted in a dual-labeled pBasta-Lyr with both L-Lys and Basta as selection markers.Then the recombinant pBasta-Lyr was transferred into Agrobacterium tumefaciens EHA105 for soybean transformation.2. By analyzing the shoot regeneration rate of soybean cotyledon nodes in different concentration bud medium of L-Lysine to determine selection pressure of two kinds soybean varieties(Jiyu72, Williams82) in the bud medium(SI) L-Lysine, the results showed that L-Lysine selection pressure which can lead to death of all cotyledon node for two kinds of soybean: Jiyu72 was16 mM; Williams82 was 22 mM.3. To compare the selection efficiency of L-Lys and Basta for the pBasta-Lyr, soybean(Jiyu 72, Williams82) was infected by Agrobacterium which contain pBasta-Lyr plasmid, and transformed plants were screened respectively by using L-Lysine and Basta as selection agent. The results showed that the regeneration rate of resistant shoots by selection agent L-Lys was 4% higher than that of selection agent Basta for the same varieties. In SE medium, resistant seedlings with selection agent L-Lys have more developed root system and sturdier stems than that of the screening agent Basta.4. Transformed plants of T0 and T1 generation were identified by PCR technology, and the corresponding transformation efficiency was caculated. The result showed that the transformation efficiency of T0 generation of Jiyu 72 soybean was 72 4.33% and 3.17% which selected by L-Lys and Basta, respectively; while the transformation efficiency of T1 generation of Williams82 was 5.33% and 0.83% which selected by L-Lys and Basta. Transformation efficiency of T1 generation of Jiyu 72 soybean was 1.50% and 0.67% which selected by L-Lys and Basta, respectively; while the transformation efficiency of T1 generation of Williams82 was 1.50% and 0.17% which selected by L-Lys and Basta. Furthermore, the Southern bloting results of partial T2 generation transgenic soybean agained implicated that the transformation efficiency of soybean selected by L-Lys was better than that by Basta. Taken together, theses results showed that L-Lys selection marker system superior to Basta selection marker system, which provided reference for the selective mark security issues on soybean transgenic technology.