Transformation Of Cry1Ia8Gene And Gshlp2Gene In Maize Inbred Lines

Maize (Zea Maya L.) is an importment cereal crop in the world today, and china is the second largest corn-producing country in the world.However, because the low content of lysine in the maize, the actual utilization of protein in the maize is only40%-50%.With the improvement of people living standards and structure changes in dietary, the demand to improve the puality of people of maize protein is increasing. On the other hand, because of the occurrence of pests, result in a great loss to the maize production, and the Pyrausta nubilalis (Hubern) is the main pest in order to improve the quality of maize and enhance it’s produetion, it’s important to use the genetic engineering method to transfer target gene into maize. It’s applicable to use Agrobacterium-mediated systerm to obtain transgenic plants of maize.In this study, seven corn inbred lines were cultured to induction the callus. Choose the good embryogenic callus as the receptor of Agrobacterium-mediated genetic transformation.And the double T-DNA expression vector that with crylla8and bar or with GsHLP2and bar was used in the transformed of maize callus, Screening the plants of co-transformationed. Provide material for the cultivate of marker-free transgenic maize by genetic segregation of the offspring of transgenic plants.The major results were as follows:1. Construction of plant expression vector of cryⅡa8gene.Constructed the plant expression vectors of crylla8gene that controled by the Constitutively strong Ubi promotor and it’s intron.2. Establish the system of genetic transformation of Agrobacterium-mediated of maize inbred lines.Seven combination of maize inbred lines and media were filted, select the most appropriate combination for callus induction inbred lines and media, the initial callus induction rate of B73(N6)和HiⅡB (NB) is82.3%、73.9%and the embryogenic callus induction rate of B73(N6)和HiⅡB (NB) is70.5%、63.8%, the combination of B73(N6) is better than HiⅡB (NB),so choose the callus of B73as the acceptor of genetic transformation The systerm of tissye culture of maize and transformation has been optimized and established a optimal condition of Agrobacterium-mediated transformation of embrygenic callus of maize inbred line.the optimized condition is:the numerical value of OD600of Agrobacterium is0.6, the time of infect is20min and the temperature of co-culture is23℃. The bialaphos screen pressure of immature maize calli of B73is2mg/L。3. The screening and identification of Transgenic maize plantsThe embryonic calli of B73were transformationed by Agrobacterium-mediated method, obtained160regenerated plants, PCR analysis showed that there were34regenerated plants with the intergration of bar gene, including4plants with the intergration of cry1Ia8gene,3plants with the intergration of GsHLP2gene and1plant fructify in the3plants.