Research Of The Agrobacterium-mediated Transformation Condition Optimization And Transformation Of BCA Gene In Lentinus Edodes

In this research,we successfully construct Lentinus edodes expression vector of BCA gene, pBHg-BCA.And then we explore the Agrobacterium-mediated transformation system of Lentinus edodes. Though optimizing the conditions of transformation,we establish the Agrobacterium-mediated transformation system of Lentinus edodes. On this basis, we transform BCA gene into Lentinus edodes. The main research results are as follows:1,Amplified BCA gene from plasmid pCB-BCA, and added restriction site KpnⅠat 5'terminus and XbaⅠat 3'terminus.After recovery, it was inserted into plasmid 1300-pEGFP, replacing EGFP gene. The recombinant plasmid was named as 1300-BCA.Then on this basis, we amplified BCA gene expression cassette from plasmid 1300-BCA, including Agaricus bisparus GPD promoter,BCAgene and 35S terminator, and added restriction site BamHⅠat 5'terminus and SalⅠat 3'terminus. After recovery, it was inserted into the multiple cloning site of plasmid pBHg. The recombinant plasmid was named as pBHg-BCA.This is the Lentinus edodes expression vector of BCA gene.2,Through the experiment on hygromycin B resistance of Lentinus edodes, determined that the hygromycin B lethal concentration of Lentinus edodes L0071 is between 5μg/ml and 10μg/ml.According this,we make 10μg/ml the primary screening concentration of Hyg B in Agrobacterium-mediated transformation,and 20μg/ml the secondary screening concentration.In the experiment on Cefotaxime Sodium toxicity on Lentinus edodes,we make 400μg/ml the best bacteriostasis concentration.3,Using the experiment on Agrobacterium-mediated transformation of Agaricus bisporus by Chen et. for reference, we explore the Agrobacterium-mediated transformation system of Lentinus edodes from six aspects, Agrobacterium, tissues, infection concentrations, infection time, co-cultivation time and concentrations of AS,in order to find the best transformation conditions.Finally we determined that the Agrobacterium strain EHA105 is the better one.The best transformation material is the young and tender lamella tissue of fruiting body.The best infection concentrations of bacterial liquid is OD600=0.5.And the best co-cultivation time is 3 days.The best induced concentration of AS is 300μM. But for the infection time of bacterial liquid,we find no significant differences.So in the following research,we will use the same infection time as Chen used. Consequently we built the Agrobacterium-mediated transformation system of Lentinus edodes.4,On the basis of frontal research,we used the Agrobacterium-mediated transformation system of Lentinus edodes we built to transform recombinant plasmid pBHg-BCA into Lentinus edodes.And then the BCA gene expression cassette can integrate into the genome DNA of Lentinus edodes.Through the verification of the transformants which have hygromycin B resistance,we finally determined that we successfully obtain the BCA gene transgenic Lentinus edodes.