Preliminary Study Of Genetic Transformation Of Two Species Of Microalgae By Agrobacterium Tumefaciens

With the rapid development of genetic engineering for microalgae, the use ofgenetic engineering methods to conduct the thorough research and geneticmodification to the algae has been increasingly need. However, genetictransformation system for algae is still a limiting step, hasn’t been resolvedcompletely. Agrobacterium tumefaciens is a kind of natural genetic transformationsystem with simple operation, low cost and high transformation efficiency andstability.Therefore it becomes a good choice in the genetic transformation of higherplants. In recent years, some studies reported the successful transformation formicroalgae mediated by Agrobacterium, using the method transplanted from higherplants.Chlamydomonas reinhardtii as well as Chlorella ellipsoidea are commonly usedspecies in microalgal genetic engineering. In this experiment, we are trying to realizethe foreign gene expression in these algal cell using Agrobacterium-mediatedmethod by constructing the binary vector of Agrobacterium with gfp as the reportorgene.The specific content and results of this experiment are as follows:1. The prokaryotic expression vector pET-28a-gfp was constructed and andtransferedinto E. coli BL21. Then the expression of GFP protein was induced byIPTG. The results show that the GFP protein is successfully expressed in thisprokaryotic expression system with the specific green fluerescence observed under thefluerescent microscope, which gets ready for the transformation of microalgae fornext step using Agrobacterium-mediated system.2. The cultural suspensions of Chlamydomonas reinhardtii CC-124andChlorella ellipsoidea FACHB-40were spread to the plates with a serial differenthygromycinconcentration to test the sensitivity of these two algal strains to thisantibiotics Thus we select30μg/ml and80μg/ml as the selective pressure respectively for the genetic transformation of Chlamydomonas and Chlorella in thisexperiment.3.The report gene gfp and selection marker gene hpt are inserted in series intoagrobacterium binary transformation vector pSB130to build efficient geneticexpression vector. We add different eukaryotic promoter and terminator to both endsof gfp and hpt gene, to ensure the expression of each of the two gene. The promotersinclude the eukaryotic viral promoter CaMV35S which is commonly used in theexpression of foreign genes in higher plants and eukaryotic algae, and the highefficient promoter R which we cloned from soil unicellular green algae CocoomycaC-169. We have constructed a agrobacterium binary transformation vectorpSB130-35S-gfp-35S-hpt which starts the expression of GFP by promoter CaMV35S,and the other vector pSB130-35S-hpt-R-gfp which starts the expression of GFP bypromoter R. And these two vectors have been transferred into agrobacterium strainEHA105by electroporation successfully, and get final agrobacterium transformationstrainwith both reportor gene gfp and selective marker gene hpt.4.The genetic transformation is conducted by co-cultivation of theagrobacterium cells containing the constructed vector together with these microalgamentioed above. Then these alga were re-transfered to solid plates with hygromycin Bto screen. However, because of the time limitation, we hasn’t got the positivecolonies.Through the experiments, we will set up a set of efficient, practical, and easymicroalgae transgenic system mediated by Agrobacterium Ti plasmid in ourlaboratory. We expect the first success will be realized in Chlamydomonasreinhardtii and Chlorella ellipsoidea with the reporter gene, which will lay thefoundation for the study of gene function of algae in the future.