High-frequency Electroporation and Expression of Human Interleukin 4 Gene in Chlamydomonas reinhardtii Chloroplast

ZHAO Ya-kun~(1)) SHI Xian-ming~(1)) ZHANG Zhong-ming~(2))(~(1)) School of Agriculture & Biology,Shanghai Jiaotong University,Shanghai 201101,China;~(2))State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China)

human interleukin 4(hIL4);C.reinhardtii;chloroplast genetic engineering;electroporation

An electroporation-mediated technique for the research of hIL4 gene expression within Chlamydomonas reinhardtii Chloroplast was developed.Human Interleukin 4(hIL4) is a key immunomodulatory factor importantly related with autoimmune disease.In order to enhance the stability of hIL4 gene expression,hIL4 gene was fused with C.reinhardtii chloroplast rbcL 5'-and 3'-untranslated regions(UTRs) to construct the 5'UTR-hIL4-3'UTR fragment.The fused fragment was then introduced into the expression vector p322-BTM to obtain homologous recombinant plasmid pXhIL4,which contains a homologous fragment(5.7 kb) with C.reinhardtii chloroplast.Cells of C.reinhardtii were cotransformed by electroporation with plasmid pXhIL4 and the selectable marker plasmid p228 conferring the resistance to spectinomycin.Transformants were screened on the plates containing 100 μg/ mL spectinomycin.The positive transformants were picked and firstly confirmed by PCR,followed by western blotting analysis for the accumulation of hIL4 protein.Results showed that electroporation is an effective approach for the cotransformation of C.reinhardtii chloroplast with the efficiency of cotransformation up to 90%.The western blotting analysis indicated that hIL4 gene is successfully expressed in C.reinhardtii strains.