The Construction Of High Efficient Expression Of Polycistronic Vectors In Chlamydomonas Reinhardtii Chloroplast

Chlamydomonas reinhardtii is always regared as a good bioreactor because of its unique characteristics. The chloroplast is superior to many systems, like the E.coli, yeast expression system. However, the low expression level of polycistronics hinders the mass production of recombinant proteins.In this work, we have sought to define an intercistronic expression element (IEE), which can effectively trigger the polycistronic mRNAs further processing into stable monocistronic transcripts. The IEE is a short sequence element, that is often located in the intercistronic cleavage sites and has the potential to fold into stable secondary structures. It provides a novel tool for transgene expression from chloroplast genome. Separation of foreign genes by this IEE will facilitate foreign genes stacking in operons, and thus will help to expand the applications of transplastomic technology.The major contents and results are as following:(1) The total DNA of Chlamydomonas reinhardtii was extracted, and the endogenous psbA promoter, terminator, Homologous sequences were a cloned by PCR. Then the psbA promoter was used to regulate the expression of the selectable marker gene aadA. Here the "aadA cassette" was also used as a negative control.(2) Two potential IEEs were amplified by using the chloroplast genome as template, and the two testing plasmids containg the IEE, spectinomycin resistance gene aadA and kanamycin resistance gene nptll were successfully constructed.(3) The "aadA cassette" and the two testing plasmids were transferred into c. reinhardtii using the method of glass beads, electroporation, and biolistic particle bombardment. In addition, electroporation parameters, the suitable concentration of antibiotics were also explored. The results showed the highest efficiency was obtained under 1000V pulse electric field. At the same time, the sutitable concentration of spectinomycin and kanamycin was 100μg/mL and 50μg/mL, respectively.(4) After several rounds of selection on spectinomycin plates, the recombinant transformants were obtained. The transformants grew well on kanamycin plates, which preliminary indicates the IEEs worked. However, it still remains some problems with its analysis on molecular level, the existence form of the plasmid DNA in chloroplasts remains unkown, further research is still needed.