Construction Of Nuclear Transformation System Of Chlamydomonas Reinhardtii And Expression Of Two Exogenous Proteins

Chlamydomonas reinhardtii is a mature model organism whose nuclear,mitochondria and chloroplast genomes can be genetically transformed,and it has many advantages such as simple culture conditions,fast growth and high photosynthetic efficiency.Based on the nuclear transformation system,“Chlamydomonas reinhardtii bioreactor” can be constructed to produce high value-added proteins,which has broad application prospects.In this study,we first constructed the nuclear transformation system of Chlamydomonas reinhardtii,studied the transformation of circular plasmids and successfully expressed human epidermal growth factor(EGF)protein,secondly,we studied the transformation of linear plasmids,analyzed the conditions of single digestion and co-transformation of linear plasmids.The above research provides theoretical basis and experimental guidance for “Chlamydomonas reinhardtii bioreactor”.(1)By studying the key factors affecting the nuclear transformation of Chlamydomonas reinhardtii,the nuclear transformation system of Chlamydomonas reinhardtii was successfully constructed.The culture conditions of Chlamydomonas reinhardtii were as follows: the inoculation amount was 1%,the culture temperature was 20?,the light-dark period was 16h/8h,the illumination intensity was 12000 Lx,the shaking speed was 120r/min,after 5 days of culture,Chlamydomonas reinhardtii reached the middle logarithmic growth stage and its color was dark green.The transformation conditions of Chlamydomonas reinhardtii were as follows: Chlamydomonas reinhardtii was cultured to logarithmic medium,the optimum plasmid dosage was 3?g,the optimum votrex time was 20 s,the screening concentration of zeocin resistance in TAP solid medium was 15?g/mL,and the screening concentration of zeocin resistance in TAP liquid medium was 5?g/mL.The results of PCR and RT-PCR detection showed that ble gene had been successfully integrated into the nuclear genome of Chlamydomonas reinhardtii and expressed at the transcriptional level.(2)Based on the construction of nuclear transformation system of Chlamydomonas reinhardtii,the transformation and expression of human epidermal growth factor(EGF)gene was explored.Firstly,the EGF gene was reconstructed and synthesized according to the codon preference of Chlamydomonas reinhardtii nucleus,and linked to construct the pSP108-EGF expression vector.The pSP108-EGF vector was transformed by glass-beads method,the transformation results were detected by 15?g/mL zeocin resistance screening,PCR,Southern blot and ELISA.The results showed that three positive transformants were obtained,both EGF and ble genes had been successfully integrated into the nuclear genome of three positive transformants.The EGF genes of three positive transformants existed in a single copy and were integrated at different sites in the nuclear genome.The concentration of EGF protein was respectively 19.4 pg/mL,17.1 pg/mL and 19.7 pg/ mL.(3)The transformation of linear plasmids by glass-beads method was studied.The single enzyme cutting test of pSP108 plasmid was carried out,the linear plasmid was transformed by glass-beads methold and key transformation conditions were optimized,finally,the transformation result was verified by resistence screening,PCR and RT-PCR detection.The results showed that the pSP108 linear plasmids single-enzymed by BamH?,Xba ?,EcoR ? and Sac?could be successfully transformed,and the transformation efficency of BamH I was the highest.The ble gene had been successfully integrated into the nuclear genome of Chlamydomonas reinhardtii and expressed at the transcriptional level.The key conditions for the transformation of linear plasmids by glass-beads method were as follows: using Chlamydomonas reinhardtii in logarithmic medium growth stage,the optimum dosage of plasmids was 4?g,the optimum vortex time was 25 s,and the optimum screening concentration of zeocin was 15?g/mL.(4)Based on the study of transformation of linear plasmid by glass-beads method,the C-reactive protein(CRP)gene was reconstructed and synthesized according to the codon preference of Chlamydomonas reinhardtii.The pCRP vector was successfully constructed.The pCRP and pSP108 linear plasmids were prepared by single digestion of BamH I and co-transformed by glass-beads method.The transformant DNA of Chlamydomonas reinhardtii was extracted through resistance screening with 15?g/mL zeocin.The results of PCR detection showed that both ble and CRP genes had been successfully integrated into the nuclear genome of Chlamydomonas reinhardtii.RT-PCR detection showed that ble gene was expressed at transcriptional level but CRP gene maybe appeared "gene silencing" phenomenon.