Establishment Of Chlorella Pyrenoidosa Chloroplast Gene Transformation System

Chlorella pyrenoidosa is a photosynthetic microorganism and an edible microalga.In the development of genetically engineered food,compared with other microbial hosts,using C.pyrenoidosa as host expression protein has unique advantages: avoiding host organism safety evaluation;no toxin residue compared with fungi and viruses;having high production efficiency compared with higher plants.Compared with nuclear genome recombination,chloroplast gene recombination has many advantages,including more copies of chloroplast genome,expression reliably and no gene silence.However,because of the research on the chloroplast gene transformation system of C.pyrenoidosa is lack and the chloroplast genome sequence of C.pyrenoidosa has not been completed,there are still many difficulties in transgenic engineering using chloroplast gene recombination of C.pyrenoidosa.In the early stage,our research lab had used to design the chloroplast genetic homologous recombination vector and carry out the research on chloroplast gene transformation based on the genetic information of other plants and microalgae.In order to improve the results of chloroplast transformation,the chloroplast genome of C.pyrenoidosa was sequenced and codon bias was analyzed.Based on the above data,the plasmid vector of gene transformation was reconstructed and a new chloroplast gene transformation system of C.pyrenoidosa was established.In this paper,the total DNA of C.pyrenoidosa FACHB-5 was extracted,and then the chloroplast genome was sequenced.According to the obtained gene sequence,the codon bias of chloroplast was analyzed.The capsid protein of fish nerve necrosis virus(NNV)and the ? subunit of human nerve growth factor(h NGF)were used as the target proteins,to construct the corresponding chloroplast expression vectors p-423-RMCP and p-cp-ag?NGF by combining the sequence information of chloroplast genome and the codon bias information.The vector p-423-RMCP was transferred into C.pyrenoidosa by electroporation,and the transformation results were identified by PCR.The main contents and results of this paper are as follows:(1)The results of chloroplast genome sequencing of C.pyrenoidosa FACHB-5 have been submitted to Gene Bank with the login number MN128434.(2)The codon bias of C.pyrenoidosa chloroplast genome was strong,and the number of optimal codon was 16(TTC,TTA,ATC,GTA,TCT,CCA,ACT,GCT,TAC,CAA,AAC,GAC,GAG,TGT,CGT,GGT).(3)The main capsid protein gene DNA molecule of red grouper nerve necrosis virus(RGNNV)was synthesized,and the vector p-423-RGNNV was constructed.The vector p-423-RMCP was synthesized and its correctness by PCR and enzyme digestion.(4)The length of homologous arm was optimized according to the sequence of chloroplast genome,and the sequence of human nerve growth factor ? subunit gene(?-NGF)was optimized according to codon bias analysis of C.pyrenoidosa.Then,the chloroplast expression vector P-cp-ag?NGF expressing human nerve growth factor ?subunit was constructed.(5)Vector P-423-RMCP was transfected into alga cells by electroporation.Positive cells were obtained through antibiotic resistance screening and identified by PCR,which proved that the plasmid was successfully transfected into C.pyrenoidosa cells.The NNV is one of the important pathogens in fisheries.Construction of chloroplast vector to express NNV capsid protein can provide a foundation of research for prevention and control of fish diseases and food production safety.The h NGF is a kind of nerve cell growth regulator and an important biological drug resource.Constructing a C.pyrenoidosa chloroplast vector to express ?-NGF can make a basis for the development of important drugs.