Research On The Construction Of Chloroplast Expression Vector In Pyramimonas Sp.

Medicine of protein and polypeptide with high value can not only create efficient economic value, but also bring practical effect to the health of human beings and the development of science. However, the cost of producing such medicine is very high because the main method to produce these medicine are mainly depending on the fermentation of animal cell and microorganism, which is too complicated to be improved in a short time. Generally, chloroplasts, an important organelle. exist in every plant cells, which also have the ability of photosynthesis and is very suitable for genetic transformation due to their merits and characteristics, so, expressed medicine of protein and polypeptide with high value in chloroplast. More than ten kinds of plants have been already successfully transformed in the chloroplasts transformation system of higher plants, the similar researches have been also performed on algae cell, as well as feed Chlamydomonas, Dunaliella salina, chlorella and so on. In the present study. Pyramimonas sp., which has not be reported yet, was used the research object, few work of prophase vector construction were done in order to build chloroplasts expression system of Pyramimonas sp..(1) The method of CTAB, which has been improved, was used to extract the total DNA of Pyramimonas sp., at the same time, we designed the primer and got the band that we wanted; the DNA which we got was used as the template, the primer was also designed according to the genome sequence of Pyramimonas parkeae chloroplast, Homologous clips of chlL and chlN were augmented, as a result, chlL gene was obtained.(2) The sensitivity of seven kinds of antibiotic towards Pyramimonas sp. was studied, such as Chloramphenicol, Neomycin, Streptomycin, Ampicillin, Spectinomycin, Zeocin and G-418, all of which are commonly used. Results showed that penbritin, neomycin, streptomycin and spectinomycin had no inhibitory effect on Pyramimonas sp., however, G-418had significant inhibitory effect under high concentration, zeocin and chloramphenicol also had obvious effect. In the present study, the ble gene, corresponding to zeocin, was used to screen the lable gene.(3) According to the genome sequence of Pyramimonas parkeae chloroplast, ble gene was used to screened the label gene, through synthesis of atpA promoter and rbcL terminator, promoter and terminator was constructed on each side of ble gene, the pBABL vector was constructed based on pBluescript, the Escherichia coli BL21was also transformed, penbritin and zeocin were used to screen until the transformation bacterium was obtained, which suggests that ble gene was expressed successfully under the contribution of atpA promoter and rbcL terminator.