The Induction Of The Protonema,Photosynthesis Of The Protonema, And Transformation Of The Protoplasts In Physcomitrella Patens.

The moss Physcomitrella patens has been used as a model system in molecular biology and plant genetic engineering due to its high rate of homologous recombination.The transformation was carried on with the protoplasts prepared from the protonema;and transformed protonema was cultivated sucessefully to get the producs.But it has not reported so far about researching the physiological photosynthesis and cultivaion conditions of the P.patens protonema.The studyof the expression foreign protein has became the hot point abroad,but it has no report in China up to now.In this work,the P.patens was induced by adding C₄H₁₄N₂O₆ to the solid ppNH₄ medium in room temperature,continuous light at the intensity of 350μmol/(m²Â·s).Then,its protonema appeared and was taken into liquid ppNH₄ medium.It has been found that refresh the liquid medium every week can prevent the differentiation of protonema.This is may be a new way to keep the protonema stage longer.The photosynthetic characteristics of P.patens in different cultivation conditions were assayed by Pulse-Amplitude-Modulation(PAM) and oxygen electrode techniques.The best conditions of P.patens protonema was as follow:light intensity of 350μmol/(m²Â·s); 24℃;pH5.5.This researching can provide the basis for the cultivation of the P.patens protonema.Granulocyte colony stimulating factor(G-CSF) gene was used for the targeting DNA to research the expression of forgien gene in P.patens.Our researching group have been successfully expressed the G-CSF in cyanophytes.Now the signal peptide sequence was added to the G-CSF to make sure the protein can be secreted well and also set up suitable restrict sites on two ends by PCR technique.The plasmid pBAtTPme is 895 1bp long.It has a tta fragment which was regulared by tetracycline.When constructing the transformation vector,at first,the EcoR V site was used,but show a low efficiency during ligation.Then the PmeI site was used,turn out to be well,and the vector pBAtTPme-GCSF was used to transform E.coli.Driselase enzymolysis was used to produce protoplasts from the protonema.The polyethyleneglycol(PEG)-mediation method was used for transforming the protoplasts.After that the transformants of the protoplasts P.patens were selected and cultivated on the solid ppNH₄ solid medium with 50ug/ml ampicillin.After one month,the transformant protoplasts regenerated to the protonema.This primerial success may becom the beginning to use P.patens as a useful bioreactor in China.The next identification of the expression of G-CSF gene in P.patens will be carried on when there are enough gene-transferred protonema of P.patens.