Screening The Rhodospirillum Rubrum Mutants Of High Hydrogen Production And Analysis The Insertion Sites Of Transposon

Global energy crisis comes now! More and more countries try to exploit alternative energy.In recent years,hydrogen energy has become a hot choice which would be an alternative energy. Hydrogen energy has many advantages.First,many kinds of meterials can produce hydrogen,so we don't need to worry about its stockpile.Second,hydrogen energy possesses high combustion heat. Third,the final product is water after combustion and have almost no pollution.Photosynthetic bacteria(PSB)can perform the photosynthesis and produce H₂.Rhodospirilum rubrum as the model bacteria of PSB has the great research value and they can grow under aerobic or anaerobic environments.Its characters that can utilize light enery,digest organic waste and release H₂ make people study it with much passion.Transposon as the important tool in genetics research is used extensively for insertional mutagenesis.It can make stable insertion in the genome and does not need additional steps to stabilize the insert.We can harvest the transposon flanking sequence of mutant and isolate new genes which has relationship with high hydrogen yield.Then,it will be helpful to research hydrogen yield mechenism of PSB.Palladium Chloride(PdCl₂)can be converted into black metallic palladium(Pd)by deoxidized gas such as H₂.According to this truth,we tried to set up a quickly screening method to search higher hydrogen production PSB mutants.We used 96-well deep microtiter plates to incubate PSB in hydrogen yield medium and sealed it with a piece of absorbent cotton which dipped in PdCl₂ solution dissolved in hydrochloric acid.We identified the higher hydrogen mutant by the different color of cotton.In this study,we established a Tn5 transposon random mutagenesis library of R.. rubrum and use PdCl₂ quickly screened the high hydrogen yield mutants.On the other hand,we fermented these mutants in 500-ml conical flask and tested these capacity of hydrogen yield by gas chromatography.But we saw the two methods have different conclusions.In this study,we discussed about the condition,and suggested the improve points.These experiments and suggestion provided a good basis for establish the PdCl₂ quickly screen method for high hydrogen production mutants.In this study,we picked up a higher hydrogen production mutant than the wild strain(R. rubrum UR2)and named it RM4.The H₂ yield of R..rubrum RM4 under continuous light is 1.89-fold that of R..rubrum UR2.There is a R6K ori and no BamH Isite in transposon.We extracted RM4 genome and digested it with BamH I,self-ligated and transformed into Escherichia coli DH5α/λpir.We sended the positive transformant to sequence by single specific primer of transposon and geted the flanking sequence.By blast in NCBI,we found Malonyl-CoA decarboxylase are broken by insertion.Nowadays,there is no article say this enzyme has important role in relationship with H₂ yield by PSB.So,RM4 genome has other insertion site possibly.