Construction Of Chlamydomonas Reinhardtii Lighting Control System And Its Application In Prod Ucing Hydrogen

Since hydrogen is known as the cleanest fuel in the world,it is widely recognized as the ideal alternative to fossil fuels.Meanwhile,green algae have high hydrogenase activity and photosynthetic hydrogen production efficiency,so hydrogen production by green algae have the brightest application prospect among biological hydrogen production methods.However,the hydrogenase of green algae is very sensitive to the oxygen released by photosynthesis,so green algae can only produce hydrogen continuously for a short time,which greatly restricts the development of photosynthetic hydrogen production industry.And nowadays,the method of using two-stage cultivation to change medium constantly is mainly utilized in the world,which induces green algae to produce hydrogen continuously by sulfur-deficient culture.Due to the difficulties of algae-liquid separation,this method cannot be spread.In our experiment,in order to make green algae be able to produce hydrogen continuously,we use heat-shock induction to regulate the approach of photosynthesis.But not only will it damage the cells,but also will affect hydrogen production efficiency.Our study has used C.reinhardtii,which is a kind of unicellular eukaryotic green algae,to build a light-mediated system for C.reinhardtii,which is based on blue light induction and used for the regulation of hydrogen production in algae cells.The system is made of many kinds of components: Firstly,the gene of CRY2,also named cryptochrome2,a photoreceptor;Secondly,the gene of CIB1,the interacting partner of CRY2;Thirdly,GAL4-BD,the DNA binding domain of GAL4 transcription factor;Fourthly,VP16,an activation domain of herpes simplex virus which has transcriptional activation ability;Fifthly,UAS,the upstream activation sequence;Finally,miRNAs,which can specifically target the functional genes of PS?.The detailed studies and results are as the following:1.Using the coding gene of D1(a core protein in PS?)as the target and then designing an amiRNA sequence which can target the coding gene successfully by using WMD3 online platform.2.Building the expression loads of p Db124-GAL4BD-CIB1 and p Dh124-VP16-CRY2-UAS-miRNA,and then transforming them into C.reinhardtii by ‘glass bead' method.After that,screening out the positive transformants.3.After blue light induction,the expression level of amiRNA-D1 is 16 times as high as that of the compared.However,the expression level of wild-type CC849 is very low.4.After blue light induction,the expression level of the target gene of amiRNA has declined by 78 %,which shows that the amiRNA inhibits its target gene successfully,while there is no obvious change in the expression level of wild-type CC849.5.After blue light induction,the hydrogen production of amiRNA-D1,the light-mediated transformant,is 5 times as much as that of wild-type CC849.Hence,we can see that this study builds a blue light-mediated system and establishes a sort of gene-modified algae which can produce hydrogen efficiently based on artificial micro RNA technology.By intermittent blue light induction,algae cells can achieve the target of continuous hydrogen production.The results provide a new technical approach for green algae bio hydrogen production technology and its industrial development.