Construction And Identification Of ZCD Transgenic Dunaliella Salina

Crocetin has extremely high health and medicinal value.Since the natural production of crocetin is extremely limited,it has a wide application prospect to use the synthetic biological means for heterologous transformation of crocetin.Dunaliella salina is a single-cell eukaryotic green alga with no cell wall.It has several advantages such as fast growth rate,simple for cultivation,and stable for genetic transformation.Especially,the content of precursor molecule of crocetin(?-carotene)in D.salina can reach up to 10% of the cell dry weight,which is the highest in all known microalgae.However,the key enzyme zeaxanthin lyase(ZCD),which uses ?-carotene as a substrate to catalyze the formation of crocetin in the carotenoid metabolic pathway of D.salina,is absent.In this study,we are going to use genetic engineering technique to express ZCD exogenous gene from saffron(CSZCD)in D.salina,with a view to construct crocetin synthesis pathway in D.salina by synthetic biological means.The research results are as follows:1.By optimizing the light intensity,the culture temperature and the proportion of medium component,the optimal growth curve of D.salina was established with improved medium;2.Based on the pCAMBIA3301 vector as the backbone,the recombinant plasmid pCAMBI3301-CSZCD has been successfully constructed and served as,the expression vector for transformation of D.salina.The recombinant vector was next transformed into the D.salina by glass bead method;3.After selected by glufosinate-resistant plate screening,the transformed algal strains that can stably inherit the foreign gene CSZCD have been further verified at molecular levels by using PCR technique and high-throughput sequencing;4.Comparative analysis of crocetin content in wild-type and transformed algal strains has been conducted by high-performance liquid chromatography(HPLC).After HPLC detection,compounds related tocrocetin in D.salina transformants were further analysed by LC-MS.These results showed that transformation of exogenous CSZCD gene could synthesize the related compounds of crocetin in D.salina;In conclusion,the expression vector of CSZCD gene has been successfully constructed in D.salina,and algal transformants with stable expression of exogenous CSZCD gene were selected.After analysis by HPLC and LC-MS,the transformants showed that the successful expression of the CSZCD gene could achieve the synthesis of crocetin-related compounds in D.salina,which may lay a technical foundation for the subsequent synthesis of crocetin in D.salina by genetic engineering.