Genetically Modified Sugar Transporter And Hexokinase To Develop The Highly Efficient Heterotrophic Chlamydomonas Reinhardtii

Glucose not only provides nutrients and energy for plants,but also acts as a signal molecule to regulate plant growth,development and metabolism and other life activities.Exogenous glucose enters the cell through a transporter,and phosphorylated by hexokinase to participate in cellular glycolysis and other pathways,thereby providing energy and intermediate metabolites for many life activities.There are many types of simple structured eukaryotic algae in lower plants,and most of them grow in different nutritional ways such as autotrophic,heterotrophic,mixotrophic.Making the full use of heterotrophic capacity is an effective way to obtain high biomass for photosynthetic organisms.Changing the nutrition pattern through genetic engineering is an effective way to increase algae biomass,and becomes one of the current research hotspots.Chlamydomonas reinhardtii,as a single-cell eukaryotic photosynthetic autotrophic model organism,cannot effectively use exogenous glucose and so it can be used as an ideal research material for studying the transport and utilization of exogenous sugar in plants.In this study,the glucose transporter gene(Cke HUP1)and the hexokinase gene(Hae HK/GK)were cloned from two single-celled eukaryotic green algae,and the plant constitutive expression vectors were constructed.Genetic transformation and phenotype identification of the transgenic strain were studied.The genetically-engineered Chlamydomonas reinhardtii CC849 strain,which can efficiently utilize exogenous sugars,was obtained by genetically modifying and establishing a new mixotrophy growth pattern.The main findings are as follows:(1)The full-length c DNA sequences of Cke HUP1 gene and Hae HK/GK genes were cloned by the combined homologous cloning and rapid-amplification of c DNA ends(RACE),and the gene sequences and phylogenetic characteristics were analyzed respectively.The results showed that the Cke HUP1 gene was 2479 bp in length and encoded a total of 534 amino acids.Cke HUP1 was a membrane-bound protein with 12 typical transmembrane domains and belonged to the major facilitator super family(MFS).The full length of the Hae HK gene c DNA was 2 047 bp,which encoded a total of 571 amino acids.The full length of the Hae GK gene c DNA was 1 750 bp,which encoded a total of 475 amino acids.Both Hae HK and Hae GK were intracellular proteins,which had the typical characteristics of the hexokinase superfamily with a "butterfly" tertiary structure.(2)The nuclear expression vector p HR13-Cke HUP1 of Chlamydomonas reinhardtii was successfully constructed,and it contained an Arg7 constitutive promoter with a Hyg resistance gene.The expression vector p HR13-Cke HUP1 was transferred into the Chlamydomonas reinhardtii CC849 by bead milling method.The target gene Cke HUP1 was integrated into the C.reinhardtii genome and effectively expressed after antibiotics screening and molecular identification.(3)The biomass,pigment content,chlorophyll fluorescence parameters,extracellular glucose content and the expression of Cke HUP1 gene of the p HR13-Cke HUP1-transformed C.reinhardtii under different culture conditions(autotrophic,heterotrophic,mixotrophic)were studied.It was found that the transformants had no significant effect on the growth and photosynthesis with wild type under autotrophic conditions,which was indicated by the low expression level of Cke HUP1 gene.However,compared with the wild strain,the transformants grow better and could absorb exogenous glucose under mixotrophic conditions.Meanwhile,the Cke HUP1 gene was up-regulated.Similar with wild strain,the transformants also could not grow under heterotrophic conditions.It is interesting that the transformants quickly resumed after dark-light recovery,and could effectively absorb exogenous glucose and the expression the Cke HUP1 gene was also significantly up-regulated.These findings showed that the transfer of Cke HUP1 gene improves the ability of C.reinhardtii to utilized exogenous glucose,and light may play an important part in the expression and the function of this gene.(4)The nuclear expression vector p DB124-Hae GK of C.reinhardtii was also successfully constructed,and it contained the Psa D constitutive promoter with Ble resistance gene.The two expression vectors p HR13-Cke HUP1 and p DB124-Hae GK were used for co-transformation.No positive transformants were obtained through double selection of hygromycin and bleomycin.But the positive transformants were obtained when the transgenic p HR13-Cke HUP1 C.reinhardtii was used as the recipient cell.Molecular identification found that the target gene Hae GK was successfully integrated into the C.reinhardtii genome,but the Cke HUP1 gene was lost.There were some reasons responsible for this question,such as the difficult co-transformation technology,low promoter efficiency,un-optimized codons,and gene silencing during the transformation.In the future,the Cke HUP1 and Hae GK gene co-transformed and expressed C.reinhardtii strains will be further cultivated and their phenotypes will be systematically identified.In summary,the glucose transporter gene(Cke HUP1)and the hexokinase gene(Hae HK/GK)were cloned successfully,and the transgenic line of C.reinhardtii with stable expression of Cke HUP1 was obtained.The Cke HUP1 gene expression was significant under dark-light recovery conditions,and the ability of C.reinhardtii to utilize exogenous glucose was enhanced.In addition,the Cke HUP1 gene can be further developed as a selection marker for microalgae genetic transformation based on exogenous sugar utilization.