Construction And Preliminary Functional Analysis Of C.reinhardtii Expressing Glut1 Protein Efficiently

Microalgae are rich in nutrients and can produce a variety of high value-added products with unique economic value,and the key issue in the development of the microalgae industry is how to achieve high-density mass production of microalgae.Through the genetic engineering technology,the microalgae's nutrition mode can be changed to make use of the exogenous carbon source for heterotrophic growth.As a strategy to solve the above key problems,Chlamydomonas reinhardtii has a fast genetic background and can express its genetic background.Features such as highly active proteins have great potential for development,but their low biomass,autotrophic growth,and other issues affecting light growth limit their development as bioreactors.Glut1,a glucose transporter,acts as a carrier for glucose transport across glucose membranes.Transport plays an important role.In this research,three expression vectors with different promoters(Hsp70promoter,PsaD promoter,and Arg7 promoter)were constructed and transformed into Chlamydomonas reinhardtii JUV to investigate the effect of different promoters on the glucose transporter gene.The expression effect was initiated,and the nutritional characteristics of the transformants were studied.The results showed that the transformants could use heterotrophic growth of glucose as a carbon source,achieving the purpose of initially changing the nutritional pattern of C.reinhardtii.The specific results are as follows:1)The pBR28-Glut1 overexpression vector,pDB124-CrGlut1 overexpression vector and pHR13-CrGlut1 overexpression vector was constructed and transferred into Chlamydomonas reinhardtii JUV.Eight strains of transformants were obtained(CrGlut1 gene:codon-optimized glucose transporter gene);2)Transcriptional expression was detected by qRT-PCR.The expression of CrGlut1 gene in the Z1,Z2 and Z4 transformants was increased by 114.69-fold,127.43-fold and 120.91-fold compared with the control group,respectively;the H5,H9 and H11 transformants CrGlut1 were detected.The gene expression levels were increased by 14.18-fold,25.66-fold and 77.24-fold,respectively,compared to the control group;the Glut1 gene expression levels of the A4 and D1 transformants were increased by 8.53-fold and 7.32-fold,respectively,compared to the control group.The results showed that all three transformants could successfully express Glut1 gene.3)The copy number of exogenous gene was detected by qRT-PCR.The copy numbers of Glut1 gene of A4 and D1 transformants were 5 and 8,respectively;the copy numbers of CrGlut1 gene of Z1,Z2 and Z4 transformants were 2,2 and 3;H5and H9 respectively.The copy number of the CrGlut1 gene of the H11 transformants was 0.30,0.38,and 0.36,respectively.4)Protein level expression was detected by Western Blot assay.The A4,D1 transformant protein was 55 kDa.The Z1,Z2,Z4 transformants and the H5,H9,H11 transformant protein were 43 kDa,and the Z1,Z2,Z4 transformants.The expression level of Glut1 protein was higher than that of H5,H9 and H11 transformants.5)Using 2-NBDG fluorescent labeling method to detect the transport of extracellular glucose by the transformants,the results showed that the intracellular2-NBDG content of the Z1,Z2,H9 and H11 transformants was increased by 7.38 times and 5.08 times,respectively,compared with the control group.10.94 times and10.01 times.6)The preliminary study on the nutritional characteristics of the transformants showed that the biomass of the transformants was greater than that of the control group in the light conditions and additional glucose as a supplemental carbon source.The transformants Z1,Z2,H9,Compared with the control group,H11 increased by26%,13%,25%,and 18%;under light conditions and glucose as the sole carbon source,the transformant biomass increased significantly with the control group,and the transformants Z1,Z2,H9,H11 increased by 45%,55%,67%,and 73%,respectively;in dark conditions and glucose as the sole carbon source,the biomass of the transformed plants was significantly higher than that of the control group,and the growth of the control group was almost stagnant.Transformants H9 and H11 were about 30% higher than Z1 and Z2.In this research,the glucose transporter gene Glut1 was optimized for codon usage and transferred into the expression of cell wall defects in Chlamydomonasreinhardtii.The Glut1 protein was successfully expressed in Chlamydomonas for the first time to achieve a preliminary change in the nutritional mode of C.reinhardtii to use glucose as a basis.The carbon source was heterotrophically grown and the ability of the three promoters to express in the nuclear expression system of Chlamydomonas sp.was compared to provide a feasible idea for the further establishment of the microalgae bioreactor.