Agrobacterium Tumefaciens Mediated Genetic Transformation Of Chlorella Vulgaris Rich In Lutein

Lutein, an oxygen-harboring caroteniod, with antioxidant activity, is capable ofpreventing cancer, cardiovascular disease, and age-related macular degeneration.Because Chlorella is rich in lutein, and has advantages of growing quickly, beingeasily cultured on a large scale, being able to be heterotrophically cultured and easilygenetically modified, biosynthesis of lutein in Chlorella has recently receivedattention.In this study, we constructed Agrobacterium tumefaciens-mediated transformation(ATMT) of Chlorella vulgaris, and transferred IPP isomerase gene (idi), Vitreoscillahemoglobin gene (vgb), glucose transporter gene (Glut1) into Chlorella vulgarisrespectively. Specific research contents and results are as followed:Firstly, we successfully constructed Agrobacterium tumefaciens mediated genetictransformation system of Chlorella vulgaris. The T-DNA region of binary vectorpCAMBIA2301was introduced into Chlorella vulgaris by ATMT, using G418resistance gene (NPTâ…¡) as selective marker which can be used to screen positiveclone. The result of PCR showed that NPTâ…¡ gene was integrated into the genome ofthe Chlorella vulgaris. And the result of RT-PCR showed that NPTâ…¡ genesuccessfully transcripted in Chlorella vulgaris. The above results suggested thatAgrobacterium tumefaciens mediated genetic transformation system was effective andcould be used to transfer exogenous gene into Chlorella vulgaris.Secondly, we successfully transferred idi gene into Chlorella vulgaris by ATMT.The T-DNA region of binary vector pCAMBIA2301-idi was introduced into Chlorellavulgaris by ATMT, using G418resistance gene (NPTâ…¡) as selective marker. Theresult of PCR showed that idi gene and NPTâ…¡ gene were integrated into the genomeof the Chlorella vulgaris. Analysing biomass, we found that the majority of thetransformants displayed similar biomass compared with the wild type strain. Whilethe highest lutein content of transformants improved by30.95%compared with thewild type. And the result of analysing lutein yield demonstrated that the highest lutein yield of transformants improved by36.77%compared with the wild type. The aboveresults demonstrated that idi gene can express in the chlorella, which improved thelutein content and yied.Thirdly, we successfully transferred vgb gene into Chlorella vulgaris by ATMT.The T-DNA region of binary vector pCAMBIA2301-vgb was introduced intoChlorella vulgaris by ATMT, using G418resistance gene (NPTâ…¡) as selective marker.The result of PCR showed that vgb gene and NPTâ…¡ gene was integrated into thegenome of the Chlorella vulgaris. By analyzing the biomass obtained in shake flasks,we found that the highest biomass of transformants was38.81%higher than that ofthe wild-type strain. And the lutein content of the transformants improved slightly.Further studies showed that the highest lutein yield of the transformants was36.77%higher than that of the wild-type strain. The above results suggested that integratedexpression of the vgb gene may improve cell growth and lutein yield in Chlorellavulgaris, which could be applied to lutein production from Chlorella duringfermentation.Fourthly, we successfully transferred Glut1gene into Chlorella vulgaris byATMT. The T-DNA region of binary vector pCAMBIA2301-Glut1was introducedinto Chlorella vulgaris by ATMT, using G418resistance gene (NPTâ…¡) as selectivemarker. The result of PCR showed that Glut1gene and NPTâ…¡ gene was integratedinto the genome of the Chlorella vulgaris. The results of analyzing the biomassshowed that the biomass of the transformants improved22.33%compared with thewide type strain. The use of glucose enhanced16.24%compared with the wide typestrain. And the lutein content of the transformants was5.78%higher than that of thewild-type strain. Further studies showed that the highest lutein yield of thetransformants was29.36%higher than that of the wild-type strain. The above resultsdemonstrated that Glut1gene could express in the Chlorella, which improved thebiomass and lutein yied.