| In order to explore the regulation of 3-ketoacyl-CoA synthase(KCS enzyme)on the fatty acid metabolism of Chlorella pyrenoidesa,enrich the content of very-long-chain unsaturated fatty acids,we utilize Agrobacterium-mediated transformation to transfer the KCS gene of Cardamine graeca to the the Chlorella pyrenoidesa,which overexpresses the stearyl carrier protein desaturase(SAD enzyme)gene,and observe the effect of exogenous gene transfer on fatty acid synthesis of Chlorella.The KCS gene was transformed with pCAMBIA1301 plasmid,and the positive transformed algae were screened by hygromycin resistance.The GUS gene was transformed with the same plasmid vector to observe the expression ability of the expression cassette of Chlorella.We validated the positive transformed algae with Cardamine KCS enzyme gene by PCR and RT-PCR.The faester acyl-CoA was analyzed by high performance liquid chromatography(HPLC),and the fatty acid composition of Chlorella was analyzed by gas chromatography-mass spectrometry(GC-MS).The gene transformation completed in this paper is a secondary transformation by Agrobacterium-mediated transformation based on the completion of SAD gene transformation in the early stage.It is the first time to obtain the Chlorella with the SAD enzyme gene and the KCS enzyme gene transformed simultaneously and explore the effect of KCS enzyme on the fatty acid synthesis of Chlorella.It provides a research reference for further study on the regulation mechanism of Chlorella fatty acid metabolism.The main contents and results of this experiment are as follows:(1)The recombinant plasmid pCAMBIA1301-KCS was constructed based on the binary vector pCAMBIA1301,which was transformed into the KCS gene by knocking out the GUS gene and combined with the KCS gene of Cardaminegraeca.The recombinant plasmid used the CaMV35 S promoter and NOS polyA terminator of the original expressing cassette as promoter and terminator to construct the new KCS gene expressing cassette.(2)The original binary vector pCAMBIA1301 and the engineered plasmid pCAMBIA1301-KCS containing GUS gene were introduced into the transformed Chlorella that overexpressed the SAD gene by Agrobacterium-mediated transformation separately,and the positive transformed Chlorella was screened by hygromycin resistance.The selected transformed Chlorella was widely cultured under hygromycin pressure.(3)GUS staining of the Chlorella introduced into the original binary vector pCAMBIA1301 showed that the GUS gene could be successfully expressed in Chlorella,that is,the expressing cassette of GUS gene could be successfully expressed in Chlorella.(4)The selected Chlorella introducted into recombinant plasmid pCAMBIA1301-KCS was examined with PCR.The results showed that the KCS gene of Cardamine graeca and the hygromycin resistance gene(Hpt)were successfully introduced into the Chlorella.And the result of RT-PCR for KCS gene showed that the KCS gene was successfully transcribed in Chlorella cells.(5)The metabolites of KCS transformed Chlorella were analyzed.Compared with the original Chlorella whose SAD gene overexpressed,it was found that the content of 6z,9z,12z-Octadecatrienoyl CoA and palmitoleoyl CoA was significantly increased.(6)The result of fatty acid analysis showed that the fatty acid composition of the Chlorella that successfully transformed the KCS gene has a significant increase in the content of very-long-chain fatty acid,docosatrienoic acid(C22:3),and its relative content in total fatty acids reached 21.27%.The relative content of the Linolenate acid(C18:3)decreased significantly,from 22.8% to only 0.23%.We also detected behenic acid(C22:0)that rarely appeared with a relative content of 0.05%.The relative content of tetracosanoic acid also increased slightly.In conclusion,exogenous KCS enzymes have a significant effect on fatty acid carbon chain elongation,but the specificity for fatty acids elongation is different from other literature reports. |
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