A Study On Genetic Transformation Of Neochloris Oleoabundans Of Foreign β-ketoacyl-co A Synthase(KCS) Gene

In this study, the Lunaria annua L. 3-ketoacyl-Co A synthase(KCS) gene was inserted into the genome of Neochloris oleoabundans(N. oleoabundans) with the method of Agrobacterium-mediated to investigate the effect of the 3-ketoacyl-Co A synthase(KCS) on the systhesis regulation of very-long-chain fatty acid in microalgae.The results of genetic transformation, the fatty acids composition of transfered microalgae and the effect of KCS gene on the metabolism regulation of fatty acids were detected and analyzed. This is the first genetic engineering study on N.oleoabundans, and the foreign gene transformation was carried out successfully. The work could lay the foundation of gene modification and application of N.oleoabundans. The main works and resuts were as follows.(1) The sensitivities of N. oleoabundans to cloramphenicol, penicillin, streptomycin,tetracycline and hygromycin were observed in liquid culture and agar plates. The results showed that in the liquid medium streptomycin and hygromycin had completely inhibited the growth of algae cells at the concentration of 10μg/m L, while cloramphenicol and tetracycline behaved inhibition when the concentration reached100μg/m L. However, penicillin exhibited only slight inhibition at the concentration of500μg/m L. The same results were obtained by the study on the solid medium.(2) Two binary vectors p CAMBIA1301-KCS and p CAMBIA1301-Rbcs2-KCS for Agrobacterium-mediated transformation were successfully constructed. In p CAMBIA1301-KCS, the KCS gene was driven by the cauliflower mosaic virus promoter Ca MV35 S, and in the vector p CAMBIA1301-Rbcs2-KCS it followed the promoter Hsp70A/Rbcs2 which was Chlamydomonas reinhardtii appreciated promoter. Meanwhile, the two vectors contain the β-glucuronidase(GUS) gene as the reporter gene and the hygromycin phosphotransferasegene(hpt) as the selective maker gene.(3) The target gene was transformed to N. oleoabundans through co-cultivation with Agrobacterium tumefaciens carried the recombinant plasmid. Firstly, recombinant plasmids were transfered into A. tumefaciens by electroporation, and the positive transferred strains were selected. Secondly, the co-cultivation was carried out to allow the T-DNA of the vector to be transferred into the genome DNA of N. oleoabundans.Finally, the positive clones of N. oleoabundans were selected on the solid medium with hygromycin 10 μg/m L and cultivated.(4) The positive clones were detected by gene detection and fatty acid analysis. The results showed that the KCS gene, GUS gene, hygromycin phosphotransferase gene and promoter Hsp70A/Rbcs2 were successfully transferred into the DNA of N.oleoabundans according to the PCR analysis. Foreign genes were proved to be successfully transcripted to m RNA as the RT-PCR analysis. GUS gene was also expressed with the exhibition of GUS staining analysis. The expression of hygromycin phosphotransferase gene was exhibited through the resistance of positive algal clones. But no change was observed on the content of very-long-chain fatty acid through the analysis of fatty acid, which need deeper analysis.