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• W43103631 abstract "Haematococcus pluvialis is a unicellular green alga which produces aketocarotenoid, astaxanthin having pharmaceutical and nutraceutical applications owingto its high antioxidant activity. Biotechnological approaches like genetic transformationmethods (Agrobacterium mediated), cloning strategies, etc are essential toimprove/regulate this ketocarotenoid in H. puvialis. For the standardization ofAgrobacterium mediated transformation procedures for algae, preliminary studies like,sensitivity of algae for different antibiotics, media selection for the co-cultivation for bothalgae and the bacteria were carried out. The sensitivity studies showed that the H.pluvialis was able to tolerate up to the concentrations of 2000 mg L-1 of antibiotics,cefotaxime and augmentin. Growth and multiplication of the algae was suppressed andultimately killed when hygromycin concentration exceeded 2 mg L-1 in solid media. Nogrowth was observed even after 4 weeks of inoculation when the hygromycinconcentration exceeds 2 mg L-1. Among the different cocultivation medium (BBM + halfstrength of LB medium, Z8 medium + half strength of LB medium, Z8 medium + 0.5%mannitol, Z8 medium only and Tris - acetate - phosphate (TAP)) medium were tested ,only the TAP medium favoured the growth of both the alga and Agrobacterium. Coloniesresistant to hygromycin at 10 mgL-1 expressed β-glucuronidase (GUS) and greenfluorescence protein (GFP). PCR was used to successfully amplify fragments of the hpt(407 bp) and GUS (515 bp) genes from transformed cells while southern blots indicatedthe integration of hygromycin gene into the genome of H. pluvialis. Scanning electronmicroscopy indicated that the cell wall of H. pluvialis was altered on infection withAgrobacterium. The transformation achieved here by Agrobacterium does not needtreatment with acetosyringone or the wounding of cells. The carotenoid profile of thetransformed H. pluvialis showed no difference between the control cells.The amplification of b-carotene ketolase (BKT) was observed for the different primerssynthesized. No amplification was observed for the any of the BKH primers studied. The1.8 kb amplicon of BKT gene was cloned to a cloning vector pRT100 in between theCaMV 35S promoter and the poly A region. The b-carotene ketolase gene from clonedplasmid pRT100 was further transferred to a binary vector pCAMBIA1304. Sequenceanalysis of cloned BKT for nucleotide and amino acid showed 99% similarity of thereported BKT gene accession number D45881. Six exons and five introns were observedfor the BKT gene cloned from the H. pluvialis. Even though there were few nucleotideviipolymorphism, there is no shift in the reading frame. The cloned plasmid pRT100 andpCAMBIA1304-BKT were further confirmed by restriction digestion. StandardizedAgrobacterium mediated genetic transformation protocol was followed to transform thecloned binary vector having BKT (pCAMBIA1304-BKT) gene to the H. pluvialis.Confirmation of the transformation of cloned BKT to the H. pluvialis was studied byanalyzing the GUS, GFP expression. PCR analysis for the CaMV 35S primers showed2.5 kb of the BKT gene with the promoter and poly A region. For the BKT forwardprimer and the CaMV 35S reverse primer also the exact size of amplicon was observed.Southern blotting also showed the difference in the banding pattern of the enzymedigested plasmid and the transformed H. pluvialis. No bands were observed for thecontrol cells. Transcript level analysis of the BKT showed 3 to 4 fold higher expressionof BKT in transformants than the control cells. The transformed cells were subjected todifferent stress condition for the induction of secondary carotenoids. Total carotenoidsand astaxanthin content in transformed cells showed 2-3 folds higher when compared tothe control. Astaxanthin was found to be higher (7.96 mg/g) in sodium acetate (4.4 mM)treated culture followed by sodium acetate 4.4mM and NaCl 0.25% (4.99 mg/g). Theintermediates like canthaxanthin and echinenone were the major carotenoids which arepresent more in the transformants but not in the control H. pluvialis. It shows thatechinenone and canthaxanthin content were approximately 8 to 10 fold higher in sodiumacetate 4.4mM and NaCl 0.25% treated culture. The NaCl + sodium acetate treatedculture in transformed cells showed higher level of expression for all the enzymesstudied. It was observed that the expression levels of PSY, PDS, LCY, BKT and BKHwere 8.8, 5.8, 11.7, 8.1 and 6.4 fold higher respectively when compared to the controlgreen cells. But when compared to the transformed green cells it is 6.1, 1.7, 22.7, 4.0 and2.9 fold higher respectively.This first successful Agrobacterium mediated transformation in green micro algaHaematococcus pluvialis would pave the way for manipulation of many importantpathways relevant to food, pharmaceutical and nutraceutical industries. Further thecloned BKT gene from H. pluvialis will be used for higher production of carotenoidsthrough transformation in the heterologous host like Duanliella sp, Daucus sp,Lycopersicum sp to regulate the carotenoid biosynthesis. Therefore this study helps inproduction of higher level of carotenoids through the Agrobacterium mediatedtransformation system in carotenoid producing organisms using the cloned BKT gene." @default.
• W43103631 created "2016-06-24" @default.
• W43103631 creator A5047974650 @default.
• W43103631 date "2009-01-01" @default.
• W43103631 modified "2023-09-27" @default.
• W43103631 title "Genetic transformation of green alga Haematococcus pluvialis for the regulation of carotenoid biosynthesis" @default.
• W43103631 hasPublicationYear "2009" @default.
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