Genetic Transformation In Oleaginous Fungus Rhizopus Arrhizus Of Antisense VeFAD₂ Gene

Developing microbial oils is a new way of improving biodiesel industry.However,complicated composition of microbial oils can't meet the requirement to monounsaturated fatty acid of biodiesel.â–³12 fatty acid desaturase(oleate desaturase,FAD2) is one of the pivotal enzyme which control the biosynthesis from monounsaturated fatty acid to polyunsaturated fatty acid. Oleic acid 18:1(â–³9)dehydrogenate to linoleic acid 18:2(â–³9,12)at the action of FAD2. With the developing of molecular biotechnology,antisense FAD2 gene is widely introduced into oil plants by genetic manipulation of fatty acid metabolism.These studies obtained high oleic acid content of transgenic maize,soybean,peanut,rapeseed,et al.But,antisense FAD2 gene introduced into Oleanous fungus has not been reported as yet.It's a totally new research fields and has an important practical significance.Compared with protoplast transformation methods,Agrobacterium tumefaciens mediated transformation (AtMT) has advantages of easier operation,higher efficiency,higher genetic stability,higher repeatability,et al.We have already successfully cloned VeFAD2 gene from woody oil plant Vernicia Montana. We assume that antisense VeFAD2 gene has bee??troduced into oleanous fungus via AtMT,hoping to obtain transgenic fungus of high oleic acid,low linoleic acid content by genetic manipulation of fatty acid metabolism.Based on the antibiotic concentration experiments, oleanous fungus Rhizopus arrhizus and Mortierella isabellina were selected as transgenic recepor materials .The ideal concentration of selective agent kanamycin was 300-500 ug/ml.Cefuroxime sodium was selected as bacteriostatic agent at the concentration of 150ug/ml.In this study,the antisense expression vector pBI121-fad2 with VeFAD2 gene was cloned into A. tumefaciens strain AGL-1 by freeze-thaw method.We have transformed filamentous fungus R. arrhizus and M. isabellina via AtMT.As a result, R. arrhizus get transformants.PCR-Southern analysis showed that VeFAD2 gene has been integrated into the genome of R. arrhizus.The hereditary stability investigation showed that kanamycin-resistant characteristic of transformants can stable inherit on non-selection medium. We studied the key factors influencing transformation,such as A.tumefaciens strains, bacterial cell volume initially used, co-cultivation time, effective period and concentration of AS .The results showed that:both A.tumefaciens EHA105 and AGL-1 are suitable for transformation of R. arrhizus ,the ideal A.tumefaciens strain is AGL-1;the optimum bacterial cell volume initially used is 50-100 ul and co-culture 24 hours will get the most transformants . At co-culture period , AS induction is indispensable. It is beneficial to improve transformation ratio of EHA105 by adding to 200 umol/L AS at preincubate period and increasing AS concentration to 400umol/L at co-culture period.In addition,we improved some experiment operation segment.By dilutting liquid mixture of fungi and agrobacterium,the problem of agrobacterium pollution has been solved.And we obtained two monoclonal positive clones by exploring on different adding methods of select medium.In this paper, antisense VeFAD2 gene has been successfully introduced into oleanous fungus R. arrhizus via AtMT and obtained transgenic fungal strains. We primarily set up a genetic transformation system of heterologous gene originated from plant into R. arrhizus by AtMT .These findings should provide some reference for genetic transformation in other...