Optimization of protoplast formation,regenerationin and transformation in Aspergillus niger

For improving the mycelial morphology and citric acid production of A.niger,we construct a gene silencing vector by RNA interference technology to silence the chitin synthase gene. The fragments of chs gene were amplified by PCR,which was ligated to plasmid p JL43- RNAi to generate the RNAi vector.In order to increase the transformation frequency,the effects of some factors on protoplast formation and regeneration from citric acid-producing fungus A.niger were investigated.The silencing vectors were introduced into purificatory protoplasts by a polyethylene glycol( PEG)- mediated transformation method. The results showed that the mycelia incubated for16 h at 36.5 ℃ were most suitable for protoplast release,which digested by enzyme combination of 5 mg / m L lysozyme,10 mg / m L snailase and 10 mg / m L cellulase for 3 h as 100 r / min at 30 ℃,and a high yield of protoplasts( 5.11 × 106/ m L) were obtained.In addition,the maximum regeneration rate was 35.33%,while the complete medium as the regeneration media. After conversion,three morphological mutant strains are smoother and have less dispersed mycelia,which were distinct from the original strain in morphology.The transformants chs-1,chs-2 and chs-3 where citric acid production rise by 12.33%,24.17% and 19.61%,respectively.The morphological mutants of chs exhibited the excellent production potential during submerged culture,which had important significance for the development of the citric acid fermentation industry.