Cold Adaptation Of Mortierella Isabellina And Construction Of An RNAi Expression Plasmid Targeting D12D Gene

Species of genus Mortierella are the main fungi used for the research of the biothesynthesis of polyunsaturated fatty acids such as linoleic acid (LA) and y-linolenic acid (GLA) in and outside of China. Mortierella isabellina, which only synthesizes n-6PUFAs, is the most potential filamentous fungus for commercial production of PUFAs in china. All the genes that are responsible for the biothesynthesis of PUFAs in Mortierella isabellina have been identified, providing a moderate model to study the effects of PUFAs on the low-temperature adaptability of the fungi.△12-fatty acid desaturase (D12D), namely oleate desaturase, is a rate-limiting enzyme that control the biosynthesis of PUFAs in low eukaryotic filamentous fungi. It catalyse the convertion of oleic acid (OA) to LA, and LA the common precursor of n-6and n-3PUFAs.In this study, M. isabellina M6-22was used to study the effects of low temperatures ranged from0to35℃on the strain growth, PUFAs biosynthesis and membrane fluidity to analyze its cold adaptability. The resutls showed that M6-22could grow at low temperature and its PUFAs content increased, although the biomass decreased. Furthermore, membrane fluidity was also observed.Red fluorescent protein was used as screening marker for genetic transformation of M. isabellina to generate a expression vector pS-DsRED. Based on the pS-DsRED, a RNAi expression plasmid pSREDMID12RNAi was constructed by inserting1-500bp of D12D gene in forward and reverse direction, respectively, into cloning sites upstream and downstream of cutinase intron in pS-DsRED. Restrictive analysis and DNA sequencing domenstrated its correctness.A protoplast regeneration system of M. isabellina M6-22was estabilished in this study. Although the regeneration rate was as low as15.7%, it could be used for further protoplast transformation. pS-DsRED and pSREDMID12RNAi were transformed into M6-22by PEG/CaCl2mediated method. Fluorescent microscopy and morphology results showed the expression of DsRED gene. GC analysis showed that the expression of DsRED gene did not affect the content of PUFAs in M6-22, and transformation of pSREDMID12RNAi did not significantly reduced the content of LA and GLA. The regulatory mechanism for PUFAs biosynthesis in M. isabellina M6-22at low temperature will be explored, which will contribute to uncover the interaction of PUFA biosynthesis regulation with cold adaptation in filamentous fungi. Based on these results, genetic modification will be used to improve the production of PUFAs in filamentous fungi for the industrial application. This study also will be helpful for the development and application of microbial resources in Yunnan province, and will has a further contribution to the development of fungal melocular biology in China.