Construction And Analysis Of A Suppression Subtractive Hybridization Library From Mortierella Isabellina High-yield Mutant Strains

Arachidonic acid (Arachidonic Acid, ARA) is an important human unsaturated fatty acids, hasa unique biological activity and health functions. Someone will be listed as a growth factor, thecurrent ARA and other unsaturated fatty acids normally interact made for the prevention andtreatment of dermatitis, hypertension, eczema, cerebral thrombosis, coronary heart disease,arteriosclerosis and other soft capsules cardiovascular and cerebrovascular diseases. In addition,metabolic derivatives ARA also has strong biological activity, and therefore involve a number ofareas ARA infant, adult food, drugs, cosmetics, etc., and has great value and prospects.ARA is mainly extracted from animal tissues, but its content is very poor, with limitedresources, the use of ARA prices of these resources in the preparation of animal tissuesexpensive, can not meet people’s needs. Fungal fermentation production technical disadvantagesexist ARA undesirable bacteria smell gas, and the lower percentage of total fatty acids ARA oils.Therefore, this study from the deep Mortierella ARA biosynthesis of functional genes start,screened and ARA yield performance-related functional genes, and accurate quantification ofgene expression differences, bi-level and metabolism studies the molecular mechanism, thefurther address the fermentation production defects, to obtain the following results:Identification of differences in the use of reverse dot blot hybridization gene expression,excluding cDNA library established in the false-positive clones, get real differentially expressedgenes.200randomly selected positive clones were further identified168positive clones for thereal and sequenced, selected more than60%homologous gene sequence analysis were obtained50expressed sequence tags (EST), through homology obtained after48than the names andfunctions of specific genes are known ESTs, unknown New ESTs, after bioinformatic analysis,48were classified as a known function of ESTs in protein transport, the biological activity of theenzyme, the energy metabolism, metabolic pathways, transcriptional regulation and otheraspects.In this study, the difference will be positive transformants access PDA medium, usingreal-time quantitative PCR screening technique to detect the expression of mRNA expressionlevels of functional genes. According to the results of cluster analysis on the impact of fatty acidsynthesis enzymes eight genes that gene cytochrome C,△5-desaturase, COA essential coenzyme, fatty acid elongase, fatty acid desaturase, glucose6-phosphate dehydrogenase,elongation factor3,△6-desaturase enzyme gene ODEUG authenticate an unknown, indicatingthat the gene is expressed in high yield nine strains were significantly increased.