Cloning And Expression Analysis Of △6 Desaturase Gene In Mortierella Alpina

Gamma linolenic acid, one of n-6 class polyunsaturated fatty acid, is precursor of prostaglandin E1 and prostaglandin E2 synthesis and plays an important role in keeping humans'normal physiological function. Mortierella alpina is highly productive strain of arachidonic acid.△6 fatty acid desaturase is a key enzyme of gamma linolenic acid synthesis in fatty acid biosynthetic pathway of Mortierella alpina. In this paper,△6 fatty acid desaturase gene full length sequence and△6 fatty acid desaturase gene cDNA have cloned from Mortierella alpina and expressed in Pichia pastoris GS115.△6 fatty acid desaturase gene consists of 2202 base pairs, which is the newest report in Mortierella alpina of home and abroad.. This sequence had the highest similarity with Mortierella alpina which had been reported with percent identities being 99. Structural analysis indicated this sequence contained two introns, locating in 17-229bp and 410-1024bp respectively. Total RNA was prepared from Mortierella alpina W15, possible cDNA fragment encoding△6 fatty acid desaturase gene was amplified by RT-PCR. The△6 fatty acid desaturase gene contained 1374 bp and codes 457 amino acids, sharing a 95% homology with other Mortierella alpina reported. The encoded protein consisted of typical cytochrome b5 domain in the N-terminal. The possible△6 fatty acid desaturase gene was cloned into plasmids pPIC9K and transformed into Pichia pastoris GS115 strain by the electroporation.. Induced by 1% methanol, exogenous gene was expressed and the converter oil was determined by GC. The results indicated that the sample hasγ-linolenic acid peak, comparing with positive control ofγ-linolenic acid standard, which certificated that the inserted gene was△6 fatty acid desaturase gene. The successful clone and expression of△6 fatty acid desaturase gene provide premise and foundation not only for sieving high-yieldγ-linolenic acid producing strains and further applying to industrialization, but also for transferring into oil grains, such as rape and soybean, in order to alter fatty acid composition and cultivate new type crops.