Cloning Of Delta 6 Desaturase CDNA And Effects Of Ambient Salinity And Dietary Lipids On Its MRNA Expression In Rabbitfish, Siganus Oramin

High unsaturated fatty acids (HUFA) are a group of fatty acids with twenty or more carbonatoms and three or more double bond, mainly including eicosapentaenoic (EPA), arachidonic(AA) and docosahexaenoic acid (DHA). They play pivotal roles during the growth anddevelopment of marine larval and juvenile fishes. HUFA are produced through elongating anddesaturating of linoleic andα-linolenic, and elongase and desaturse play an important role duringthis process. It is generally thought that freshwater fish could bioconvert linoleic andα-linolenicinto HUFA. By contraries, marine fish are generally presumed to have a low capability orinability to bioconvert linoleic and linolenic acids into HUFA. Therefore, HUFA are consideredas essential fatty acid (EFA) required in their diets for normal growth and survival. The reasonsfor this deficiency of bioconverting capacity in marine species remain unclear. The deficiency ofâ–³6- andâ–³5-desaturase activity in marine fish could be due to a repression of desaturase activityby the high levels of HUFA usually present in the diet. As to the euryhaline and migrating fishes,it remains unknown whether the growth performance, the capacity of converting linoleic andα-linolenic into HUFA, and EFA requirement vary with the environment salinity and the changeof feed fat ingredient. If they change, it is worth while to clarify the enzymatic and molecularbiological mechanisms.In this study, a growth experiment was conducted with euryhaline Siganus oramin. Fishwere reared in water with salinity 10%o or 32%0 for 9 weeks, and fed four artificial formulateddiets with similar proximate compositions but different in lipid sources (diet 1, fish oil rich inHUFA; diet 2, safflower oil rich in linoleic acid but no HUFA; diet 3, perilla oil with linoleicacid:α-linolenic acid=1:1 but no HUFA; diet 4, safflower oil and perilla oil with linoleicacid:α-linolenic acid=3:1). The cDNA of delta 6 desaturase, a key enzyme for the biosynthesisof HUFA, was cloned in S. oramin. The tissue expression characteristics were analysized. Themolecular mechanisms were studied on the environmental salinity and dietary lipids affecting thecapacity of HUFA biosynthesis, by comparing the mRNA contents in different liver samplesfrom the growth experiment. The results were shown as follows: 1. Characterics of the full length cDNA and amino acids sequence of Siganus oramindelta6 desaturaseThe full length cDNA sequence of Siganus oramin delta 6 desaturase is 1872 bp in size andencodes a polypeptide of 445 amino acids. It contains a 1338 bp open reading frame from the146 bp to 1483 bp, 145 bp of 5'-UTR and 388 bp of 3'-UTR. The predicted protein sequence of S.oramin delta 6 desaturase displayed a tapical structure of fatty desaturase. It contains aconservative domain of fatty desaturase, a delta 6 desaturase-like domain and a cytochrome b₅domain. The predicted protein sequence of S. oramin delta 6 desaturase reveal a high percentageidentity to other fish, such as Sparus aurata (78%), Gadus morhua (74%), Salmo salar(73%),Oncorhynchus mykiss (72%) and Oncorhynchus masou(72%).2. The tissue distribution of delta 6 desaturase of Siganus oraminThe delta 6 desaturase of S. oramin showed a high mRNA expression in liver and brain, andlower mRNA expression in gill. In intestines and muscle, its mRNA expression wasundetectable.3. Effects of environmental salinity on the expression of delta 6 desaturase mRNAIn the liver samples of the 6th week and 9th week of diet 1 and diet 2 groups, the delta 6desaturase mRNA demonstrated high expression in salinity 10 than in salinity 32; In the liversamples of diet 3 and diet 4 groups, its mRNA was higher at 6th week than 9th week in salinity10. In contrast, it was lower at 6th week than 9th week in salinity 32. By comparing theexpression of delta 6 desaturase mRNA in all the samples from four dietary groups between thesalinities, it was found that the mRNA level in salinity 10 was about 1.5 times of that in salinity32.4. Effects of dietary lipids on the expression of delta 6 desaturase mRNAIn two different salinitys, the expression of delta 6 desaturase mRNA showed the sameexpression tendency. In diet 1 group, the expression of delta 6 desaturase mRNA was very low.Furthmore, the samples at 9th week had a lower expression than those at 6th week. These resultssuggested that high dietary EPA and DHA may suppress the expression of delta 6 desaturasemRNA. In diet 2 group, the expression of delta 6 desaturase is lower at 6th week, but rised at 9thweek. This suggested the linoleic may promote the expression of delta 6 desaturase mRNA.Because of the different proportion of linoleic andα-linolenic in the diet 3 and 4, delta 6 desaturase mRNA had reversed expression level. In diet 3 group, the expression of delta 6desaturase mRNA was higher at 6th week than at 9th week. However, it was higher at 9th weekthan at 6th week in diet 4 group. The desaturations of linoleic andα-linolenic are catalysed bydelta 6 desaturase, lin01eic andα-linolenic have a fierce competition. The above resultssuggested thatα-linolenic had a higher competition ability and could surpress the expression ofdelta 6 desaturase when its content is equal to linoleic as in diet 3, and promote its expressionwhen its content was low as in diet 4.The present study showed that the expression of delta 6 desaturase mRNA in livers of S.oramin reared in the low ambient salinity was higher than that in the high ambient salinity. Theexpression of delta 6 desaturase mRNA was surpressed by high dietary HUFA. Dietary linoleiccould promote the expression of delta 6 desaturase mRNA. If the diet contained both linoleic andα-linolenic, the content ofα-linolenic was lower, and the desaturating functions of linoleic weresurpressed by the desaturating functions ofα-linolenic. Thus the delta 6 desaturase mRNAexpressed lower. If the diet had high perctent of linoleic, the expression of the delta 6 desaturasemRNA would be promoted.This is for the first time to clarify the relationship between the biosynthetic capacity ofHUFA and ambient salinity or dietary lipids from the molecular biology level in fish. The resultswill be of important theoretic and academic significance for enriching fish nutrition physiology,as well as important practical significance for directing the selection of dietary fatty sources andEFA addition for the aquiculture of euryhaline fish.