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Functional Analysis of Vernicia fordii FAD2and DGAT2

Author: CuiQinQin
Tutor: WangYangDong
School: Chinese Academy of Forestry
Course: Tree Genetics and Breeding
Keywords: Vernicia fordii FAD2 DGAT2 function identification fatty acids
CLC: S794.3
Type: Master's thesis
Year: 2013
Downloads: 34
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Abstract


Tung tree (Vernicia fordii Hemsl) is a world-famous industrial crop, the oil content andconstitute are the key indicator of tung production. Fatty acid desaturase (FAD2) catalyzes thefirst step of polyunsaturated fatty acids biosynthesis in the plant. Diacylglycerol acyltransferase(DGAT2) catalyzes the last and most committed step of triacylglycerol (TAG) biosynthesis,which plays an important regulation role in the accumulation of fatty acids in tung seeds. Inthis paper, we characterized the time course of oil accumulation during tung seed developmentand maturation. The expression profiles of FAD2, DGAT2in tung different tissues/organs andseeds at different developmental stages were also tested. Using the expression vectorsconstructed early, the transgenic yeast and Arabidopsis containing FAD2or DGAT2wereobtained. The FAD2/DGAT2co-expressed yeast and Arabidopsis were also got. Further, theexpression patterns of FAD2, DGAT2in transgenic yeast and Arabidopsis were investigated,and their functions in manipulating the fatty acids accumulation were also detected. The majorresearch conclusions were as follows:1) The results showed that July is the slow formation period during the seed maturation,the end of July to the beginning of September is the rapid accumulation period, afterMid-September, the seeds reaches the accumulation completion period. We got the oil contentof37.64%in this paper. Also the fatty acids profiles indicated that only the eleostearic acidexhibited rising trend, which was consistent with the tendency of tung oil accumulation. Theeleostearic acid accounted for75.81%of total fatty acids in the end.2) FAD2and DGAT2expression were investigated in all tested tissues, including leaves,petioles, stems, petals, pistils, fruitlets and developing seeds at six different developmentalstages using the real-time quantitative PCR methods. The results indicated that both FAD2andDGAT2were highly expressed in developing seeds which was much higher than those in othertissues. During the seed maturation, FAD2and DGAT2showed temporal specific expression,which was found to be consistent with tung oil accumulation and eleostearic acid production, predicting that the genes were involved in manipulating fatty acids synthesis of tung.3) FAD2and DGAT2expression vectors were transferred into yeast and Arabidopsisrespectively. The linoleic acid (C18:2) of transgenic yeast enhanced102.86%,81.95%and21.80%seperately compared with control. While the linoleic acid of transgenic Arabidopsisenhanced10.05%,8.60%and8.54%seperately. The results showed that over-expression ofFAD2can promote the conversion of oleic acid to linoleic acid. Further, the total fatty acids oftransgenic yeast were enhanced0.55-fold,2.86-fold,6.24-fold compared with control. The oilcontent in the seeds of transgenic Arabidopsis were raised18.29%,14.90%and18.53%compared with wild-type Arabidopsis, and the total fatty acids have enhanced6.76%,7.52%and5.65%. All the results indicted that DGAT2can improve the seed oil content of Arabidopsis,promote fatty acids accumulation into lipid of yeast and Arabidopsis.4) FAD2/DGAT2co-expressed vector was transferred into yeast and Arabidopsis. Theexpression patterns of FAD2and DGAT2in transgenic yeast and Arabidopsis were bothdeclined compared with single-gene transgenic line. The effect of enhancing linoleic acidcontent in co-expressed yeast and Arabidopsis was inferior to that in FAD2-transgenic line.While the effect of improving total fatty acids was lower than that in DGAT2-transgenic line.However, the amount of oil content improved by FAD2/DGAT2in Arabidopsis seed was closeto that in DGAT2-transgenic plants. The results above indicated that synergistic effect of FAD2and DGAT2was not apparent, which might be related with the relatively lower genesexpression in FAD2/DGAT2transformants than that of single gene transformants. Neverthelesswe can improve the oil content and a small amount of linoleic acid in the meantime.

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CLC: > Agricultural Sciences > Forestry > Forest tree species > Special use of broad-leaved class > Tung
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