Studies On Genetic Regulation Of Fatty Acid Composition In Rapeseed And Outcrossing Stability Of Fatty Acid Contents In Transgenic Lines

Rapeseed is the most important oil crop, and rapeseed oil occupies a decisive position in supply of edible vegetable oils in China. Further development of rapeseed production, therefore, is the fundamental way to remit shortage and ensure supply security of edible oils. So far, prominent achievements have been made in double-low rapeseed (DLR) breeding and the popularization rate of DLR varieties has reached more than70%in China. On this basis, increasing attention is being paid to breeding for high oleic acid (OA) content. On the other hand, however, rise in erucic acid (EA) content and decrease in OA content in rapeseed caused by outcrossing with foreign pollens have become a practical problem awaiting solution in DLR production. To construct a low erucic and high oleic acid mechanism in rapeseed from the perspective of breeding, by which low EA and high OA contents could be kept essentially constant under outcrossing condition, is undoubtedly a good strategy of solving this problem.According to above research background, establishment of an outcrossing-insensitive low erucic and high oleic acid mechanism, which was based on RNA interference expression of genes encoding for fatty acid elongase1(FAE1) and delta-12fatty acid desaturase (FAD2), was carried out in our laboratory. Centering on this goal and based on the existing research foundation, the ability of FAE1-RNAi transgenic lines to keep outcrossing stability of low EA content was verified, molecular identification and fatty acid composition analysis of FAD2-RNAi transgenic lines were conducted to screen transgenic plants with low contents of linoleic and linolenic acids and verify their ability to maintain outcrossing stability, and regulation effect of synergistic interference expression of the two target genes on fatty acid composition in rapeseed was investigated by means of hybridization of two kinds of transgenic lines in this study. The main results were as follows:1. After crossing of FAE1-RNAi transgenic low EA lines with three conventional high EA varieties, EA contents in hybrid seeds were similar to those of transgenic lines and OA contents changed little too, indicating that FAE1-RNAi transgenic lines had strong ability to keep outcrossing stability of low EA contents, and therefore, basic stability of OA contents.2. PCR assay showed that a specific target band could be amplified in most plants of FAD2-RNAi transgenic lines in T2generation. Southern blotting analysis demonstrated single-and multi-copy insertion of the transgene. RT-PCR analysis indicated that expression of the target gene FAD2was significantly inhibited. These results showed that the transgene had been integrated into genome of the receptor parent and could be stably inherited. Fatty acid composition analysis indicated that linoleic and linolenic acid contents in transgenic seeds harvested from T2plants were2～3%and about3%(11.97%and6.49%for the control).3. After crossing of FAD2-RN Ai transgenic lines with three conventional high linoleic and linolenic acid varieties, the average content of these two kinds of fatty acids in hybrid seeds was6.09%, close to5.47%of transgenic plants, and far lower than18.65%-22.82%of three conventional varieties, indicating that FAD2-RNAi transgenic lines could well maintain stability of low linoleic and linolenic acid contents under outcrossing condition.4. Analysis and comparison of fatty acid composition showed that inhibition of FAE1gene expression via RNA interference reduced EA and eicosenoic acid contents from38.92%and12.95%of the control to1.76%and4.56%, and increased OA content from24.28%to64.05%. RNA interference expression of FAD2gene decreased linoleic and linolenic acid contents by78.5%and51.8%respectively, and enhanced OA content by10percentage points. Syntheses of linoleic, linolenic, eicosenoic and erucic acids were effectively inhibited by synergistic interference expression of the two target genes, resulting in the average OA content of75%in F2seeds. In summary, the ability of FAE1-and FAD2-RNAi transgenic lines to respectively keep outcrossing stability of low EA and low polyunsaturated fatty acid contents was verified, and transgenic plants with high OA, low EA and low polyunsaturated fatty acid contents were preliminarily obtained by means of synergistic interference expression of the two target genes in this study. It is believed that these results are of great practical significance in developing new rapeseed varieties with outcrossing-insensitive low EA and high OA contents, ensuring edible quality, nutritive value and supply security of rapeseed oil, as well as in promoting sustainable development of rapeseed production in China.