The Effection Of The Fatty Acid Composition By Inhibition Of Fad2 Gene Expression With RNAi

Oleic acid is one of the high-quality component in rap oil, it is not only because its Oxidative Stability which bring on long-term preservation in edible, but also the high value in industrial Production. So improving oleic acid content is becoming one of the most important ways in Rape Quality Improvement. The technology of genetic engineering is becoming one of the main Methods to improve the Rape quality.Fad2 is a key enzyme in the metabolism of fatty acids, which function is inhibiting the formation of linoleic acid and a-linolenic acid and improving the Oleic acid content.This study is aimed at blocking Linolic acid biosynthesis by RNAi strategy and limiting multi-unsaturated fatty acid synthesis in rapeseed(Brassica napus)during the Seed development period. I construct an ihpRNA vector for Seed-specific expression—two segments of fad2 in the antisense and sense orientations were inserted between the intron of vector pKANNIBAL, and Napin promoter replaced the 35S promoter intrinsic. The ihpRNA vector pG-fad2i is constructed by cutting the whole expressing sequence down and inserting it into the Expression Vector pGreen0229. The vector is aimed to transforming the Brassica napus and silencing the post-transcription expression of fad2 gene during the seed development. The main study results are as follows:1. I designed the Napin promoter's Primer according to the published sequence AF420598 in GeneBank, and then cloning its sequence from the Brassica napus L by PCR, which is being inserted into the vector pKANNIBAL to replace the 35S promoter intrinsic. And then I designed the fad2 gene's Primer according to the published sequence AF243045 in GeneBank and cloned it by RT-PCR. At the end I choosed a 310bp Conservative segment from fad2 gene and inserted them between the intron of the vector pKA-N. The ihpRNA vector pG-fad2i was constructed by cutting the whole expressing sequence down and inserting it into the Expression Vector pGreen0229.2. The FAD2gene is expressed in E. coli DE3, and the syncretic protein detected by SDS-PAGE electrophoresis is accordant with the forecast.3. The ihpRNA vector pG-fad2i were transformed into Brassica napus cultivars: Zhong shuang 9,61077 and 61078 by Pollen tube pathway and Agrohacterium-mediated method. There are 30 plants with PPT resistance. Among them, There are 9 plants gotten from Zhongshaung 9,16 plants gotten from 61077 and 4 plants gotten from 61078.4. The PCR analysis showed that the ratio of negative transgenic plants was 50%.5. The result of analysising the fatty acid component of the seed oil of Zhong shuang 9: the Oleic acid content increases nearly 7%, Linoleic acid content decreases nearly 6%, Linolenic acid content decreases nearly 1%; The result of analysising the fatty acid component of the seed oil of 61077: The Oleic acid content increases nearly 4%, Linoleic acid content decreases nearly 3%.