Obtaining Transgenic Plant Of Brassica Napus By RNA Interference Of Fad2 Gene

In recent years, the deep study on molecular biology about fat metabolism related gene separation, clone and the organ specificity promoter and so on, has opened the possibility to change rape seed pathways and improve rape quality using gene engineering. From the fatty acid biosynthesis chain, we may see that the oleic acid is an important metabolism branching point in fatty acid synthesis, which can change the fatty acid composition and their proportion in the seed. Delta-12 oleate desaturase gene (fad2) is a rate-limiting gene for multi-unsaturated fatty acid synthesis in rapeseed (Brassica napus), so blocking linolic acid biosynthesis by RNAi strategy targeting fad2 gene will increase the content of oleic oil in the low erucic acid rape. If in the high erucic acid rape, blocking linolic acid biosynthesis may increase the content of erucic acid and expected to obtain the rape whose content of erucic acid is close to the theory value 66%.Recently, the double strand RNA interference technology (RNA interference, RNAi) has obtained the unprecedented progress in the research about improvement of oil-bearing crop's fatty acid composition. The present research is to introduce the silence of fad2 gene in Brassica napus using ihpRNA (intron splicing hpRNA, ihpRNA) whose silencing efficiency is higher, then to achieve the genetic improvement of oil in rapeseed. And also this work may provide technology and material foundation for further breeding high erucic acid or high oleic acid rape, and develop the theory and practical application of gene silence technology in plant quality improvement.In the present study, we have constructed RNAi plant expression vector-pCAMBIA3301-fad2i targeting against fad2 gene by RNAi strategy. With the cotyledons with petiole of a high erucic acid cultivar Y14-1 and a low erucic acid R02 as materials, the fad2 RNAi was introduced into plant by A. tumefaciens-mediated method. Thereinto, we expected to obtain some PPT-resistant green shoots with increased content of erucic acid by infecting the high erucic acid rape Y14-1; some PPT-resistant green shoots with high oleic acid by infecting the low erucic acid rape R02.The results listed as follow:1. Cloning and analysis of five fragments for constructing the fad2 RNAi vectorThis study had amplified five fragments for constructing the fad2 RNAi vector-napin promoter, fad2-1, fad2-2, rbcS-3C intron, CaMV 3'UTR. These fragments were then ligated into pMD18-T vector and transformed into the E.col competent cell. The sequencing results had been compared to the sequence which published in NCBI. Results showed that sequence of five cloned fragments was consistent with above 90% of the sequence which published in NCBI. And it is feasible to use the total genome DNA of rape as template to amplify fad2-1and fad2-2 fragments.2. Creating fad2 RNAi plant expression vectorThe fad2 RNAi plant expression vector in this research is an ihpRNA construct, containing anti-sense/sense arms and an intron, which has consistently enhancing effect. The gene-specific sequences of fad2 in the antisense and sense orientations interrupted by a spliceable intron from pea rbcS-3C gene, were cloned into the plant expression vector pCAMBIA3301. We chose the napin promoter in order to keep its expression time consist with target gene. The terminator from pCAMBIA1301 had high terminating function.3. Transforming the fad 2 RNAi constructs into rapeseed Brassica napus by A. tumefaciens-mediated methodUsing the cotyledons with petiole of the high erucic acid cultivar Y14-1 and the low erucic acid variety cultivar as materials, the fad2 RNAi constructs were introduced into rapeseed by A. tumefaciens-mediated method. We had obtained 83 PPT-resistant green shoots by infecting the cotyledons with petiole of cultivar Y14-1. After GUS histochemistry dyeing examination and PCR amplification survey, 14 transgenic plants were obtained. Using the near-infrared reflectance method (NIR) analyzer, we examined the content of fatty acid of rapeseeds. The results indicated that: in plants of PPT-resistant green shoots obtained by transforming the fad2 RNAi fragment into the rape Y14-1, the content of erucic acid increased, whenas the content of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid decreased, in contrast to the rapeseeds which weren't infected. This showed that it was possible to obtain the rape whose content of erucic acid is close to the limit in theory. Because the content of total acids in T1 generation is under the usual value resulted from immaturity, so the experiment data is only as a reference. We should detect the fatty acid content of T2 generation again, and expect to obtain the more exact analytic results. In the all, the present work has paved a way for breeding high erucic acid rapeseed variety in the near future.