Studies On Inheritance And Improvement Of Oleic Acid Content Via IhpRNA Silencing Fad2 Gene In Brassica Napus L.

Oleic acid is an important fatty acid composition of rapeseed oil. The nutritional and economic value of rapeseed oil is determined by content of oleic acid in the oil. How to improve oleic acid content in rapeseed oil is one of the important breeding goals in oilseed rape breeding program. High oleic acid material had been obtained through EMS chemical mutagenesis and RNAi gene silencing technology as well as traditional breeding methods. The inheritance of oleic acid content was studied in this research by applying major gene plus polygene mixed genetic models and joint analysis method of multi-generation.And some transgenic plants with fad2 gene were created by RNAi gene silencing technology. The main results from this research were as follows:1. Inheritance of oleic acid content(OAC) was investigated in P₁,P₂,F₁,B₁,B₂andF₂ generations derived from the cross of 8087×8108(Brassica napus L.).Results showed that: (1)OAC were controlled by two major genes with additive-dominance-epistatic effects plus polygenes with additive-dominance- epistatic effects (the E-0 model). (2)Heritability values of the major genes (h²_mg) of B₁,B₂ andF₂ population was estimated as 43.83%, 94.06%and 94.71%respectively, while those of polygenes(h²_pg) was 0.65%,0.86%and30.85%.It demonstrated that OAC level in the cross was controlled by two major genes mixed with the effect of polygenes, but mainly governed by two major genes.(3)Additive effects and epistatic effects contributed largely to OAC, while dominance effects was relatively smaller.2. The full-length sequence (1147bp) of napin promoter and a conserved region (537bp) of microsomalΔ¹²-desaturase gene (fad2) were amplified from B.napus L. and cloned into the pGEM-T easy vector. To develop a seed-specific expression ihpRNA targeted the fad2 gene in B. napus L.,an inverted repeated unit of the 537bp segment of the fad2 gene was first cloned into a medium vector, the pHurrican Vector, interrupted by a spliceable fad2 intron sequence.Then the seed-specific promoter, napin promoter, was placed to the 5'-end of the invert repeat cassette, and a nos polyA to the 3'-end of it. Finally, an ihpRNA expression vector pCNFIRnos was constructed by inserting the whole invert repeat cassette into the multiple done sites in the binary vector pCAMBIA2300.The pCNFIRnos was confirmed by the digestion of restriction enzymes.3. Agrobactedum-mediated transformation procedure for fad2 gene was established in this research using the hypocotyl and petiole of Ningyou 12(B.napus L.) as an explant. Results demonstrated that: (1)The hypocotyl of Ningyou 12 was cultured in the base medium suitable for differentiation of MS+I.Smg/L 6-BA+0.15mg/L NAA+0.01mg /L GA3+0.5g/L Mes+0.Sg/L PVP+40mg/L Adenine+3.Smg/LAgNO3.Th rate of regenera- tion and differentiation was 25.71% and 31.43% respectively.(2)The polarization frequency of hypocotyl and petiole of Ningyou 12 precultivated in MS medium with 1mg/L 2,4-D was enhanced significantly.(3)With the increasing concentration of AgNO₃, the frequency of regeneration and polarization increased gradually.Smg/LAgNO₃ was the optimum concentration for the high frequency regeneration of hypocotyl of NingYou 12.(4)With the kanamycin concentration increasing, the shoot regeneration frequency of hypocotyl and petiole of Ningyou 12 decreased gradually.In our experiment,the optimum concentration of kanamycin was 20mg/L for screening transformed shoot.(5)The appropriate concentration of carbentcillin was 250mg/L which was good enough to inhibit the growing of agrobacterium excessively.(6)The regeneration frequency of kanamycin-resistant green shoot obtained from hypocotyl explants were 0.45%,while that from petiole explants reached 6.23%.By result of PCR amplification,more than 90% of the kanamycin-resistant plants showed positive.Preliminary evidence showed that the fad2 inverted repeats expression cassettes have been integrated into the nuclear genomes of NingYou 12.