Creation Of A High Erucic Acid Mutant And Cloning Of Related Genes In Brassica Napus

Rapeseed(Brassica napus L.,genome AACC,2n=38) is today the most widely cultivated crop species in crucifer(Brassicaceae) family.As one of the most important oil crops,rapeseed is annually planted 7.0×10⁶ hectares in China which takes the first place in planting area and yield among rapeseed-growing countries. Rapeseed fatty acid composition determines the quality and the use of vegetable oil, the level of erucic acid content playing a key role in rapeseed edible consumption and industrial use.Erucic acid can be used as paints,cosmetics,plastics,pharmaceuticals and industrial lubricants,and other products.High erucic acid rapeseed is known as the 21st century chemical raw materials because of the importance of industrial uses.The seeds of Brassica napus M13(erucic acid content 48%) and 742(erucic acid content 1.6%) were irradiated by ⁶⁰Coγray.SSR markers were used for analysis of the F₂ populations from the cross M13×742.The genes FAE1 and FAD2 were cloned from 742,M13 and a high erucic acid mutant H27.The main results were as follows:(1) Seeds of Brassica napus lines M13 and 742 were irradiated by ⁶⁰Coγray at doses of 800,1000 or 1200Gy.The treatment of 800～1000Gy irradiation increased oil content to various degrees,with erucic acid and oleic acid content of rape seeds having greater variability.Increased oleic acid variants were found in advanced progenies of irradiated 742 line while increased erucic acid mutants in M₃ progenies of irradiated M13 line.(2) Three high erucic acid mutants 55.51%(06 A-8),55.21%(06A-991),55.09% (06A-1256)) were found in M₂ population of M13 irradiated by 800 Gy;Their stability in erucic acid content was detected in M₃ population.Although the erucic acid content of 06 A-991 and 06 A-1256 reverted to 48%,06A-8 is 55.8%on average. H27,a mutant highest in erucic acid content in this group(06A-8),had erucic acid content of 57.9%,as confirmed by gas chromatography analysis.(3) Segregation of erucic acid content in the F₂ generation from M13×742 showed that the erucic acid content is controlled by two loci,which one locus playing a major role.QTLs for erucic acid content have been previously mapped on the linkage groups N3,N8,N11 and N13 and therefore the 76 SSR markers were chosen for mapping from these four linkage groups.It is found that the SSR marker CB10364 can distinguish the plants with＜6%erucic acid content from the plants with＞36%in the F₂ population.(4) FAE1 and FAD2 fragments amplified by PCR using the genomic DNA extracted from leaves of M13,H27 or 742 were sequenced.Comparison of FAE1 sequences showed that each sequencing map had a single peak,indicating that direct sequencing can accurately reflect the original information.The amplified FAE1 fragments were 1521 bp long and had homology of 99%in nucleotide sequence among the three lines.M13 and H27 had three nucleotide substitutions,but no amino acid change.M13 and 742 not only had four nucleotide substitutions but also four amino acid changes.Sequencing maps FAD2 fragments were bimodal,meaning that two kinds of base likely exist at the same position.FAD2 direct DNA sequencing will not accurately reflect the differences between materials.(5) The FAD2 amplified products of M13,H27 and 742 were cloned with T-vector and then sequenced.Analysis showed that there are two FAD2 copies in each line.Their lengths were 1155(FAD2.1) and 1140 bp(FAD2.2).The three lines shared the nucleotide sequence of 98.99%in FAD2.1 copy,without difference between M13 and H27.However,35 single nucleotide substitutions and three amino acid changes have been found in FAD2.1 between M13 and 742.Comparison of FAD2.2 sequence with FAD2(1155bp) published in NCBI database showed loss of 15 nucleotides(TCCCTCACCCTCTCT) from nt230 to nt244 in all the three lines. Substitution of T for A at nt409 of FAD2.2 of the line H27 nucleotide 409 produced a premature stop codon(TGA).Nucleotide have 21 single-nucleotide and 11 amino acid differences have been found in FAD2.2 between M13 and 742.The two copies of FAD2 had difference in nucleotide and amino acid sequence of M13 and 742,which may indicate that different erucic acid content will be differences in FAD2.(6)It can be speculated from the above results that the increase of erucic acid content in the mutant H27may be due to both non-sense mutation of FAD2.2 leading to FAD2.2 gene inactivation and block of conversion of oleic acid into linoleic acid, and synonymous mutation of FAE1 enhancing FAE1 activity which converts more oleic acid into erucid acid.