Isolation Of Key Genes For Fatty Acid Biosynthesis And Study Of Fatty Acid Accumulation Pattern In Rapeseed

With the completion of DNA sequencing of whole genomes in model plants such as Arabidopsis and Rice, the golden oppotunity for genetic improvement of oil crops has arrived. The cloning and further studying of the key genes involved in agricultural production, and the application of the research outcome to molecular breeding has become more and more imperative. In order to provide some scientific reference for molecular breeding of fatty acid composition and oil content in Brassica napus, this research is designated to clone several key genes involved in the biosynthesis of fatty acids, to analyse the relationship between their sequence properties and functions, to study the accumulation pattern of the fatty acids during seed development, and to construct plant transformation platforms for further research.In the study of gene isolation and sequence analysis, the author first cloned the genes for fatty acid elongase (fael), fatty acid desaturase (fad2), and biotin carboxyl carrier protein (bccp) from the genomic DNA of different cultivars of Brassica napus. The sequences of the genes were characterized in both DNA and protein levels using softwares such as VectorNTI. For fael gene, 4 isoforms were isolated from A and C genomes of Zhongyou 821 and Zhongshuang No.9. Isoforms from Zhongyou 821 are of 1521bps in length and encodes 506aa, while isoforms from Zhongshuang No.9 are of 1517bps in length and encodes 466aa. No intron was found in any of the fael isoforms. For fad2 gene, 4 isoforms with integrate ORF were isolated from the same two cultivars. It was shown that fad2 gene is 1152bps in length, encoding a 384aa protein and containing no intron in the coding region. For beep gene, 3 isoforms were obtained from 3 cultivars, of which only one isoform encodes the integrate protein and contains the domain for biotinylation, while the other two isoforms encode proteins that are incomplete and lack the domain for biotinylation. Further analysis revealed that the shortened protein of the two bccp isoforms is arisen from a frame-shift mutation which is caused by an adenine insertion between intron 3 and intron 4 in the gene. This research may provide some scientific basis for genetic engineering and molecular marker-assistant breeding of the isolated genes.In the investigation of accumulation pattern of major fatty acids in Brassica napus, the author analyzed the biosynthetic patterns of seven key fatty acids during seed development of HEAR Zhongyou 821 and LEAR Zhongshuang No.9. The data were processed by correlation analysis and principal component analysis. Results showed that the biosynthetic patterns of saturated (c16:0,c18:0) and polyunsaturated (cl8:2,cl8:3) fatty acids were of highly similarity, all exhibited an "ascent-descent-flat" pattern. They accumulated predominantly during the first 15 days after pollination (DAP). Oleic acid (c18:1) exhibited a similar "ascent-flat-descent" pattern in both varieties, but the rapid ascending phase, prolonged and the final content doubled in Zhongshuang No.9 in comparison with Zhongyou 821Eicosenoic acid (c20:l) and erucic acid (c22:l) showed an "uneven wave" and "double ascents" pattern respectively, and the their accumulation mainly occured after the 15th DAP. Highly significant difference was detected in the accumulation of VLCFAs (c20:l,c22:l) between the two cultivars as their synthesis in Zhongshuang No.9 was nearly blocked. The correlation among main fatty acids in Zhongshuang No.9 had remarkably changed due to the mutation of fael compared with Zhongyou 821. According to the principal component analysis, fatty acids in Zhongshuang No.9 could be assorted into 3 groups, saturated and polyunsaturated fatty acids as the first and third group respectively, and oleic acid as the second, in which accumulation patterns of fatty acids from the same group had no serious difference, and presented certain negative relationship if from the first and third group.This research has also successfully constructed several plant transformation platforms including Arabidopsis, Tobacco, and Brassica napus, and also verified the function of Cre/lox site-specific system in Arabidopsis. It has provided the down-stream platforms for further study in fatty acid biosynthesis and the application of the theoretical outcome to practice.