Cloning Of Novel Genes Relative To Fatty Acid Synthesis From Brassica Napus L.

6 SNPs that can discriminate the resource of Fad2 gene from A genome (Brassica rapa) or C genome (Brassica oleracea) were found out through sequencing and aligment. By use of the SNPs, Two pairs of allele-PCR primers have been designed to detect the expression of Fad2 gene from A genome or C genome in Brassica napus seeds rapidly. In this study, the expression pattern of Fad2-A and Fad2-C genes were investigated. The reluts shows that Fad2-A gene expression pattern is similar with HO2, and Fad2-C is similar with HOI. It was reported that HOI and HO2 gene are the two key gene sites related to synthesis of high oleic acid in Brassica napus L. So, it's the first time giving evidences to prove that oleic acid's formation in Brassica napus is related to gene's genotype.To further study on the fatty acid synthesis regulation,20 DAP developing seeds and 35 DAP developing seeds were chosen for estabolishing suppression subtractive hybridization (SSH) library, which SSH is an effective method for picking out specific expression genes among different samples. Two libraries,20 DAP SSH library derived from 20 DAP seed cDNA as tester and 35 DAP seed cDNA as driver and 35 DAP library from 20 DAP seed cDNA as driver and 35 DAP seed cDNA as tester were constructed. The two SSH libraries had a high quality with high suppression subtractive efficiency after tested by PCR and RT-PCR. A total of 489 clones were randomly selected from the two libraries for sequencing and 452 high quality sequences tags were obtained. Blast analysis and functional annotation showed that most of the genes in 20 DAP SSH library were relative to carbohydrate metabolism, while those in 35 DAP library relative to fatty acid metabolism. Significantly, five function-unknown genes in 20 DAP library and seven in 35 DAP library were found out. In summary, this work adds an extra layer of complexity to the regulation of starch-to-oil transition and at the same time the different genes, especially the function-unknown genes shed light on studies of molecular mechanism of fatty acid metabolic regulation in seeds of Brassica napus L.BnaLCR78 gene is function-unknown in rapeseed. Biology information tell us that BnaLCR78 gene could translate a low-molecular-weight cysteine rich secreted protein with 79 AA residues. Through BLAST in NCBI and found that BnaLCR78 gene's sequence and structure property are similar with LCR gene family in Arabidopsis Thaliana. There are 86 members in this gene family. According to the alignment and cladogram, we divided the whole family into 6 classes. Almost all the 11 proteins relative to defense function were divided into the first class based on published papers. BnaLCR78 gene shared a higher homology with the fourth class that their function are not clear yet.To confirm the function of BnaLCR78 gene, we transformed the full BnaLCR78 gene into Arabidopsis Thaliana and make the gene expression constitutively by 35S promoter or specially in seed by NapinA promoter. For gene silence, the T-DNA mutant of LCR23 gene was orderd from ABRC and the homozygous mutant was obtained through PCR screening. RT-PCR test of whole plant showed that both LCR23 gene and BnaLCR78 gene were expressed in seed.Some other members of LCR gene family expressed as a pollen coat protein (PCP), whose function is to help stigma and pollen recognize each other. When plant is lack of PCP gene, pollen tube couldn't find micropyle accurately and make pollination difficult. In this study, both mutant BnaLCR78 and mutant LCR23 could complete fertilization in two hours which indicated that neither BnaLCR78 gene nor LCR23 gene took part in the pollination process of Arabidopsis Thaliana.Fatty acid constituent content in Arabidopsis seed were detected by GC-MS. Compared with wild types, eicosenoic acid's content in mutant LCR23 decreased by about 14%, erucic acid content increased by about 7% and eicosadienoic acid content decreased to zero. Two carbon elongation of eicosenoic acid and eicosenoic acid desaturation produces eicosadienoic acid. So LCR23 gene just like a molecular switch regulated the content alteration between erucic acid and eicosadienoic acid at the same time. This is a new discovery about the function of LCR gene family.