Mapping Of Lipid Genes And The Molecular Mechanism Of Temperature Effect On Seed Fatty Acid Accumulation In Oilseed Rape (Brassica Napus L.)

Oilseed rape(Brassica napus L.) is one of the most efficient oil crops throughout the world, it is not only a major source of edible oil but also can be used as biodiesel to solve the shortage of petrolic oil. Oil accumulation and fatty acid metabolism during seed development exist in large variations, including a large number of genes response to environmental and endogenous stimuli. Researchers and breeders have spent a lot of efforts to identify key genes related to oil formation, but so far not suffcient knowledge reflecting the genetic control of seed oil has been acuqired. This study is aim to:(1) develop a series of molecular markers of functional genes involved in oil accumulation based on the comparation of the Arabidopsis and Brassica genomes; (2) map molecular markers involved in oil accumulation on the genetic linkage map using DH lines and to identify key candidate genes contributing to certain QTL effct; (3) investigate the molecular mechanisms of the interaction between QTL and environmental factors using NILs whose oil content was in response to growth temperature during seed development. The main results are as follows:1. The genome of Arabidopsis has been searched for sequences of genes involved in acyl lipid metablism(http://lipids.plantbiology.msu.edu/). According to the sequence information of Arabidopsis lipid gene database and the analysis of the distribution of ESTs in organs,sequences of 75 seed-specific lipid genes were selected to do BLAST for homologous Brassica ESTs, a search for intron polymorphisms within the EST database was conducted. A set of 150 PCR primer pairs was designed according to Brassica ESTs. For each gene we designed 2 primer pairs, of which 51 showed polymorphisms between Tapidor and Ningyou 7. The polymorphsim rate was over 30%.2. A total of 15 markers on the seed expressing function genes were linked to the TN-DH map, which was made up by a total of 19 linkage groups and 700 markers that span over a 2060 cM distance with an average interval of 3.3 cM. Of these,5 unique markers were detected in the QTLs region contributing to oil seed oil cntent. The genes located on the 1,3,10,11,12 linkage group. Bioinformatic analysis suggested that these candidate genes involved in oil formation.3. In order to confirm the function of the candidate genes putatively contributing to the QTLs, we analysized the allelic variation of the respective loci among Arabidopsis T-DNA insertional mutant. The results indicated that the T-DNA insertion on three of the five loci resulted in signifcant change of seed oil content, whereas, the T-DNA insertion on the rest loci did not give rise to the change of total seed oil content, but instead to the change of specific fatty acid ratio.4. Temperature had a clear effect on seed oil content as well as fatty acid composition. Overall, the NIL plants responded to increasing temperature with decreasing levels of total seed oil, linoleic acid (C18:2), linolenic acid (C18:3), and erucic acid (C22:1). NIL-9 had higher seed oil content than did NIL-1 in all three growth chambers. However, the degree of surplus varied. The highest surplus was found in Chamber T3 and the lowest in Chamber T2.5. Statistical calculations indicated that the effect of genotype, temperature, and the interaction between genotype and temperature on the expression of the genes in 25 DAF seeds were all significant at a 0.01% false discovery rate, generated 19 111,4 982 and 839 DEGs (Diffenentially expressed genes, DEGs) respectively. The smallest expression differences were found under T2, under which NIL-1 differed from NIL-9 with the expression of 251 genes globally and 39 genes at the qOC.C2.2 region. More genetic divergences were observed under T1, under which the NILs differed from each other with 2 933 DEGs globally and 460 DEGs at the qOC.C2.2 region. The greatest difference was caused by T3, under which the NILs differed from each other with 3 499 DEGs globally and 558 DEGs at the qOC.C2.2 region. This results were consistant with the oil content phenotype.6. The 246 DEGs resulting from genotype can be grouped into various GO categories. Of these, a high proportion of GO slims were specifically related to embryo development, DNA-dependant transcription regulations, stress responses (such as oxidation reduction; salt stress response; heat response; wound response; cadmium stress response), and photosynthesis (photorespiration; photosystem stoichiometry adjustment; glycolysis; malate metabolism). These were over represented on the list of DEGs arising from genotype. A number of DEGs also belonging to the GO categories are involved in more general biological processes. Of these, some are associated with protein biosynthesis, protein folding and protein intercellular transport, translational initials, jasmonic acid response.7. Either increasing the temperature to T3 or decreasing the temperature to T1 resulted in the up-regulation of the genes related to DNA-dependent transcription regulations, embryo development, stress response (bacterium defending response; cadmium stress response), protein biosynthesis, amino acid phosphorylation, and protein folding. Increasing the temperature to T3 caused the up-regulation of the genes involved in heat response and ubiquitin-dependent protein catabolism, but the down-regulation of several genes associated with fatty acid biosynthesis, red light response, photosynthesis, gibberellic acid stimulus response, and translational elongations. Decreasing the temperature to T1 led to the down-regulation of some genes regulating glycolysis, malate metabolism, abscisic acid stimulus response, the tricarboxylic acid cycle, and water deprivation response. Either increasing temperature to T3 or decreasing temperature to T1 resulted in the down-regulation of the genes of the cold response category and the up- and down-regulation of the genes of the oxidation reduction category.8. The genetic difference between NIL-9 and NIL-1 had a significant effect on the expression of the genes BnLACS1, BnOLEO1, BnCLO1, BnFatA, and BnLHY. Relative to NIL-1, NIL-9 had a higher expression level of BnLACS1, BnCLO1, and BnLHY, but a lower expression level of BnOLEO1 and BnFatA. Increasing temperature led to a higher expression of BnAB13, BnFUS3, BnTAG1, BnOLEO1, BnCLO1, and BnFaTA, but a lower expression of BnLEC1, BnWRI1, BnFAD2, BnFAD3, and BnLPAT2. The G×T interaction significantly affected the expression of BnFAB2. Treated only under T1, NIL-9 had a higher expression of BnLPAT2 than NIL-1.