Genetic Analysis Of Seed Oil Content And Function Analysis Of Gene Involved In Lipid Biosynthesis In Brassica Napus L.

Rapeseed is one of the most important oil crops in the world.And in China,the acereage and total production accounts for about 1/4,the highest in the world.High-oil breeding is a primary improvement strategy for oil-yield in oil crops,and it is an essential goal in Brassica napus in China as well.As seed oil content(OC)is a key measure of rapeseed quality,better understanding the genetic basis of OC would greatly facilitate the breeding of high-oil cultivars.Our study thoroughly investigated and reliably quantified various genetic factors associated with OC of rapeseed by using a full diallel and backcross and reciprocal backcross.This findings lay a foundation for future genetic studies of OC and provide guidance for breeding of high-oil rapeseed cultivars.Here,we investigated the components of genetic effects and genotype × environment interactions(GE)that govern OC using a full diallel set of nine parents,which represented a wide range of the Chinese rapeseed cultivars and pure lines with various OCs.Our results from an embryo-cytoplasm-maternal(GoCGm)model for diploid seeds showed that OC was primarily determined by genetic effects(VG)and GE(VGE),which together accounted for 86.19% of the phenotypic variance(VP).However,the influence of GE should not be neglected,since GE accounted for 51.68% of the total genetic variance.Furthermore,maternal variance explained 75.03% of the total genetic variance,embryo and cytoplasmic effects accounted for 21.02% and 3.95%,respectively.We also found that the OC of F1 seeds was mainly determined by maternal effect and slightly affected by xenia.In addition,general combining ability(GCA),specific combining ability(SCA)significantly contributed to the OC variation over the years,indicating of both additive and non-additive effects on OC.Maternal effect was significant,whereas non-maternal effect was not,suggesting that OC was not under strict nuclear control and could also be influenced by cytoplasm.Thus,the OC of rapeseed was simultaneously affected by various genetic components,including maternal,embryo,cytoplasm,xenia and GE effects.All of the genetic effects of parent H1 were positive,suggesting that it might be an ideal maternal parent for breeding high-oil rapeseed.For breeding high-oil rapeseed,we determined that the selection of parents,especially the maternal parent,is important and that the parental pollen and the maternal cytoplasm and embryo should also be evaluated.To gain insights into the transcriptional regulation of OC in rapeseed,a microarray analysis was conducted to identify the genes potentially involved in the regulation of OC.The results showed that BnCIPK9 was one of the significantly down-regulated genes in high-oil lines compared with in low-oil lines.DNA sequence analysis of this gene suggested that it had four copies and was conserved.High BnCIPK9 transcription levels were observed in the stem,leaf,and 24 DAP silique wall,whereas low transcription levels were found in flower,bud,and 24 DAP seed,especially in the root.The expression of BnCIPK9 was relatively higher at the middle stage of seed development.These results indicated that BnCIPK9 has a similar expression pattern as AtCIPK9: both of them are expressed in various organs,including photosynthetic and non-photosynthetic tissues.We cloned two approximately 3050bp(promoter1)and 3372bp(promoter2)fragments of the 5'-untranslated region proximal to BnCIPK9 genomic locus,which contained several putative cis-elements for gene expression and regulation.Both BnCIPK9 promoter1 and promoter2 regions contained two putative sugar-responsive elements,TATCCA,found in the ?-amylase gene of rice.Four and six I-BOX motifs were found in promoter1 and promoter2 fragments,respectively.In addition,the promoter1 and promoter2 fragments respectively include four and sive E-BOX motifs,which are likely involved in seed-specific expression.In order to specifically control the lipid accumulation in the mature seeds without ectopic effects,the BnCIPK9 transgene was placed under the control of the Bnnapin promoter,which is seed-specific.Compared with the control(CK: non-transgenic line)plants,all tested transgenic lines showed decreased total OC.Therefore,BnCIPK9 might be a negative regulator of OC.Results showed that the average OC of WT was 24.06%,whereas that of cipk9 was 26.25%.In cipk9 seeds,there was an obvious increase in the relative proportion of C20:1?11,whereas the relative proportion of C18:2 was clearly decreased.The complementation lines had similar oil content to wild-type plant and the overexpression lines exhibited decreased in OC compared with cipk9 mutant.These results showed that the increase of cipk9 seed oil content was caused by the disruption of AtCIPK9.Here,we also examined the role of AtCIPK9 in the seedling establishment comparing cipk9 mutant,transgenic lines and wild-type.The cipk9 mutant seedling failed to establishment on the sugar-free medium and this phenotype would be rescued by sucrose or glucose.The phenotype test suggested that both the overexpression and complementation transgenic lines displayed a similar phenotype to wild-type when they were grown on sugar-free medium.Because of CBL2 and CBL3 may play roles in seedling establishment under sugar-free condition by interacting with CIPK9,the phenotype of cbl2 and cbl3 mutant under sugar-free condition were analyzed.Only cbl3 mutants exhibited a similar sugar-free phenotype to cipk9 mutant,suggesting that AtCBL3 and AtCIPK9 might work together and play roles in seedling establishment under sugar-free condition.