The Role Of Safflower Transcription Factor CtNF-YB12 In Regulating Seed Oil Content

Safflower(Carthamus tinctorius L.),a Compositae herb,is an economic crop with multiple uses such as oil and medicine.Safflower seed oil is a precious high-quality edible oil extracted from safflower seeds,which is called "healthy oil" and "nutritional oil".With the improvement of people’s living conditions,the demand for high-quality safflower edible oil has greatly increased.Judging from the existing safflower planting area,it is far from meeting the market demand.Therefore,it is imminent to cultivate high-quality new safflower strains by means of genetic engineering.The LEC1-type transcription factors of the NF-YB gene family exist in eukaryotes and can increase the oil content of seeds by regulating fatty acid(FA)synthesis and glycolysis processes.In recent years,there have been many studies on LEC1-type transcription factors involved in the regulation of oil synthesis,but no report has been reported on the role of LEC1 in the accumulation of oil in safflower.In this study,the safflower NF-YB family transcription factor(Ct NF-YB)was screened from the safflower genome database,and Ct NF-YB12 was identified as a LEC1-type transcription factor by bioinformatics analysis.The tissue-specific expression patterns of the NF-YB gene family were clarified by quantitative real-time PCR(q RT-PCR).The full-length c DNA fragment of Ct NF-YB12 gene was cloned by PCR technology,and the yeast expression vector p PICZαA-Ct NF-YB12 was constructed to transform Pichia pastoris X33,and the effect of Ct NF-YB12 gene on yeast oil content was preliminarily verified.A plant expression vector was constructed,and the Flora dip method was used to infect Arabidopsis thaliana,and the T3 generation overexpressed Arabidopsis thaliana and mutant complements Arabidopsis thaliana were obtained,and then identify the biological function of Ct NF-YB12 gene in Arabidopsis thaliana.The specific research results are as follows:1.Fourteen high-confidence Ct NF-YB genes were screened from the safflower genome database,which were divided into class I(non-LEC1),class II(LEC1)and class III(Dr1-like).By analyzing the cis-regulatory elements of the Ct NF-YB gene promoter,it was found that it contained cis-regulatory elements related to abiotic stress,biotic stress and plant growth and development.Most of Ct NF-YB was enriched to molecular functions and biological processes by Blast 2 go analysis.2.The results of q RT-PCR showed that class I(non-LEC1)genes were highly expressed in roots,leaves and flowers.Class II(LEC1)genes were highly expressed in the early stage of seed development.Class III(Dr1-like)genes were expressed in roots,stems and leaves.The subcellular localization experiments of Ct NF-YB12 gene proved that Ct NF-YB12 gene was located in the nucleus.3.By constructing the yeast expression vector p PICZαA-Ct NF-YB12,and transforming the yeast strain,it was found that the yeast transgenic Ct NF-YB12 gene significantly increased the oil content.4.By constructing a plant expression vector and transforming Arabidopsis thaliana,the T3 generation overexpression and mutant complementing Arabidopsis thaliana were obtained,and three high expression lines were obtained in mutant complements Arabidopsis thaliana and overexpresses Arabidopsis thaliana.The seed pods of Arabidopsis thaliana overexpressing Ct NF-YB12 were longer,the number of seeds was larger,and the thousand-seed weight was significantly increased.Mutant Arabidopsis thaliana recovered to the level of wild-type seeds.5.Compared with wild-type seeds,the oil content and triacylglycerol(TAG)content of overexpressed Arabidopsis thaliana seeds increased significantly,and the fatty acid content increased.The oil,TAG and fatty acid contents of the replenished Arabidopsis thaliana seeds recovered to the levels of wild-type seeds.The expression of Ct NF-YB12 increased the soluble sugar,glucose,fructose and sucrose of seeds through the analysis of glucose metabolism and lipid metabolism,and also positively regulated the expression of key enzyme genes in glycolysis and fatty acid synthesis.6.By constructing the yeast expression vector p GBKT7-Ct NF-YB12,it was verified that p GBKT7-Ct NF-YB12 has no transcriptional autoactivation activity.It was confirmed by yeast two-hybrid experiment that Ct NF-YB12 may interact with Ct NF-YC2.