| Fengdan(Paeonia ostii)is a perennial woody deciduous shrub belonging to the genus Paeoniae in the Paeoniaceae family.It is an important new oil crop with high ornamental value,medicinal value and oil value.The seeds are rich in oil and high in unsaturated fatty acids,especially linoleic acid and linolenic acid,so they have great development potential.Plant transcription factor WRINKLED1(WRI1),a member of AP2/EREBP,is involved in the regulation of glycolysis and the expression of genes related to the denovo synthesis of fatty acids in plastids,and is an important regulator of seed oil synthesis and accumulation.So far,gene sequences encoding transcription factor WRI1 have been cloned and functionally analyzed from model plants such as Arabidopsis thaliana,Zea mays and Brassica napus.In order to explore the genetic information related to oil and to analyze the mechanism of high-level synthesis and accumulation of oil in the seeds of Paeonia ostii,analysis of Paeonia ostii seed transcriptome with high-throughput sequencing technology,is conducive to the identification of fatty acid biosynthesis related genes.On this basis,the key transcription factor gene was cloned and transformed into Arabidopsis for functional identification.The phenotypic observation,lipid assay and gene expression of transgenic Arabidopsis seeds were analyzed in order to study the regulation function of transcription factor PoWRI1 on seed lipid accumulation.The study would be advantageous to the variety improvement and utilization of Paeonia ostii,of which the obtained results summarized as follows:(1)The cDNA library was constructed by utilizing Illumina HiSeq platform for transcriptome sequencing.212.9 Gb of data information was obtained.After assembly clustering and redundancy removal,94,976 Unigenes were obtained,and 38,457 differentially expressed genes(DEGs)were identified,and 18 DEGs were selected randomly to verify the accuracy and reliability of RNA-seq data by real-time quantitative PCR(qRT-PCR).Compared and annotated Unigene with public database,it was found that the similarity between P.ostii and Vitis vinifera in NR database was the highest(23.63%);Pathway analysis showed that Glutathione metabolism,Photosynthesis-antenna proteins,Phenylpropanoid biosynthesis,alpha-Linolenic acid metabolism were enriched together and significantly in the top 20 metabolic pathways with the lowest Q-value(Q-value≤0.05);Then the specific Unigenes related to the oil accumulation were determined.(2)Base on the analysis of transcriptome sequencing,the transcription factor gene PoWRI1 related to the regulation of seed oil accumulation was cloned.The full-length sequence of PoWRI1 was 1413 bp,and the open reading frame was 1269 bp which encoded 422 amino acids.The homology of WRI1 protein sequence with Syzygium jambos,Eucalyptus grandis,Vernicia fordii and Manihot esculenta was 53%,55%,55%and 58%.The amino acid sequence encoded by PoWRI1 was analyzed by biological information.The relative molecular mass of this protein was 47.0 kDa,no transmembrane structure was formed.Also,there was no signal peptide,and the protein was located outside the membrane.The two conserved AP2 domains of PoWRI1 are located between amino acids 56 and 125 and between amino acids 159 and 222.(3)The eukaryotic expression vector pCAMBIA11301-PoWRI1 containing the complete reading frame of PoWRI1 gene was constructed,and transformed into Arabidopsis thaliana mediated by Agrobacterium.The expression level of PoWRI1 in transgenic Arabidopsis thaliana lines was analyzed by qRT-PCR,and the results showed that the expression level of PoWRI1 in different transgenic lines was different,but compared with the wild type,the expression level of PoWRI1 was higher;The phenotype of transgenic plants was observed,and it was found that transgenic Arabidopsis seeds were larger in morphology,length,width and dry weight than wild-type plants.(4)The expression level of WRI1 downstream target gene in transgenic Arabidopsis thaliana was detected by qRT-PCR.Compared with wild-type Arabidopsis thaliana,the expression levels of WRI1 downstream target genes of oil synthesis in transgenic plants were significantly increased.Fatty acid determination results showed that the total fatty acid content of Arabidopsis thaliana seeds with overexpression of PoWRI1 was more than doubled compared with that of wild-type seeds,while the contents of long-chain fatty acids and unsaturated fatty acids were significantly increased.The proportion of unsaturated fatty acids in seeds of transgenic plants was increased compared with that of wild-type seeds.The study above could lay an important foundation for the further in-depth identification of PoWRI1 gene function and for its application in genetic improvement of Paeonia ostii seed oil content. |
PDF Full Text Request