| Cyperus esculentusis,also known as tiger nut,is native to the Mediterranean coast of Africa.It has the advantages of wide adaptability,strong stress resistance,and high yield.Its oil production per unit area is 4 times that of soybeans,and it is known as a new oil crop.The content of unsaturated fatty acids in Sesame oil is as high as over 80%,which is rich in nutritional value.The study of the biosynthetic pathway of oil in the tubers of Cyperus esculentusis on the one hand lays the foundation for the study of the mechanism of oil synthesis in the tubers of plants,and on the other hand,it provides candidate genes for the use of genetic engineering methods to improve the germplasm of Cyperus esculentusis and increase the amount of oil in vegetative organs.However,the previous literature revealed that the researches related to Cyperus esculentusis mainly focused on introduction,cultivation,and tuber oil composition analysis,and molecular biology research was relatively scarce.In this study,we integrated the developmental morphology of Cyperus esculentusis tuber,the detection of oil content and other indicators,and took 5 different developmental stages(35DAS(35 days after sowing),50 DAS,70 DAS,90 DAS and 120 DAS)tubers as the research object and carried out as follows Research content: First,transcriptome sequencing of tuber tissues at different developmental stages was carried out using RNA-seq technology to screen fatty acid synthesis-related genes;liquid chromatography-mass spectrometry(LC-MS)was used to detect fatty acid types in tubers at different stages And lipid difference molecules.Second,the Pearson correlation analysis was carried out by combining the differential gene expression and the content of differential lipid molecules,and the phosphatidic acid phosphatase(CePAP2)gene(Pearson r = 0.9637)related to the synthesis of diacylglycerol(DAG)was screened out and performed Gene function identification.The main results of this study are as follows:1.After filtering and assembling the transcriptome sequencing data,a total of 50260 Unigenes were obtained.Perform GO database comparison,get 15034 Unigenes,which are annotated to biological processes,molecular functions and cells constitute 3 large subclasses,53 small subclasses;perform KOG database comparison,get 11071 Unigenes and be annotated to 26 in functional classification;KEGG metabolic pathway database comparison was performed,and 10842 Unigenes were obtained and annotated into 19 metabolic pathways.2.Randomly select 12 differentially expressed genes related to oil synthesis for q RT-PCR verification,and the results are consistent with the trend of the transcriptome data results.3.Carry out oil extraction from tubers at different developmental stages,and use LC-MS to carry out lipidomics analysis.A total of 28 lipids and 167 differential lipid molecules were identified.Pearson’s correlation analysis was carried out based on the differential gene expression level and the relative content of differential lipid molecules in the transcriptome data,and the phosphatidic acid phosphatase(CePAP2)gene related to the synthesis of diacylglycerol(DAG)was screened.4.Successfully cloned the CePAP2 gene with a full length of 1014 bp.Construct the p ESC-URA-FLAG-CePAP2 recombinant expression vector,and verify the function of CePAP2 gene in Saccharomyces cerevisiae INVSc1.The results of SDS-PAGE and Western blot showed that the expression of CePAP2 was the highest after 48 hours of induction by galactose;microscopic observation of intracellular oil droplets staining showed that the number of oil droplets in yeast cells increased significantly under this condition;and the detection of DAG and total oil content found that: compared with the control,the content of DAG and total oil in yeast cells increased by 21.86% and 43.75%,respectively.5.Construct plant expression vector p CAMBIA3301-CePAP2,successfully transform Arabidopsis thaliana and obtain 6 transgenic plants of generation.The detection of seed oil content found that compared with the wild type,the oil content of the transgenic lines increased by 9.31%,8.48%,9.33%,9.81%,9.05% and 7.95%,respectively. |
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