| Peanut(Arachis hypogaea L.)is an important oilseed and economic crop in China,rich in oil and protein,with high nutritional and economic value.At present,the molecular mechanisms of lipid metabolism during peanut development and germination are not yet clear,and the genetic basis of seed growth and development is weak,which restricts the development of high nutrient and high-value peanut varieties.Conducting research on peanut lipids and lipid droplets is beneficial for deciphering the mechanisms of lipid synthesis and degradation,improving the functions of peanut seed lipid droplet related proteins,and providing reference for peanut genetic breeding.This article comprehensively applied multidisciplinary methods such as peanut lipidomics,transcriptomics,proteomics,bioinformatics,and molecular biology experiments to analyze the rules and characteristics of lipid metabolism in ZP06 seeds of peanuts at different development and germination stages,and constructed a network map of peanut lipid synthesis and degradation processes.Simultaneously,this study optimized the extraction method of peanut seed lipid droplet related proteins and analyzed the impact of lipid droplet related proteins on peanut seed germination.The main results of this paper are as follows:1.Analysis of main quality traits of various peanut seedsSelect 20 different peanut materials for seed quality identification and analysis,and use qualitative and quantitative methods such as acid hydrolysis and gas chromatography to determine the main quality traits.The results showed that the oil content of peanut seeds from different materials ranged from 45% to 53%,and the protein content ranged from 20%to 28%,with a significant negative correlation between seed oil content and protein content;Peanuts mainly contain 8 types of fatty acids,among which oleic acid and linoleic acid have the highest content,accounting for about 80% of the total fatty acid content.There is a significant positive correlation between hundred kernel weight and individual plant weight,and a significant positive correlation between seed length,width,and hundred kernel weight.Nine materials belong to large fruit peanuts,among which ZP06 has a hundred kernel weight of 130.02 g and a protein content of 24.81%,belonging to super large fruit materials.Subsequently,ZP06 was selected for lipidome and transcriptome correlation analysis at different stages of seed development and germination.2.Lipidome analysis of peanut seeds at different developmental and germination stagesThe ultraperformance liquid chromatography uadrupole time-of-flight mass spectrometry technology was used to detect lipid metabolites of ZP06 seeds during 7 stages of development and germination.A total of 322 lipids were detected.Among them,the number of Triacylglycerol(TAG)was 95,accounting for 28.61%,and their content at the late stage of seed development accounted for 77.90%.It is the main nutrient storage substance for seeds and affects the content and composition of peanut seed lipid components.In addition,a potentially beneficial lipid HO-TAG was found to have a rapid increase in content during the early stages of germination,with a growth rate of approximately 16.79.C16:0,C18:1,C18:2,and C18:3 are the main branched fatty acid structural types of lipids.Free fatty acid(FFA)increased sharply on the 5th day of seed germination,with a total growth rate of313.73%.Among them the growth rate of C18:1 was 3628.04%,and C18:2 was 3478.01%.3.Transcriptome and joint analysis of peanut seeds at different development and germination stagesUsing RNA-seq technology,transcriptome sequencing was performed on 7 stages of ZP06 seed development and germination,and a total of 67124 gene data were obtained.857 genes were screened for significant expression differences compared to mature seeds.Among them,differentially expressed genes during the developmental stage are mainly involved in sphingolipid metabolism,inositol phosphate metabolism,and fatty acid degradation pathways.During the germination stage,they are mainly involved in glycerophospholipid metabolism,fatty acid degradation,and glyceride metabolism pathways.Weighted gene co expression network analysis(WGCNA)was performed on lipid and transcriptome data from different reproductive stages to construct a molecular network map of peanut lipid accumulation and degradation processes,and 10 candidate genes were screened.Among them,FAD2,FAD3,PLC,and DGAT affect the accumulation of lipid PC(18:1/18:2),DAG(18:2/18:2),and TAG(18:1/18:1/18:2)during seed development;GDSL,LOX2 S,PLD1,and TAGL coordinate the degradation of lipid TAG(18:1/18:2/18:2)and DAG(18:2/18:3)during peanut seed germination to form C18:1,C18:2,and C18:3.4.The optimization of peanut seed protein extraction methodDifferent protein extraction techniques were conducted on peanut seeds at different stages.The results were compared with each other through electrophoresis experiments.Then,SDS extraction method was selected and used to solve the impurity interference,and to determine the 7 key time points for peanut seed development and germination.The optimization of peanut seed lipid droplet related protein extraction methods mainly includes:(1)reducing sucrose concentration(20%)to obtain high-purity lipid droplets,(2)high-temperature heating(95 °C)to promote lipid droplet and lipid decomposition,and(3)increasing the ratio of chloroform to SDS extraction solution(1:1).As a result,the optimizated method could effectively remove the influence of oil and enrich lipid droplet related proteins.5.Analysis of lipid droplet related proteins and their functions during peanut seed germinationUsing TMT protein labeling technology to quantitatively analyze lipid droplet related proteins in peanut seeds during germination process,a total of 2050 lipid droplet related proteins were matched.Among them,1558 proteins belonged to 21 protein functions such as energy metabolism,and they had interactions with each other.Analysis of differential protein abundance at different germination stages revealed that the enriched differential abundance proteins in seeds with germination disorders mainly existed in the transcriptional translation during the early stages of seed germination,and the proteins were not processed,which hindered the lipid metabolism pathway.E3(2KIY95)as a hub protein in the sub network had interactions with 10 lipid droplet related proteins.A total of 7 types of lipid droplet related proteins were identified,and rubber elongation factor(BZTS91)showed the maximum difference multiple of 5.72 and 8.24 compared to other stages.Protein family and molecular verification showed that the interaction between BZTS91 and 2KIY95 may affect peanut lipid droplet degradation.The cultivation of high-value and nutritious large fruit varieties is one of the main directions in peanut breeding.The above results analyzed the main quality traits of peanut seeds,analyzed the biological basis of lipid metabolism during peanut seed development and germination,studied the functions of lipid droplet related proteins during peanut seed germination,and provided a reference for molecular genetic breeding to cultivate new peanut varieties. |
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