Study On The Mechanism Of Cone Development And Oil Accumulation In Pinus Koraiensis

Pinus koraiensis is an evergreen tree of Pinaceae.It is a national second-class protected plant with ecological and economic importance.The oil content of Pinus koraiensis kernels can reach more than 60%,especially rich in unsaturated fatty acids such as oleic acid,linoleic acid and linolenic acid.It also contains various nutrients such as protein and vitamins,which can be used as nuts.In this study,Pinus koraiensis cones were used as materials.By collecting samples at different development stages of cones,the morphological changes during development were dynamically observed,and phenotypic traits and nutrient content were measured.By analyzing the variation of each index and the correlation between the indexes,the key period of cone development and nutrient accumulation were explored.At the same time,combined with a wide range of targeted metabolomics and transcriptomics studies,the dynamic changes of primary metabolites in different development stages of Pinus koraiensis cone were analyzed to explore the biosynthesis process of main metabolites in each stage of Pinus koraiensis kernel development,and to determine the interaction between related metabolites and genes.It provides a theoretical basis for further elucidating the molecular mechanism of Korean pine cone development,and provides a scientific basis for the commercialization and comprehensive utilization of Pinus koraiensis nuts.The main research results are as follows:（1）The whole growth cycle of Pinus koraiensis cones is 15 months.The fast-growing period of cone development was from mid-May to early July of the second year,and the fast-growing period of seed development was from June 20^th to early July of the second year.There was a rapid growth period after July 20^th of the second year.The shape of cones was long oval in the first year and became oval in the second year.The kernel is colorless and transparent at the early stage of development,and turns into light yellow inverted egg triangle at maturity.When the seeds of Pinus koraiensis were mature,the kernel moisture content was about 20.5%,the oil content 70.96%,the protein content 10.47%,and the total polysaccharide content 3.48mg/g.Correlation analysis showed that there was a significant positive correlation between the six traits of cone length,cone width,cone weight,seed length,seed width and 100-seed weight（P<0.01）.The oil content of kernel was significantly negatively correlated with water content（P<0.01）and significantly positively correlated with total polysaccharide content（P<0.01）.（2）Absolute growth and relative growth rate of Pinus koraiensis cone growth and development at different developmental stages were analyzed.It showed that the absolute growth of cone weight and cone volume reached the maximum value on June 10^th（7.92g,2.82mm³）,of which relative growth rate reached the maximum value on May 20^th（100.63%,77.41%）.The relative growth rate of 30-kernel weight and kernel volume reached the maximum value on July 1^stin the second year,of which the relative growth rate reached the maximum value（141.86%,50.99%）.The cone weight,cone volume,30-seed weight,seed volume,30-kernel weight and kernel volume of Pinus koraiensis were fitted with the development time,and the regression equations were as follows:y=104.794-55.023 X+6.235 X²-0.146 X³,y=44.364-22.291 X+2.651 X²-0.066 X³,y=0.366 X^1.805,y=0.109X^1.254 and y=0.0764 X^1.867.（3）The transcriptome data showed that there were 172,282 Unigenes obtained by assembly.The obtained Unigenes were annotated into the NR database,and 43.36%of the Unigenes were found to have similar sequences with North American spruce.The number of differentially expressed genes in the early stage of kernel development was the highest at27,356 compared with that in the late stage,including 13,764 down-regulated genes and 13,592up-regulated genes.Cluster analysis of differentially expressed genes showed that the gene expression patterns between the two groups of samples at 810 and 901 were similar.K-means clustering analysis was performed on the obtained DEGs,and DEGs were divided into two types,including 15,769 and 34,507 DEGs,respectively.（4）The functional annotation and enrichment results of differential genes in different comparison groups showed that differential genes were mainly enriched in plasma membrane anchoring components,β-glucan metabolism process,glucan biosynthesis process,tubulin binding,microtubule binding and oxidoreductase activity.The differential genes in each comparison group were mainly distributed in metabolic pathways,biosynthesis of secondary metabolites,plant hormone signal transduction,flavonoid biosynthesis,flavonoid and flavonol biosynthesis and circadian rhythm（plant）.The analysis of transcription factors involved in the development of Pinus koraiensis kernels showed that the number of AP2/ERF-ERF,MYB,NAC,b HLH and m TERF transcription factors was relatively large.（5）A total of 500 primary metabolites were identified from different development stages of Pinus koraiensis kernels.A total of 148 lipid compounds,28 phenolic acids,107 amino acids and their derivatives,70 organic acid metabolites,and 68 nucleotides and their derivatives were detected.Differential genes and differential metabolites were mainly enriched in hormone signal transduction,phenylpropanoid metabolism,pentose and glucuronic acid conversion,fatty acid synthesis,glycerophospholipid metabolism,α-linoleic acid metabolism,zeatin biosynthesis and unsaturated fatty acid biosynthesis.Through functional gene identification,three metabolites were annotated in the plant hormone signal transduction pathway before and after kernel development,including jasmonic acid,abscisic acid and jasmonic acid-L-isoleucine.The content of these metabolites was down-regulated.There were35 enzyme-coding genes related to plant hormone signal transduction,of which 6 genes were up-regulated,2 genes were down-regulated,and 27 genes were both up-regulated and down-regulated.（6）Based on the results of transcriptomics and metabolomics,a total of 286 differentially expressed genes related to oil were identified,of which 129 were related to fatty acid biosynthesis,149 were related to triacylglycerol assembly,and 8 were related to oil body formation.Among them,CC（BC）,KAS I,ACP and LACS were mainly expressed in 510-710.The expression levels of ACC（BCCP）,SAD and PHD genes were higher at the late stage of kernel development.Gene interaction network analysis identified 10 core genes:gene-Cluster-728.76612,gene-Cluster-728.44250,gene-Cluster-728.59549,gene-Cluster-728.79523,gene-Cluster-728.42748,gene-Cluster-728.92774,gene-Cluster-728.79239,gene-Cluster-728.54336,gene-Cluster-728.79978 and gene-Cluster-728.92739.These 10 core genes are mainly involved in the biosynthesis ofα-amylase,glycogen phosphorylase and glucose-1-adenosyl transferase.