Explore the gene network regulating the composition of fatty acids in cottonseed

Abstract

Background: Cottonseed is one of the major sources of vegetable oil. Analysis of the dynamic changes of fatty acid components and the genes regulating the composition of fatty acids of cottonseed oil is of great significance for understanding the biological processes underlying biosynthesis of fatty acids and for genetic improving the oil nutritional qualities.

Results: In this study, we investigated the dynamic relationship of 13 fatty acid components at 12 developmental time points of cottonseed (Gossypium hirsutum L.) and generated cottonseed transcriptome of the 12 time points. At 5-15 day post anthesis (DPA), the contents of polyunsaturated linolenic acid (C18:3n-3) and saturated stearic acid (C18:0) were higher, while linoleic acid (C18:2n-6) was mainly synthesized after 15 DPA. Using 5 DPA as a reference, 15,647 non-redundant differentially expressed genes were identified in 10-60 DPA cottonseed. Co-expression gene network analysis identified six modules containing 3275 genes significantly associated with middle-late seed developmental stages and enriched with genes related to the linoleic acid metabolic pathway and α-linolenic acid metabolism. Genes (Gh_D03G0588 and Gh_A02G1788) encoding stearoyl-ACP desaturase were identified as hub genes and significantly up-regulated at 25 DPA. They seemed to play a decisive role in determining the ratio of saturated fatty acids to unsaturated fatty acids. FAD2 genes (Gh_A13G1850 and Gh_D13G2238) were highly expressed at 25-50 DPA, eventually leading to the high content of C18:2n-6 in cottonseed. The content of C18:3n-3 was significantly decreased from 5 DPA (7.44%) to 25 DPA (0.11%) and correlated with the expression characteristics of Gh_A09G0848 and Gh_D09G0870.

Conclusions: These results contribute to our understanding on the relationship between the accumulation pattern of fatty acid components and the expression characteristics of key genes involved in fatty acid biosynthesis during the entire period of cottonseed development.

Keywords: Co-expression network; DEGs; Fatty acid components; Gossypium hirsutum; Transcriptomic analysis.

Fig. 1

Gene expression network of oil accumulation in developing cottonseed. The expression patterns of each differentially expressed unigene was shown on the relative log2 (FPKM value) from left to right is 0 ~ 60 DPA. ACC, acetyl-CoA carboxylase gene; FAS, Fatty Acid Synthase; KASII, β-ketoacyl ACP synthase II gene; SAD, stearoyl-ACP desaturase gene; FATA/B, fatty acyl-ACP thioesterase A/B gene; FAD2, fatty acid desaturase 2 gene; FAD3, fatty acid desaturase 3 gene; C16:0, palmitic acid; C18:0, stearic acid; C18:1n-9, oleic acid; C18:2n-6, linoleic acid; C18:3n-3, α-linolenic acid. This model was modified based on references of  Zhao et al. (2018) [22]

Fig. 2

Heatmap showing the relative expression level of the 12 selected genes at the 12 cottonseed developmental stages determined by RNA-seq analysis (a) and qRT-PCR (b). The selected genes include: ACC (Gh_A01G1574 and Gh_D05G2554), KASII (Gh_D13G2493and Gh_A08G2201), FATA (Gh_A08G1740), FATB (Gh_A06G0514), SAD (Gh_D02G1097 and Gh_D05G3162), FAD2 (Gh_D13G2238 and Gh_D11G3169), FAD3 (Gh_A09G0848 and Gh_A07G0946). S 5, S10, S15, S20, S25, S30, S35, S40, S45, S50, S55 and S60 represent 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 DPA, respectively

Fig. 3

Gene ontology classification of DEGs at various developmental stages of cottonseed. a. Venn diagram showing the number of enriched GO terms at the six cottonseed developmental stages. b. Analysis of GO enrichment at 10 DPA. c. Analysis of GO enrichment at 20, 25 and 30 DPA. d. Analysis of GO enrichment at 10, 15, 20, 25 and 30 DPA. S10, S15, S20, S25 and S30 represent 10, 15, 20, 25 and 30 DPA, respectively

Fig. 4

KEGG analysis of DEGs associated with cottonseed developmental at 20 and 25 DPA. a. KEGG categories of DEGs associated at 20 DPA. b. KEGGs categories of DEGs at 25 DPA. The vertical axes show logarithm (−log₂) of the corrected P value

Fig. 5

WGCNA of DEGs at different cottonseed developmental stages. a Hierarchical cluster tree showing co-expression modules identified by WGCNA. The major tree includes 10 modules according to calculation of eigengenes; each module is highlighted in a designated colour. b Module-sample relationships. Each row corresponds to a different module showing on the left side, each column represents a sample, and the correlation coefficient and evalue of each sample-module relationship are displayed. Red represents high expression, and blue represents low expression. S5, S10, S15, S20, S25, S30, S35, S40, S45, S50, S55 and S60 represent 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 DPA, respectively

Fig. 6

Co-expression network analysis. Red circle represented the hub genes. Co-expression network analysis of the brown module