Elsevier

Plant Science

Volume 175, Issue 5, November 2008, Pages 663-673
Plant Science

Identification of differentially expressed genes in soybean seeds differing in oil content

https://doi.org/10.1016/j.plantsci.2008.06.018Get rights and content

Abstract

Characterization of gene regulation is fundamental for achieving an understanding of the complex processes for oil synthesis. Up to now, it is still unknown how many genes are involved in the oil synthesis in soybean (Glycine max L.) seeds. In this study, we examined the gene expression of different seed maturity using two contrasting cultivars (Zhongdou 32, a high-oil cultivar and Youchun 02–6, a low-oil cultivar). Results indicated that seed oil content varied with maturing stages. Two significant increases of oil content were observed at 22 and 43 days after flower (DAF) in Zhongdou 32. To explore their molecular basis, differentially expressed gene clones in two soybean cultivars and two development stages were analyzed by SSH. All SSH clones were arrayed and screened by dot-blotting hybridization, followed by semi-quantitative PCR analysis for selected clones, and finally determined by real-time quantitative RT-PCR. A total of 59 genes have been examined successfully in two cultivars. At 36 DAF, 33 genes were found with high expression in Zhongdou 32, while the other 26 genes in Youchun 02–6. In Zhongdou 32, a total of 33 genes showed differential expression at 15 and 36 DAF. 13 genes showed a high expression at 15 DAF, while the other 20 genes at 36 DAF. A homology search for their nucleotide sequences revealed that the above genes are highly homologous to known sequences of genes for oil biosynthesis, energy metabolism, signal transduction, and stress response, and some genes have not been implicated previously in oil synthesis. Tissue-specific analysis indicated that most of genes expressed in seed, and 10 genes expressed specifically in seeds. Time-course analysis of transcript accumulation indicated that the expression of seed-specific genes changed with development stages, which were probably associated with oil biosynthesis. The results provide information on gene-encoding factors responsible for oil synthesis. Functional studies of the seed-specific genes would shed new light on both the molecular basis of oil metabolism and the regulatory pathways.

Introduction

Soybean is one of the most important leguminous seed crops among the oil crops, which plays a major role in supplying protein and amino acids for human and animal nutrition. The stored components including carbohydrate, oil and protein contribute to 90% or more of the dry seed weight of soybean. A recent survey of oil content in the seed of ∼360 known Arabidopsis ecotypes revealed a range from 34 to 46% of seed dry weight. According to Zhou et al. [1], oil accumulation in dry seed of soybean might range from 12 to 26%. In vitro and in vivo studies showed that seed oil biosyntheses were catalyzed by a set of enzymes located in plastids [2], [3]. Jaworski et al. [4] overexpressed a ketoacyl-acyl-carrier protein synthase in plants and reduced the rate of lipid synthesis [5]. But Roesler et al. [6] overexpressed an Arabidopsis cytosolic ACCase gene in rape (Brassica napus), and found that the oil content was not increased significantly in despite of a 10- to 20-fold increase of enzyme activity. Van et al. [7] enriched cDNA clones associated with storage lipid synthesis, and found that an oil-desaturase gene was highly expressed in developing seeds than in leaves [8]. Then they sequenced the cDNAs derived from developing castor bean seeds, and identified a set of the enzymes involved in ricinoleic acid biosynthesis. In a similar manner, Cahoon et al. [9] identified some genes essential for the biosynthesis of conjugated oil in Momordica charantia and Impatiens balsamina. Wang et al. [10] found GmDof4 and GmDof11 genes increased the content of total fatty acids and lipids by activating the acetyl CoA carboxylase gene and long-chain-acyl CoA synthetase gene in soybean seeds. Taken together, the above findings indicate that oil biosyntheses were conferred by multi-biosynthesis mechanisms or pathways. Although the pathways for lipid biosynthesis have been identified, the factors that regulate the biosynthetic pathways at the transcript level are largely unknown. It is still unclear how many genes in soybean seeds are involved in oil synthesis and how the products of these genes interact with each other in terms of lipid biosynthesis. To isolate the genes governing oil biosynthesis, we monitored the genes that were differentially expressed in seeds of soybean cultivars differing in oil content at different growth stages using SSH and real-time RT-PCR. Using these approaches, we identified 59 genes with differential expression in the seeds of Zhongdou 32 and Youchun 02–6 at 36 days after flower (DAF), and 33 genes with differential expression in Zhongdou 32 at 15 and 36 DAF, respectively. 10 seed-specific genes were also isolated. These genes may be suitable for genetic improvement of seed oil accumulation and may also provide molecular markers for studies of soybean breeding.

Section snippets

Material cultivation

Soybean cultivars (Zhongdou 32, a high-oil cultivar and Youchun 02–6, a low-oil cultivar) were selected as experimental materials in this study. Soybean seedlings of the same maturity were cultivated by Dr. Xin-An Zhou (Institute of Oil Crops, Chinese Academy of Agriculture Sciences, Wuhan, China). Soybean plants were grown in soils under standard greenhouse conditions. Immature seeds were sampled at different DAF for subsequent mRNA extraction and construction of cultivar- or stage-specific

Accumulation of oil and fatty acids

To explore variation of storage product accumulation, we examined the contents of protein, oil and fatty acids in soybean seeds of two cultivars as shown in Fig. 1. In Zhongdou 32 protein concentration of declined obviously from 15 to 22 DAF, and then remained stable from 22 to 43 DAF. While in Youchun 02–6 protein increased smoothly from 22 to 43 DAF. On 43 DAF, Zhongdou 32 had a lower protein concentration in comparison to Youchun 02–6 (Fig. 1A). Oil accumulation was different from that of

Fatty acid and oil synthesis

In plants, fatty acid and oil synthesis requires the spatial and temporal activity of many gene products. Many efforts have been made to characterize the biosynthesis of oil [13], [14], [15]. More than 200 genes encoding oleosins have been identified. In this study, we cloned three genes related to oleosins, which were p24 oleosin isoform A (C443), B (A445) and oleosin 3 (D448). In soybean p24 oleosin isoform A and B only expressed in seeds, while oleosin 3 expressed in all tissues of soybean

Acknowledgements

This work was supported partially by the National Natural Science Foundation of China (30671312; 30771294), the Presidential Foundation (200602) and the Opening Scientific Research Foundation (200705) of the Institute of Oil Crops, Chinese Academy of Agricultural Sciences.

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