Identification and analysis of the germin-like gene family in soybean

Abstract

Background: Germin and germin-like proteins constitute a ubiquitous family of plant proteins. A role of some family members in defense against pathogen attack had been proposed based on gene regulation studies and transgenic approaches. Soybean (G. max L. Merr.) germin genes had not been characterized at the molecular and functional levels.

Results: In the present study, twenty-one germin gene members in soybean cultivar 'Maple Arrow' (partial resistance to Sclerotinia stem rot of soybean) were identified by in silico identification and RACE method (GmGER 1 to GmGER 21). A genome-wide analyses of these germin-like protein genes using a bioinformatics approach showed that the genes located on chromosomes 8, 1, 15, 20, 16, 19, 7, 3 and 10, on which more disease-resistant genes were located on. Sequence comparison revealed that the genes encoded three germin-like domains. The phylogenetic relationships and functional diversity of the germin gene family of soybean were analyzed among diverse genera. The expression of the GmGER genes treated with exogenous IAA suggested that GmGER genes might be regulated by auxin. Transgenic tobacco that expressed the GmGER 15 [corrected] gene exhibited high tolerance to the salt stress. In addition, the GmGER mRNA increased transiently at darkness and peaked at a time that corresponded approximately to the critical night length. The mRNA did not accumulate significantly under the constant light condition, and did not change greatly under the SD and LD treatments.

Conclusions: This study provides a complex overview of the GmGER genes in soybean. Phylogenetic analysis suggested that the germin and germin-like genes of the plant species that had been founded might be evolved by independent gene duplication events. The experiment indicated that germin genes exhibited diverse expression patterns during soybean development. The different time courses of the mRNAs accumulation of GmGER genes in soybean leaves appeared to have a regular photoperiodic reaction in darkness. Also the GmGER genes were proved to response to abiotic stress (such as auxin and salt), suggesting that these paralogous genes were likely involved in complex biological processes in soybean.

Figure 1

Unrooted phylogenetic tree was constructed using the coding sequences of the GmGER genes and those of different plant species. Bootstrap values were placed at the nodes and the scale bar corresponded to 0.1 estimated nucleic acid substitutions per site. Five major classes (I to V) were shown.

Figure 2

The alignment of the germin domain in soybean germin-like genes and schematic diagram of soybean GmGER genes. (A) The alignment of soybean germin-like protein domain was performed using the ClustalW program. (B) The rectangular boxes indicated the domains and their localization in each protein sequence.

Figure 3

Genomic arrangement and orientation of soybean germin-like protein genes on linkage groups (chromosomes) A2 (8), D1a (1), E (15), I (20), J (16), L (19), M (7), N (3) and O (10). Arrows indicated the transcription orientation of the genes. The numbers represented the exact positions of each gene. The QTL name and position referred to the Soybean Breeders Toolbox. The lines indicated the discrepancies of marker alignments between the physical map and genetic map.

Figure 4

Unrooted Bayesian tree of soybean GmGER genes. Bootstrap values were placed at the nodes and the scale bar corresponded to 0.2 estimated nucleic acid substitutions per site. Three major classes (I, II and III) were shown.

Figure 5

Expression analysis of the GmGER gene by quantitative real-time RT-PCR. The analyse is in response to IAA (100 u M) treatment at 0, 4, 8, 12, 16, 20, 24, 48 hours.

Figure 6

Expression level of GmGER mRNA in four photoperiodic treatments. Continuous darkness (up to 48 h), continuous light (up to 48 h), SD (short-day, 8 h light and 16 h dark), LD (long-day, 16 h light and 8 h dark).

Figure 7

Seedlings of transgenic tobacco with GmGER gene and WT tobacco under different concentration of NaCl stress. A: normal growth condition; B: 150 mM NaCl stress; C: 250 mM NaCl stress; D: 350 mM NaCl stress; E: comparison of average stem length between transgenic and WT plants; F: comparison of average fresh weight between transgenic and WT plants. The first lines in the A, B, C, D were WT tobacco; the second lines in the A, B, C, D were the tobacco plants transformed with GmGER gene.