Genome-wide mining seed-specific candidate genes from peanut for promoter cloning

Abstract

Peanut seeds are ideal bioreactors for the production of foreign recombinant proteins and/or nutrient metabolites. Seed-Specific Promoters (SSPs) are important molecular tools for bioreactor research. However, few SSPs have been characterized in peanut seeds. The mining of Seed-Specific Candidate Genes (SSCGs) is a prerequisite for promoter cloning. Here, we described an approach for the genome-wide mining of SSCGs via comparative gene expression between seed and nonseed tissues. Three hundred thirty-seven SSCGs were ultimately identified, and the top 108 SSCGs were characterized. Gene Ontology (GO) analysis revealed that some SSCGs were involved in seed development, allergens, seed storage and fatty acid metabolism. RY REPEAT and GCN4 motifs, which are commonly found in SSPs, were dispersed throughout most of the promoters of SSCGs. Expression pattern analysis revealed that all 108 SSCGs were expressed specifically or preferentially in the seed. These results indicated that the promoters of the 108 SSCGs may perform functions in a seed-specific and/or seed-preferential manner. Moreover, a novel SSP was cloned and characterized from a paralogous gene of SSCG29 from cultivated peanut. Together with the previously characterized SSP of the SSCG5 paralogous gene in cultivated peanut, these results implied that the method for SSCG identification in this study was feasible and accurate. The SSCGs identified in this work could be widely applied to SSP cloning by other researchers. Additionally, this study identified a low-cost, high-throughput approach for exploring tissue-specific genes in other crop species.

Fig 1. Characterization of the top 108 SSCGs from A. duranensis and A. ipaensis.

(A) Chromosomal distribution of the 108 SSCGs. The chromosome numbers are shown at the top of each chromosome (black bars). The names on the left of each chromosome correspond to the approximate location of each SSCG. (B) Numbers of SSCGs on each chromosome of A. duranensis and A. ipaensis. (C) Functional classification of the 108 SSCGs.

Fig 2. Expression profiles of the top 108 SSCGs in 20 different tissues of A. duranensis and A. ipaensis.

The FPKM data of 20 distinct tissues for the top 108 SSCGs were retrieved from the work of Clevenger et al. [31]. The FPKM value of each gene was log2-transformed and displayed in the form of heat maps by HemI. The color scale in the lower right represents the relative expression level: green represents a low level, and red indicates a high level. Twenty different tissues are shown on top of the heat map. The SSCGs are listed on the right of the heat map.

Fig 3. Semiquantitative RT-PCR analysis of the top 108 SSCGs in different tissues of cultivated peanut.

Orthologous genes were considered a single gene. Expression patterns were detected in the roots (Rt), stems (St), leaves (Lf), pegs (Pg), pod shells (Ps) and seeds (Sd) using the Actin gene as an internal control.

Fig 4. Functional characterization of the putative promoter AHSSP29 in transgenic Arabidopsis.

(A) Mature seed wrapped in a testa. (B-C) Germinating seed without a testa. (D) Young seedlings with two true leaves. (E) Adult plant. (F) Stem and flower of adult plants.(G-H) Siliques.