Yanan He

Identification and Characterization of microRNAs from Peanut (Arachis hypogaea L.) by High-Throughput Sequencing

Background MicroRNAs (miRNAs) are noncoding RNAs of approximately 21 nt that regulate gene expression in plants post-transcriptionally by endonucleolytic cleavage or translational inhibition. miRNAs play essential roles in numerous developmental and physiological processes and many of them are conserved across species. Extensive studies of miRNAs have been done in a few model plants; however, less is known about the diversity of these regulatory RNAs in peanut (Arachis hypogaea L.), one of the most important oilseed crops cultivated worldwide. Results A library of small RNA from peanut was constructed for deep sequencing. In addition to 126 known miRNAs from 33 families, 25 novel peanut miRNAs were identified. The miRNA* sequences of four novel miRNAs were discovered, providing additional evidence for the existence of miRNAs. Twenty of the novel miRNAs were considered to be species-specific because no homolog has been found for other plant species. qRT-PCR was used to analyze the expression of seven miRNAs in different tissues and in seed at different developmental stages and some showed tissue- and/or growth stage-specific expression. Furthermore, potential targets of these putative miRNAs were predicted on the basis of the sequence homology search. Conclusions We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library. This study of the identification and characterization of miRNAs in peanut can initiate further study on peanut miRNA regulation mechanisms, and help toward a greater understanding of the important roles of miRNAs in peanut.

Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR

Quantitative real-time reverse transcription PCR (qRT-PCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on the reference genes have been done with peanut to date. In the present study, 14 potential reference genes in peanut were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms. Expression stability was assessed by qRT-PCR across 32 biological samples, including various tissue types, seed developmental stages, salt and cold treatments. The results showed that the best-ranked references genes differed across the samples. UKN1, UKN2, TUA5 and ACT11 were the most stable across all the tested samples. A combination of ACT11, TUA5, UKN2, PEPKR1 and TIP41 would be appropriate as a reference panel for normalizing gene expression data across the various tissues tested, whereas the combination of TUA5 and UKN1 was the most suitable for seed developmental stages. TUA5 and EF1b exhibited the most stable expression under cold treatment. For salt-treated leaves, TUA5 and UKN2 were the most stably expressed and HDC and UKN1 for salt-treated roots. The relative gene expression level of peanut Cys2/His2-type zinc finger protein gene AhZFP1 was analyzed in order to validate the reference genes selected for this study. These results provide guidelines for the selection of reference genes under different experimental conditions and also a foundation for more accurate and widespread use of qRT-PCR in peanut gene analysis.

Genome-Wide Analysis of Fatty Acid Desaturases in Soybean (Glycine max)

Fatty acid desaturases can introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In the present study, 29 full-length desaturase genes were identified from soybean genome by a thorough annotation exercise. A comprehensive analysis was performed to characterize phylogeny, chromosomal locations, structures, conserved motifs, and expression patterns of those genes. The soybean genes were phylogenetically clustered into nine subfamilies with the Arabidopsis counterparts, FAB2, FAD2, FAD3, FAD5, FAD6, FAD7, FAD8, SLD1, and DES1. Twenty-nine desaturase genes were found to be distributed on at least 15 of the 20 soybean chromosomes. The gene structures and motif compositions were considerably conserved among the subfamilies. The majority of desaturase genes showed specific temporal and spatial expression patterns across different tissues and developmental stages based on microarray data analyses. The study may provide new insights into the origin and evolution of fatty acid biosynthesis pathways in higher plants. Additionally, the characterization of desaturases from soybean will lead to the identification of additional genes for genetic modification of plants to produce nutritionally important fatty acids.

Cloning of Six ERF Family Transcription Factor Genes from Peanut and Analysis of their Expression during Abiotic Stress

Several ethylene-responsive element binding factor (ERF) family proteins have been demonstrated to play important roles in transcriptional regulation of a variety of biological processes, including responses to environmental conditions such as drought, salt, and cold. To date, there have been no reports on this family in peanut. In this study, 40 ESTs in a peanut cDNA library, whose amino acid sequences contained AP2/ ERF domain, have been identified. Of these, full-length sequences of six genes were cloned. Sequence analysis indicated that all six proteins contained only one AP2/ ERF domain and should belong to the ERF family. The six genes were designated Arachis hypogaea ERF 1–6 (AhERF1–6). The expression patterns of AhERF1–6 were analyzed under cold, salt and drought stress. The results indicated that the expression of AhERF4 and AhERF6 were rapidly and substantially enhanced under abiotic stress. The expression of AhERF1 and AhEERF5 were slightly enhanced under certain stress conditions. Some genes were down-regulated when under stress, such as AhEERF3 in leaves under salt stress and AhERF2 in leaves under drought stress. Interestingly, the expression of AhERF3 and AhERF5 exhibited contrary expression patterns in peanut leaves and roots upon PEG treatment. These results suggested that different ERF proteins may have different functions in peanut abiotic stress acclimation.

AhZEP1, a cDNA Encoding C2H2-Type Zinc Finger Protein, Induced by Salt Stress in Peanut (Arachis hypogaea L.)

The plant C2H2-type zinc finger proteins play important roles in plant tolerance to abiotic stresses. In this study, we isolated the full-length cDNA of a C2H2-type zinc finger protein gene AhZFP1 from peanut (Arachis hypogaea L.). AhZFP1 encodes a 24.75kDa protein with two C2H2-type zinc finger domains. Phylogenetic analysis showed that the AhZFP1 was close to Arabidopsis zinc finger protein AZF2. The semi-quantitative RT-PCR assay revealed that the expression of AhZFP1 was induced by salt stress in peanut roots, stems and leaves. AhZFP1 was the first C2H2-type zinc finger protein gene reported in peanut.