Qingsong Yang

Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear

AbstractRT-qPCR is a widely used method in gene expression and transcriptome studies. Normalization based on reference genes is necessary to accurately analyze RT-qPCR data. Thus, an accurate and systematic evaluation of these reference genes before experiments are conducted is necessary. In this study, 18 candidate reference genes were evaluated under various experimental conditions covering a range of tissue types and cultivars, NaCl, CaCl2 and temperature treatments, hormones (6-BA, ABA and NAA) and a set of osmotic stress (mannitol and PEG6000) treatments. Gene expression across 48 pear samples was evaluated using geNorm, NormFinder and BestKeeper statistical algorithms. Actin2/7 (ACT2/7), ubiquitin extension protein (UBI) and Yellow-leaf-specific gene 8 (YLS8) exhibited the most stable expression across all the pear samples tested. While in the other experimental groups, different sets of samples had their own best reference genes. In addition, the gene expression of PbCBL7, a member of the calcineurin B-like protein, was measured across all the 48 samples using the best three reference genes, it displayed variation in gene expression across different tissues and cultivars, and exhibited diverse up- or down-regulated expression patterns under various treatments, which indicate that PbCBL7 may play a role in response to specific abiotic stress in pear. These results are valuable for future research on gene expression and abiotic stress tolerance in pear.

Transcriptome sequencing and analysis of major genes involved in calcium signaling pathways in pear plants ( Pyrus calleryana Decne.)

BackgroundPears (Pyrus spp. L.) are an important genus of trees that produce one of the world’s oldest fruit crops. Salinity stress is a common limiting factor for plant productivity that significantly affects the flavor and nutritional quality of pear fruits. Much research has shown that calcium signaling pathways, mediated by Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs), are closely associated with responses to stresses, including salt. However, little is known about the molecular mechanisms that govern the relationship between salt stress and calcium signaling pathways in pear plants. The available genomic information for pears has promoted much functional genomic analysis and molecular breeding of the genus. This provided an ample foundation for characterizing the transcriptome of pear under salt stress.ResultsA high-throughput Illumina RNA-seq technology was used to identify a total of 78,695 unigenes that were successfully annotated by BLASTX analysis, using the publicly available protein database. Additionally, 2,855 novel transcripts, 218,167 SNPs, 23,248 indels and 18,322 alternative splicing events occurred. Assembled unique sequences were annotated and classified with Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, which revealed that the main activated genes in pear are predominately involved in functions such as basic physiological processes, metabolic pathways, operation of cellular components, signal transduction mechanisms, and other molecular activities. Through targeted searches of the annotations, the majority of the genes involved in calcium signaling pathways were identified, among which, four genes were validated by molecular cloning, while 11 were validated by RT-qPCR expression profiles under salt stress treatment.ConclusionsThese results facilitate a better understanding of the molecular genetics and functional genomic mechanisms of salt stress in pear plants. Furthermore, they provide a valuable foundation for additional research on the molecular biology and functional genomics of pear and related species.

Gene characterization and transcription analysis of two new ammonium transporters in pear rootstock ( Pyrus betulaefolia )

Abstract Ammonium is the primarily nitrogen source for plant growth, but the molecular basis of ammonium acquisition in fruit species remains poorly understood. In this study, we report on the characterization of two new ammonium transporters (AMT) in the perennial tree Pyrus betulaefolia. In silico analyses and yeast complementation assays revealed that both PbAMT1;3 and PbAMT1;5 can be classified in the AMT1 sub-family. The specific expression of PbAMT1;3 in roots and of PbAMT1;5 in leaves indicates that they have diverse functions in ammonium uptake or transport in P. betulaefolia. Their expression was strongly influenced by ammonium availability. In addition, the transcript level of PbAMT1;5 was significantly affected by the diurnal cycle and senescence hormones. They conferred the ability to uptake nitrogen to the yeast strain 31019b; however, the 15NH4+ uptake kinetics of PbAMT1;3 were different from those of PbAMT1;5. Indeed, PbAMT1;3 had a higher affinity for 15NH4+, and pH changes were associated with this substrates’ transport in yeast. The present study provides basic gene features and transcriptional information for the two new members of the AMT1 sub-family in P. betulaefolia and will aid in decoding the precise roles of AMTs in P. betulaefolia physiology.

Characterization of CIPK Family in Asian Pear (Pyrus bretschneideri Rehd) and Co-expression Analysis Related to Salt and Osmotic Stress Responses

Asian pear (Pyrus bretschneideri) is one of the most important fruit crops in the world, and its growth and productivity are frequently affected by abiotic stresses. Calcineurin B-like interacting protein kinases (CIPKs) as caladium-sensor protein kinases interact with Ca2+-binding CBLs to extensively mediate abiotic stress responses in plants. Although the pear genome sequence has been released, little information is available about the CIPK genes in pear, especially in response to salt and osmotic stresses. In this study, we systematically identified 28 CIPK family members from the sequenced pear genome and analyzed their organization, phylogeny, gene structure, protein motif, and synteny duplication divergences. Most duplicated PbCIPKs underwent purifying selection, and their evolutionary divergences accompanied with the pear whole genome duplication. We also investigated stress -responsive expression patterns and co-expression networks of CIPK family under salt and osmotic stresses, and the distribution of stress-related cis-regulatory elements in promoter regions. Our results suggest that most PbCIPKs could play important roles in the abiotic stress responses. Some PbCIPKs, such as PbCIPK22, -19, -18, -15, -8, and -6 can serve as core regulators in response to salt and osmotic stresses based on co-expression networks of PbCIPKs. Some sets of genes that were involved in response to salt did not overlap with those in response to osmotic responses, suggesting the sub-functionalization of CIPK genes in stress responses. This study revealed some candidate genes that play roles in early responses to salt and osmotic stress for further characterization of abiotic stress responses medicated by CIPKs in pear.

Comprehensive analysis of differentially expressed genes under salt stress in pear (Pyrus betulaefolia) using RNA-Seq

Pear is one of the most important fruit trees in temperate zones, and is cultivated widely throughout the world. Salt stress affects the normal growth of pear, and further affects fruit yield and quality. Pyrus betulaefolia is a common rootstock in pear orchards, which can improve salt tolerance by grafting pear onto it. However, limited availability of P. betulaefolia genomic information has hindered research on the mechanisms underlying this tolerance. Consequently, we comprehensively analyzed P. betulaefolia salt tolerance using RNA-Seq under NaCl and NaCl + LaCl3 treatments in leaf and root. Based on mapping analyses, 3796 novel transcripts were identified, which contained 18 differentially expressed genes (DEGs). There were 90,752 alternative splicing events identified, with transcription start site and transcription terminal site as the major splicing patterns. In addition, we identified 583 differential expressed exons. A total of 276 DEGs were identified among all six comparisons, and 237 of these were up-regulated and 39 were down-regulated. One DEG (Pbr038831.1) was detected in all treatments, and was up-regulated. All DEGs were divided into three clusters according to hierarchical clustering. Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that 18 DEGs were located in six significantly enriched terms, and specific enriched categories and DEGs were identified for NaCl and NaCl + LaCl3 treatments. All of these enriched genes may be related to salt stress in P. betulaefolia. This transcriptome analysis will provide a rich genetic resource for gene discovery related to salt tolerance in P. betulaefolia and closely related species. The data will serve as an important public information platform to further understanding of the molecular mechanisms involved in salt tolerance in P. betulaefolia.

A γ-Glutamylcysteine Synthetase Gene from Pyrus calleryana Is Responsive to Ions and Osmotic Stresses

Pyrus calleryana Decne. is widely used as a pear rootstocks in Asia. Glutathione (GSH) can induce the phytochelatin synthase gene’s (PcPCS1) transcription in this plant when exposed to cadmium. This phenomenon indicated that GSH plays a vital role in protecting P. calleryana from cadmium stress. Here, we have isolated and characterized a gene encoding γ-glutamylcysteine synthetase (PcγECS), the key enzyme of GSH biosynthesis, from the leaves of P. calleryana, with the aim of expanding our knowledge of the role of GSH at the molecular level. P. calleryana plants treated with cadmium, salt, PEG6000, or mannitol responded by increasing transcription of the PcγECS gene. The response was specific for cadmium, which causes a toxicity that is thought to be mitigated through phtochelatins, and other treatments that cause damage that can be relieved by the synthesis of GSH. Feeding experiments suggested that cysteine contributed to activating the transcription of PcγECS and GSH synthesis. However, the gene’s expression was inhibited in the presence of GSH. Recombinant Escherichia coli carrying PcγECS had higher GSH synthesis levels and grew better than the control cells under cadmium, salt, or osmotic stresses. These results suggest that PcγECS may participate in responses against multiple environmental stimuli in P. calleryana.

Identification and characterization of circRNAs in Pyrus betulifolia Bunge under drought stress

Circular RNAs (circRNAs) play important roles in miRNA function and transcriptional control. However, little is known regarding circRNAs in the pear. In this study, we identified circRNAs using deep sequencing and analyzed their expression under drought stress. We identified 899 circRNAs in total, among which 33 (23 upregulated, 10 downregulated) were shown to be dehydration-responsive. We performed GO and KEGG enrichment analysis to predict the functions of differentially expressed circRNAs. 309 circRNAs were predicted to act as sponges for 180 miRNAs. A circRNA-miRNA co-expression network was constructed based on correlation analysis between the differentially expressed circRNAs and their miRNA binding sites. Our study will provide a rich genetic resource for the discovery of genes related to drought stress, and can readily be applied to other fruit trees.

Genome-Wide Identification and Functional Prediction of Novel Drought-Responsive lncRNAs in Pyrus betulifolia

Increasing evidence shows that long noncoding RNAs (lncRNAs) play important roles in developmental regulation and many other biological processes in plants. However, identification of lncRNAs in Pyrus betulifolia is limited compared with studies of functional gene expression. Using high-throughput sequencing technology, the transcriptome of P. betulifolia under drought stress was analyzed to identify lncRNAs. A total of 14,478 lncRNAs were identified, of which 251 were found to be drought-responsive. The putative target genes of these differentially expressed lncRNAs were significantly enriched in metabolic processes, organic substance metabolic processes, macromolecule metabolic processes, and heterocyclic compound binding. Real-time quantitative polymerase chain reaction validation suggested that the results of the RNA sequencing data analysis were reliable. This study will provide genetic resources for pear breeding and provide reference to other pomological studies.

The AMT1 family genes from Malus robusta display differential transcription features and ammonium transport abilities

Ammonium is an important nitrogen sources for plant growth. In this study, we report on the gene characterization of the ammonium transporter AMT1 subfamily in the apple rootstock Malus robusta Rehd. Thirteen AMT genes were comprehensively evaluated from the apple genome (version 1.0). Then the gene features and expression patterns of five AMT1 members from M. robusta were analyzed. These genes fell into four clusters in the AMT phylogenetic tree: clade I (MrAMT1;1 and MrAMT1;3), clade II (MrAMT1;4), clade III (MrAMT1;2), and clade IV (MrAMT1;5). All the AMT1s, apart from MrAMT1;4, were expressed in vegetative organs and strongly responded to nitrogen concentration changes. For example, MrAMT1;2 and MrAMT1;3 had high transcript accumulation levels in the leaves and roots, respectively. Finally, the functions of these AMT1s were studied in detail by heterologous expression in yeast. These genes allowed strain 31019b to assimilate nitrogen, but their 15NH4+ uptake kinetics varied. These results revealed the functional roles of AMT1 during ammonium absorption in the AMT-defective mutant yeast system.

Characterization and Expression Profiling Analysis of Calmodulin Genes in Response to Salt and Osmotic Stresses in Pear (Pyrus bretschneideri Rehd.) and in Comparison with Arabidopsis

A genome-wide identification and cloning of CaM genes in pear was conducted and in compared with Arabidopsis that indicated a conserved expansion of CaM genes in pear, and PbCaMs and AtCaMs had a similar distribution of cis-elements and expressions in response to salt and osmotic stress. In particular, PbCaM1 and PbCaM3 were both significantly upregulated in response to salt and osmotic stress in pear.