Dataset Information

Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.

ABSTRACT: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1?) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions.

SUBMITTER: Bu J

PROVIDER: S-EPMC4846040 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.

Bu Jiaodi J Zhao Jin J Liu Mengjun M

PloS one 20160426 4

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFin ...[more]

PMID: 27116123

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Project description:BackgroundAmong several TF families unique to eukaryotes, the basic leucine zipper (bZIP) family is one of the most important. Chinese jujube (Ziziphus jujuba Mill.) is a popular fruit tree species in Asia, and its fruits are rich in sugar, vitamin C and so on. Analysis of the bZIP gene family of jujube has not yet been reported. In this study, ZjbZIPs were identified firstly, their expression patterns were further studied in different tissues and in response to various abiotic and phytoplasma stresses, and their protein-protein interactions were also analyzed.ResultsAt the whole genome level, 45 ZjbZIPs were identified and classified into 14 classes. The members of each class of bZIP subfamily contain a specific conserved domain in addition to the core bZIP conserved domain, which may be related to its biological function. Relative Synonymous Codon Usage (RSCU) analysis displayed low values of NTA and NCG codons in ZjbZIPs, which would be beneficial to increase the protein production and also indicated that ZjbZIPs were at a relative high methylation level. The paralogous and orthologous events occurred during the evolutionary process of ZjbZIPs. Thirty-four ZjbZIPs were mapped to but not evenly distributed among 10 pseudo- chromosomes. 30 of ZjbZIP genes showed diverse tissue-specific expression in jujube and wild jujube trees, indicating that these genes may have multiple functions. Some ZjbZIP genes were specifically analyzed and found to play important roles in the early stage of fruit development. Moreover, some ZjbZIPs that respond to phytoplasma invasion and abiotic stress environmental conditions, such as salt and low temperature, were found. Based on homology comparisons, prediction analysis and yeast two-hybrid, a protein interaction network including 42 ZjbZIPs was constructed.ConclusionsThe bioinformatics analyses of 45 ZjbZIPs were implemented systematically, and their expression profiles in jujube and wild jujube showed that many genes might play crucial roles during fruit ripening and in the response to phytoplasma and abiotic stresses. The protein interaction networks among ZjbZIPs could provide useful information for further functional studies.

Project description:BackgroundJujube is a typical fruit tree species from China. 'Muzao', a cracking-susceptible cultivar, and 'Linhuang No. 1', a cracking-resistant cultivar, were selected in a previous study as contrasting research materials. Whole-genome resequencing and transcriptomic analysis of 'Linhuang No. 1' and 'Muzao' allowed the identification of differentially expressed genes with different gene structures between the two cultivars and could be helpful in explaining the differences and similarities between the two cultivars.ResultsResequencing identified 664,129 polymorphic variable sites between 'Linhuang No. 1' and 'Muzao'. To determine the genetic relationship among 'Linhuang No. 1', 'Muzao' and the jujube genome reference cultivar 'Dongzao', the characteristic polymorphic variable sites were analysed by principal component analysis. The genetic relationship between 'Linhuang No. 1' and 'Muzao' was closer than that of either variety and 'Dongzao'. Nineteen differentially expressed genes were identified by combining transcriptomic analysis with resequencing analysis. LOC107427052 (encoding a nitrite reductase) was identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for further study. The identified insertion was not in the domain region of the LOC107427052 gene coding sequence (CDS) region and was verified by the finding that the insertion did not affect translation of the protein. The LOC107427052 gene expression levels, nitrite reductase activities and nitrite contents of 'Muzao' were significantly higher than the corresponding values of 'Linhuang No. 1' at the young fruit stage. There was no significant difference in the quantity of the product of nitrite reductase, namely, ammonia, between the two cultivars.ConclusionsThe present study was the first to explore the differences between different jujube cultivars ('Linhuang No. 1' and 'Muzao') by combining genome resequencing and transcriptomics. LOC107427052 (encoding a nitrite reductase) was characterized by KEGG enrichment analysis. The insertion in the CDS region of the LOC107427052 gene provides a new direction for the study of nitrogen metabolism in jujube. Our study has laid a foundation for the comparative analysis of nitrite metabolism between the jujube cultivars 'Linhuang No. 1' and 'Muzao'.

Project description:Chinese jujube (Zizyphus jujuba Mill.), a member of the Rhamnaceae family with favorable nutritional and flavor quality, exhibited characteristic climacteric changes during its fruit growth stage. Therefore, fruit samples were harvested at four developmental stages on days 55 (young fruits), 76 (white-mature fruits), 96 (half-red fruits), and 116 (full-red fruits) after flowering (DAF). This study then investigated those four growth stage changes of the jujube proteome using label-free quantification proteomics. The results identified 4762 proteins in the samples, of which 3757 proteins were quantified. Compared with former stages, the stages examined were designated as "76 vs. 55 DAF" group, "96 vs. 76 DAF" group, and "116 vs. 96 DAF" group. Gene Ontology (GO) and KEGG annotation and enrichment analysis of the differentially expressed proteins (DEPs) showed that 76 vs. 55 DAF group pathways represented amino sugar, nucleotide sugar, ascorbate, and aldarate metabolic pathways. These pathways were associated with cell division and resistance. In the study, the jujube fruit puffing slowed down and attained a stable growth stage in the 76 vs. 55 DAF group. However, fatty acid biosynthesis and phenylalanine metabolism was mainly enriched in the 96 vs. 76 DAF group. Fatty acids are precursors of aromatic substances and fat-soluble pigments in fruit. The upregulation of differential proteins at this stage indicates that aromatic compounds were synthesized in large quantities at this stage and that fruit would enter the ripening stage. During the ripening stage, 55 DEPs were identified to be involved in photosynthesis and flavonoid biosynthesis in the 116 vs. 96 DAF group. Also, the fruit entered the mature stage, which showed that flavonoids were produced in large quantities. Furthermore, the color of jujube turned red, and photosynthesis was significantly reduced. Hence, a link was established between protein profiles and growth phenotypes, which will help improve our understanding of jujube fruit growth at the proteomic level.

Dataset Information

Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.

Publications

Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.

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