Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica

Global Gene Expression of Kosteletzkya virginica Seedlings Responding to Salt Stress.

Soil salinization is becoming a serious threat to crop yield all over the world. Nowadays, acquainting the specific molecular mechanisms underlying various abiotic stresses especially to salt stress should be of great importance. While the development of the high-throughout sequencing technology promoted the progress powerfully. The intricate perception, transduction and regulation mechanisms underlying salt stress are being illustrated more and more clearly. As a perennial halophytic plant, Kosteletzkya virginica is able to help us to understand the mechanisms more directly and effectively. We carried out the whole transcriptome analysis on young seedlings with or without salt treatment through high-throughout sequencing technology. The results revealed that the numbers of different expressed transcripts between control and different treatments are 4145 and 9134, respectively. The ORF prediction suggested that there were 94308 ORF out of the 103489 (91.10%) total transcripts. We also carried out further differential expression analysis through gene ontology (GO) classification, cluster of orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In a word, our transcriptome study on Kosteletzkya virginica would provide direct and effective references for researches on molecular mechanisms of salt-tolerance, extending our view of salt tolerance in plant further. Above all, the related report in this paper is the first about Kosteletzkya virginica

H (2014) Molecular Cloning and Bioinformatics Analysis of a New PlasmaMembrane Na+/H+ Antiporter Gene from the Halophyte Kosteletzkya virginica. The Sci World J 2014: 7

A new plasma membrane Na + /H + antiporter gene (named as KvSOS1) was cloned from the halophyte Kosteletzkya virginica by reverse-transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, which is a homologue of SOS1 (salt overly sensitive 1). The full-length cDNA is 3850 bp and contains an open reading frame (ORF) encoding a protein of 1147 amino acids with a molecular weight of 127.56 kDa and a theoretical pI of 6.18. Bioinformatics analysis indicated that the deduced protein appears to be a transmembrane protein with 12 transmembrane domains at the N-terminal region and a long hydrophilic tail in cytoplasm at its C-terminal region and shares 72-82% identity at the peptide level with other plant plasma membrane Na + /H + antiporters

Molecular Cloning and Bioinformatics Analysis of a New Plasma Membrane Na+/H+ Antiporter Gene from the Halophyte Kosteletzkya virginica

A new plasma membrane Na+/H+ antiporter gene (named as KvSOS1) was cloned from the halophyte Kosteletzkya virginica by reverse-transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, which is a homologue of SOS1 (salt overly sensitive 1). The full-length cDNA is 3850 bp and contains an open reading frame (ORF) encoding a protein of 1147 amino acids with a molecular weight of 127.56 kDa and a theoretical pI of 6.18. Bioinformatics analysis indicated that the deduced protein appears to be a transmembrane protein with 12 transmembrane domains at the N-terminal region and a long hydrophilic tail in cytoplasm at its C-terminal region and shares 72–82% identity at the peptide level with other plant plasma membrane Na+/H+ antiporters

Dissection of Photosynthetic Electron Transport Process in Sweet Sorghum under Heat Stress

Plant photosynthesis and photosystem II (PSII) are susceptible to high temperature. However, photosynthetic electron transport process under heat stress remains unclear. To reveal this issue, chlorophyll a fluorescence and modulated 820 nm reflection were simultaneously detected in sweet sorghum. At 43 degrees C, J step in the chlorophyll a fluorescence transient was significantly elevated, suggesting that electron transport beyond primary quinone of PSII (Q(A)) (primary quinone electron acceptor of PSII) was inhibited. PSI (Photosystem I) photochemical capacity was not influenced even under severe heat stress at 48 degrees C. Thus, PSI oxidation was prolonged and PSI re-reduction did not reach normal level. The inhibition of electron transport between PSII and PSI can reduce the possibility of PSI photoinhibition under heat stress. PSII function recovered entirely one day after heat stress at 43 degrees C, implying that sweet sorghum has certain self-remediation capacity. When the temperature reached 48 degrees C, the maximum quantum yield for primary photochemistry and the electron transport from PSII donor side were remarkably decreased, which greatly limited the electron flow to PSI, and PSI re-reduction suspended. The efficiency of an electron transferred from the intersystem electron carrier (plastoquinol, PQH(2)) to the end electron acceptors at the PSI acceptor side increased significantly at 48 degrees C, and the reason was the greater inhibition of electron transport before PQH(2). Thus, the fragment from Q(A) to PQH(2) is the most heat sensitive in the electron transport chain between PSII and PSI in sweet sorghum

Differential expression pattern of all the transcripts in the control (CK) and two salt treatments (S1, S2) libraries.

<p>Differential expression pattern of all the transcripts in the control (CK) and two salt treatments (S1, S2) libraries.</p

The expression behaviors of the related genes in KEGG pathways.

<p>The expression behaviors of the related genes in KEGG pathways.</p

Cluster analysis of the expressed genes.

<p>Every column stands for a sample and each line represents a single gene. Different colors indicate different expression levels. Red indicates up-regulation and green indicates down-regulation, while the black indicates unchanged.</p

The whole sequencing statistics for <i>Kosteletzkya virginica</i> transcriptome.

<p>The whole sequencing statistics for <i>Kosteletzkya virginica</i> transcriptome.</p

KEGG pathway visualization of starch and sucrose metabolism changes under salt stress.

<p>The enzymes circled by red frame are up-regulated in salt stress; the enzymes circled by green frame are down-regulated in salt stress in addition the more obvious variation with deeper color.</p