Validation of internal control for gene expression study in soybean by quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>Normalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean.</p> <p>Results</p> <p>All ten HKGs displayed a wide range of Ct values in 21 sample pools, confirming that they were variably expressed. GeNorm was used to determine the expression stability of the HGKs in seven series sets. For all the sample pools analyzed, the stability rank was <it>ELF1B</it>, <it>CYP2 </it>> <it>ACT11 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For different tissues under the same developmental stage, the rank was <it>ELF1B</it>, <it>CYP2 </it>> <it>ACT2/7 </it>> <it>UBC2 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>ACT11 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For the developmental stage series, the stability rank was <it>ACT2/7</it>, <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ELF1B </it>> <it>TUB </it>> <it>CYP2 </it>> <it>ACT11 </it>> <it>G6PD </it>> <it>UBQ10</it>. For photoperiodic treatments, the rank was <it>ACT11</it>, <it>ELF1B </it>> <it>CYP2 </it>> <it>TUA </it>> <it>ELF1A </it>> <it>UBC2 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>G6PD </it>> <it>UBQ10</it>. For different times of the day, the rank was <it>ELF1A</it>, <it>TUA </it>> <it>ELF1B </it>> <it>G6PD </it>> <it>CYP2 </it>> <it>ACT11 </it>> <it>ACT2/7 </it>> <it>TUB </it>> <it>UBC2 </it>> <it>UBQ10</it>. For different cultivars and leaves on different nodes of the main stem, the ten HKGs' stability did not differ significantly. ΔCt approach and 'Stability index' were also used to analyze the expression stability in all 21 sample pools. Results from ΔCt approach and geNorm indicated that <it>ELF1B </it>and <it>CYP2 </it>were the most stable HKGs, and <it>UBQ10 </it>and <it>G6PD </it>the most variable ones. Results from 'Stability index' analysis were different, with <it>ACT11 </it>and <it>CYP2 </it>being the most stable HKGs, and <it>ELF1A </it>and <it>TUA </it>the most variable ones.</p> <p>Conclusion</p> <p>Our data suggests that HKGs are expressed variably in soybean. Based on the results from geNorm and ΔCt analysis, <it>ELF1B </it>and <it>CYP2 </it>could be used as internal controls to normalize gene expression in soybean, while <it>UBQ10 </it>and <it>G6PD </it>should be avoided. To achieve accurate results, some conditions may require more than one HKG to be used for normalization.</p

Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics

<p>Abstract</p> <p>Background</p> <p>Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. <it>Superroo</it>t of <it>Lotus corniculatus </it>is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic <it>L. corniculatus </it>plants from <it>Superroot</it>-derived leaves through <it>A. tumefaciens-</it>mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification.</p> <p>Results</p> <p>In the present study, we developed an <it>A. rhizogenes</it>-mediated transformation of <it>Superroot</it>-derived <it>L. corniculatus </it>for gene function investigation, combining the efficient <it>A. rhizogenes</it>-mediated transformation and the rapid regeneration system of <it>Superroot</it>. The transformation system using <it>A. rhizogenes </it>K599 harbouring pGFPGUS<it>Plus </it>was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD₆₀₀= 0.6, and co-cultivation on medium (pH 5.4) at 22°C for 2 days enhanced the transformation frequency significantly. As proof of concept, <it>Superroot</it>-derived <it>L. corniculatus </it>was transformed with a gene from wheat encoding an Na⁺/H⁺antiporter (<it>TaNHX2</it>) using the described system. Transgenic <it>Superroot </it>plants were obtained and had increased salt tolerance, as expected from the expression of <it>TaNHX2</it>.</p> <p>Conclusion</p> <p>A rapid and efficient tool for gene function investigation in <it>L. corniculatus </it>was developed, combining the simplicity and high efficiency of the <it>Superroot </it>regeneration system and the availability of <it>A. rhizogenes</it>-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could reach 92% based on GUS detection. The combination of the highly efficient transformation and the regeneration system of <it>Superroot </it>provides a valuable tool for functional genomics studies in <it>L. corniculatus</it>.</p

Establishment of a novel experimental system for studying the photoperiodic response of short-day dicots using soybean ‘cotyledon-only plant’ as material

Soybean is an important model crop for photoperiodic response studies in plants and contributes significantly to the study of plant development and physiology in the past century. Because soybean plant is much bigger in size and longer in life cycle than Arabidopsis, it needs much more space for growth and time for investigation, which significantly hamper the efficiency of research. In the current study, we tested the photoperiodic response of a distinctive artificially-made cotyledon-only plant (COP) using a photoperiod-sensitive soybean variety Zigongdongdou (ZGDD) and other varieties with diverse sensitivity to photoperiod. ZGDD COPs flowered 39.4 ± 2.5 d after emergence under short-day conditions but maintained vegetative growth under long-day and night break conditions, which is similar to the case in the intact ZGDD plants. The COPs of early-maturing and medium-maturing soybean varieties also grew and flowered normally under natural day-length conditions. At the molecular level, the key genes in the photoperiodic pathway such as E1, GmFT1a, GmFT2a, and GmFT5a in the COPs also showed the same photoperiod sensitivity as in the intact plants. In addition, a simpler material of COP with only one cotyledon and root was generated and found to be sensitive to photoperiod as well. Notably, the COPs are only one-fifth the height of intact plants and one-third the maximum diameter of the intact plants grown in chambers 30 d after emergence. Based on COPs, we established a novel experimental system characterized by an entire photoperiodic response and longer longevity of cotyledons in addition to small plant size, ensuring the consistency, reliability, and stability of plant materials. COPs have the potential to be a novel model material for studies of the developmental biology of soybean and other dicots

Identification of differentially expressed HERV-K(HML-2) loci in colorectal cancer

Colorectal cancer is one of the malignant tumors with the highest mortality rate in the world. Survival rates vary significantly among patients at various stages of the disease. A biomarker capable of early diagnosis is required to facilitate the early detection and treatment of colorectal cancer. Human endogenous retroviruses (HERVs) are abnormally expressed in various diseases, including cancer, and have been involved in cancer development. Real-time quantitative PCR was used to detect the transcript levels of HERV-K(HML-2) gag, pol, and env in colorectal cancer to systematically investigate the connection between HERV-K(HML-2) and colorectal cancer. The results showed that HERV-K(HML-2) transcript expression was significantly higher than healthy controls and was consistent at the population and cell levels. We also used next-generation sequencing to identify and characterize HERV-K(HML-2) loci that were differentially expressed between colorectal cancer patients and healthy individuals. The analysis revealed that these loci were concentrated in immune response signaling pathways, implying that HERV-K impacts the tumor-associated immune response. Our results indicated that HERV-K might serve as a screening tumor marker and a target for tumor immunotherapy in colorectal cancer

Metabolic Disturbances Associated with Systemic Lupus Erythematosus

The metabolic disturbances that underlie systemic lupus erythematosus are currently unknown. A metabolomic study was executed, comparing the sera of 20 SLE patients against that of healthy controls, using LC/MS and GC/MS platforms. Validation of key differences was performed using an independent cohort of 38 SLE patients and orthogonal assays. SLE sera showed evidence of profoundly dampened glycolysis, Krebs cycle, fatty acid β oxidation and amino acid metabolism, alluding to reduced energy biogenesis from all sources. Whereas long-chain fatty acids, including the n3 and n6 essential fatty acids, were significantly reduced, medium chain fatty acids and serum free fatty acids were elevated. The SLE metabolome exhibited profound lipid peroxidation, reflective of oxidative damage. Deficiencies were noted in the cellular anti-oxidant, glutathione, and all methyl group donors, including cysteine, methionine, and choline, as well as phosphocholines. The best discriminators of SLE included elevated lipid peroxidation products, MDA, gamma-glutamyl peptides, GGT, leukotriene B4 and 5-HETE. Importantly, similar elevations were not observed in another chronic inflammatory autoimmune disease, rheumatoid arthritis. To sum, comprehensive profiling of the SLE metabolome reveals evidence of heightened oxidative stress, inflammation, reduced energy generation, altered lipid profiles and a pro-thrombotic state. Resetting the SLE metabolome, either by targeting selected molecules or by supplementing the diet with essential fatty acids, vitamins and methyl group donors offers novel opportunities for disease modulation in this disabling systemic autoimmune ailment

GmFT2a, a Soybean Homolog of FLOWERING LOCUS T, Is Involved in Flowering Transition and Maintenance

BACKGROUND: Flowering reversion can be induced in soybean (Glycine max L. Merr.), a typical short-day (SD) dicot, by switching from SD to long-day (LD) photoperiods. This process may involve florigen, putatively encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana. However, little is known about the potential function of soybean FT homologs in flowering reversion. METHODS: A photoperiod-responsive FT homologue GmFT (renamed as GmFT2a hereafter) was cloned from the photoperiod-sensitive cultivar Zigongdongdou. GmFT2a gene expression under different photoperiods was analyzed by real-time quantitative PCR. In situ hybridization showed direct evidence for its expression during flowering-related processes. GmFT2a was shown to promote flowering using transgenic studies in Arabidopsis and soybean. The effects of photoperiod and temperature on GmFT2a expression were also analyzed in two cultivars with different photoperiod-sensitivities. RESULTS: GmFT2a expression is regulated by photoperiod. Analyses of GmFT2a transcripts revealed a strong correlation between GmFT2a expression and flowering maintenance. GmFT2a transcripts were observed continuously within the vascular tissue up to the shoot apex during flowering. By contrast, transcripts decreased to undetectable levels during flowering reversion. In grafting experiments, the early-flowering, photoperiod-insensitive stock Heihe27 promotes the appearance of GmFT2a transcripts in the shoot apex of scion Zigongdongdou under noninductive LD conditions. The photothermal effects of GmFT2a expression diversity in cultivars with different photoperiod-sensitivities and a hypothesis is proposed. CONCLUSION: GmFT2a expression is associated with flowering induction and maintenance. Therefore, GmFT2a is a potential target gene for soybean breeding, with the aim of increasing geographic adaptation of this crop