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

A Survey on Evaluation of Large Language Models

Large language models (LLMs) are gaining increasing popularity in both academia and industry, owing to their unprecedented performance in various applications. As LLMs continue to play a vital role in both research and daily use, their evaluation becomes increasingly critical, not only at the task level, but also at the society level for better understanding of their potential risks. Over the past years, significant efforts have been made to examine LLMs from various perspectives. This paper presents a comprehensive review of these evaluation methods for LLMs, focusing on three key dimensions: what to evaluate, where to evaluate, and how to evaluate. Firstly, we provide an overview from the perspective of evaluation tasks, encompassing general natural language processing tasks, reasoning, medical usage, ethics, educations, natural and social sciences, agent applications, and other areas. Secondly, we answer the `where' and `how' questions by diving into the evaluation methods and benchmarks, which serve as crucial components in assessing performance of LLMs. Then, we summarize the success and failure cases of LLMs in different tasks. Finally, we shed light on several future challenges that lie ahead in LLMs evaluation. Our aim is to offer invaluable insights to researchers in the realm of LLMs evaluation, thereby aiding the development of more proficient LLMs. Our key point is that evaluation should be treated as an essential discipline to better assist the development of LLMs. We consistently maintain the related open-source materials at: https://github.com/MLGroupJLU/LLM-eval-survey.Comment: 23 page

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

Histopathological Observation of Immunized Rhesus Macaques with Plague Vaccines after Subcutaneous Infection of Yersinia pestis

In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 µg F1 and 10 µg rV270) and SV2 (200 µg F1 and 100 µg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×106 CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague

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

An Analytical Method for Calculating the Cogging Torque of a Consequent Pole Hybrid Excitation Synchronous Machine Based on Spatial 3D Field Simplification

Consequent pole hybrid excitation synchronous (CPHES) machines have the advantage of symmetrical bidirectional magnetomotive force increments. Compared with a traditional hybrid excitation motor (HEM), a CPHES machine improves the disadvantage of asymmetry in the adjustment range when magnetization and demagnetization occur. The calculation and analysis of the cogging torque of the CPHES machine are complex due to the complicated structure. This paper proposes an analytical method for calculating the cogging torque of a CPHES machine. This analytical method converts the complex three-dimensional magnetic field problem into a two-dimensional magnetic circuit problem and, through the accumulation method, can quickly and accurately calculate the cogging torque of the CPHES machine. In contrast with the finite element method, the calculation results basically follow each other, but the analytical method is more efficient and omits complicated meshing. This is of great significance to the preliminary electromagnetic design and performance optimization of a CPHES machine

High Density and Uniform Plant Distribution Improve Soybean Yield by Regulating Population Uniformity and Canopy Light Interception

Optimizing the spatial distribution of plants under normal conditions of water and fertilizer is widely used by farmers to improve soybean yield. However, the relationship between soybean yield and spatial plant distribution in the field has not been well studied. This study examined the effect of planting density and plant distribution pattern on soybean plant growth, yield components, canopy light interception, and dry matter accumulation. We also analyzed the relationship between photosynthetic rate, dry matter accumulation, and yield under different planting densities and plant distribution. A two year field experiment was conducted during the 2018 and 2019 soybean planting seasons. Two planting densities (1.8 × 105 and 2.7 × 105 plants ha−1) and two plant distribution patterns (uniform and non-uniform plant spacing) were tested. Higher planting density significantly increased the canopy light interception and dry matter accumulation during soybean growth, leading to increased soybean productivity. The seed yield of soybean under higher planting density was 22.8% higher than under normal planting density. Soybean planted under uniform spacing significantly reduced the differences plant-to-plant. Uniform plant spacing significantly increased the canopy light interception and dry matter accumulation of the soybean population. In addition, the coefficient of variation of seed weight per plant between individual plants under uniform plant distribution decreased by 71.5% compared with non-uniform plant distribution. Furthermore, uniform plant distribution increased soybean seed yield by 9.5% over non-uniform plant distribution. This study demonstrates that increasing planting density under uniform plant distribution can be useful to obtain higher seed yield without increasing other farm inputs

Can Soybean Cultivars with Larger Seed Size Produce More Protein, Lipids, and Seed Yield? A Meta-Analysis

Increasing soybean production and ensuring greater access to soybean protein and lipids is critical for global food security and human health. Seed size (i.e., seed weight) is one of the most important agronomic traits of soybean, which not only determines the seed yield, but can also affect the yield of protein and lipids. In China, farmers favor soybean cultivars with large seeds, which they believe produce more protein and lipids; however, experimental evidence supporting this belief is lacking. Therefore, we conducted field experiments from 2017 to 2020 at 35 locations across the Huang-Huai-Hai region (HHH) of China with 64 soybean cultivars. The seed yield, seed protein content, and seed lipids content of soybean, and their relationship with seed size were investigated. The highest seed yield (i.e., seed weight per unit area) was 2996.5 kg ha−1 in the north of HHH. However, the highest seed protein content was found in the south of HHH (42.5%) for the higher temperature, which was significantly higher than that of the middle (41.7%) and north of HHH (40.2%). In contrast, the highest seed lipids content was 20.7% in the north of HHH. Temperature, which had a path coefficient on seed yield of 0.519, can promote soybean seed yield. The correlation analysis indicated that the selection of the large seed size cultivar did not increase seed yield, and even led to a reduction of seed yield under high-yield environmental conditions. The seed protein content of soybean was not increased in the cultivars with large seed sizes. In addition, under different levels of seed lipids content (20.30%), a significantly negative relationship was found between seed lipids content and hundred seed weight. Therefore, it is recommended that farmers choose to plant cultivars with smaller soybean seed sizes, so as to ensure high and stable soybean seed yield and obtain more vegetable protein and lipids per unit area