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Transcriptome-based identification of the optimal reference genes as internal controls for quantitative RT-PCR in razor clam (Sinonovacula constricta)

Xuelin Zhao,Jianping Fu,Liting Jiang,Weiwei Zhang,Yina Shao,Chunhua Jin,Jinbo Xiong,Chenghua Li 한국유전학회 2018 Genes & Genomics Vol.40 No.6
Quantitative real-time PCR (qRT-PCR) is a standard method to measure gene expression in function exploring. Accurate and reproducible data of qRT-PCR requires appropriate reference genes, which are stably expressed under different experimental conditions. However, no housekeeping genes were validated as internal controls for qRT-PCR in Sinonovacula constricta. In this study, we classified the transcriptome data of two tissues for Vibrio infection and Cd2+ stress into ten clusters based on the gene expression patterns. Among them, cluster 5 had the most stable gene expression patterns regardless of tissues and treatments as the database for candidate reference genes. A total of 55 orthologs of classical housekeeping genes in the clam transcriptome were annotated. Combined the expression profiles and housekeeping genes in S. constricta, we chose eight candidate reference genes and validated their expression in Vibrio-infected samples and different tissues by qRT-PCR. Their expression stability was analyzed by three different algorithms geNorm, NormFinder and BestKeeper. Although the rank of the eight candidate reference genes is different in different treatments using different software, RS9 could be the best reference genes for normalization of qRT-PCR expression data in S. constricta under various treatments considering the above analysis. Meanwhile, the ranking of genes based on the CV values of transcriptomic data was similar to the validation results. This study provides for the first time a list of suitable reference genes for S. constricta and a valuable resource for further studies of clam immune defense systems.
2
Gallium(III) Nitrate Inhibits Pathogenic Vibrio splendidus Vs by Interfering with the Iron Uptake Pathway

( Tongxiang Song ),( Xuelin Zhao ),( Yina Shao ),( Ming Guo ),( Chenghua Li ),( Weiwei Zhang ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.6
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It is well known that iron is critical for bacterial growth and pathogenic virulence. Due to chemical similarity, Ga3+ competes with Fe3+ for binding to compounds that usually bind Fe3+, thereby interfering with various essential biological reactions. In our present study, gallium(III) nitrate [Ga(NO3)3] could repress the growth of V. splendidus Vs without complete inhibition. In the presence of Ga(NO3)3, the secretion of homogentisic acid-melanin (HGAmelanin) in V. splendidus Vs cells could be increased by 4.8-fold, compared to that in the absence of Ga(NO3)3. HGA-melanin possessed the ability to reduce Fe3+ to Fe2+. In addition, HGA-melanin increased the mRNA levels of feoA and feoB, genes coding Fe2+ transport system proteins to 1.86- and 6.1-fold, respectively, and promoted bacterial growth to 139.2%. Similarly, the mRNA expression of feoA and feoB was upregulated 4.11-fold and 2.71-fold in the presence of 640 μM Ga(NO3)3, respectively. In conclusion, our study suggested that although Ga(NO3)3 could interfere with the growth of V. splendidus Vs, it could also stimulate both the production of Fe3+-reducing HGA-melanin and the expression of feoA and feoB , which facilitate Fe2+ transport in V. splendidus Vs.
3
Effect of Ligustilide Supplement on Physical Fitness of Athletes

Xiaojuan Gao,Shuai Yang,Meng Liu,Yawen Jiang,Li Wenyan,Rui Ma,Zijian Zhao,Xuelin Zhang 아시아건강운동학회 2020 Journal of Asian Society for Health & Exercise Vol.2 No.2
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PURPOSE: In this study, high content ligustilide plant extract was supplemented to monitor the changes of physical fitness and physiological function of athletes, and to explore the effect of ligustilide plant extract on physical function and physical fitness of athletes. METHODS: Thirty five high-level basketball players from Zhengzhou University were selected and divided into two groups randomly: nutritional supplement group (S, n = 18) and control group (C, n=17). This study was approved by the Ethics Committee of Zhengzhou University. Informed consent was obtained from each subject. The intervention period was 8 weeks, six days per week. The training method adopted the small cycle plate training scheme. We measured changes of the scores of rating of perceived exertion (RPE), physical fitness test (15m running, 30m running, 400m running) and routine blood test (mainly including red blood cell count, hemoglobin concentration and average erythrocyte hemoglobin concentration, etc.) before and after training. RESULTS: In group S, the percentage of RPE (grade 6-11) and RPE (grade 12-18) before experiment was 37.5% and 62.5%. After 8 weeks of experiment, 62.5% of RPE (grade 6-11) and 37.5% of RPE (grade 12-18). In group C, 14% of RPE (grade 6-11), 86% of slight RPE (grade 12-18), and 100% of RPE (grade 12-18) after 8 weeks of experiment. It was found that after 8 weeks of training, the number of red blood cells, the concentration of hemoglobin and the average concentration of red blood cell hemoglobin in group S were significantly higher than those before the experiment (p <0.05). According to the comparison of the physical fitness test results of the athletes in group S, it was found that the performance of 400m after 4 weeks and 8 weeks was significantly higher compared with pre-experiment (p<0.05). The changes of 30m and 15m were not significant different (p>0.05). The 400-meter test performance of athletes in group C was significantly higher than that before the experiment only after 8 weeks of training (p<0.05). CONCLUSIONS: Ligustilide supplementation can increase the number of red blood cell and hemoglobin concentration of athletes and has a positive impact on the physical fitness of athletes and the recovery of fatigue.

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