Real-time Fluorescence Quantitative Analysis Research Articles

Comparative transcriptome analysis and identification of candidate R2R3-MYB genes involved in anthraquinone biosynthesis in Rheum palmatum L.

BackgroundRheum palmatum L. has important medicinal value because it contains biologically active anthraquinones. However, the key genes and TFs involved in anthraquinone biosynthesis and regulation in R. palmatum remain unclear.MethodsBased on full length transcriptome data, in this study, we screened the differentially expressed genes in the anthraquinone biosynthesis pathway. The R2R3-MYB family genes of R. palmatum were systematically identified based on full-length transcriptome sequencing followed by bioinformatics analyses. The correlation analysis was carried out by using co-expression analysis, protein interaction analysis, and real-time fluorescence quantitative analysis after MeJA treatment. The RpMYB81 and RpMYB98 genes were amplified by RT-PCR, and their subcellular localization and self-activation characteristics were analyzed.ResultsComparative transcriptome analysis results revealed a total of 3525 upregulated and 6043 downregulated DEGs in the CK versus MeJA group; 28 DEGs were involved in the anthraquinone pathway. Eleven CHS genes that belonged to the PKS family were differentially expressed and involved in anthraquinone biosynthesis. Twelve differentially expressed MYBs genes were found to be co-expressed and interact with CHS genes. Furthermore, 52 MYB genes were identified as positive regulators of anthraquinone biosynthesis and were further characterized. Three MYB genes including RpMYB81, RpMYB98, and RpMYB100 responded to MeJA treatment in R. palmatum, and the levels of these genes were verified by qRT-PCR. RpMYB81 was related to anthraquinone biosynthesis. RpMYB98 had an interaction with genes in the anthraquinone biosynthesis pathway. RpMYB81 and RpMYB98 were mainly localized in the nucleus. RpMYB81 had self-activation activity, while RpMYB98 had no self-activation activity.ConclusionRpMYB81, RpMYB98, and RpMYB100 were significantly induced by MeJA treatment. RpMYB81 and RpMYB98 are located in the nucleus, and RpMYB81 has transcriptional activity, suggesting that it might be involved in the transcriptional regulation of anthraquinone biosynthesis in R. palmatum.

Growth Axis Somatostatin, Growth Hormone Receptor, and Insulin-like Growth Factor-1 Genes Express and Are Affected by the Injection of Exogenous Growth Hormone in Chinemys reevesii.

In this study, to explore the effect of growth hormone changes on the related genes and regulatory roles of the turtle, PCR amplification, real-time fluorescence quantitative analysis, and enzyme cutting technology were used to clone and sequence the somatostatin (SS) gene, growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-I) sequence of Chinemys reevesii. The effects of human growth hormone on the mRNA expression of growth-axis-related genes SS, GHR, and IGF-1 in different sexes were observed. The study of the SS gene in turtles using real-time fluorescence quantitative PCR showed that the SS gene was mainly expressed in the nervous system and the digestive system, with the highest expression found in the brain, while the GHR gene and the IGF-I gene were expressed in all tissues of Chinemys reevesii. The SS gene was expressed in the brain, pituitary, liver, stomach, and intestine, with the highest expression in the brain and the lowest expression in the liver. Within 4 weeks of the injection of exogenous growth hormone, the expression level of the SS gene in the brain of both sexes first increased and then decreased, showing a parabolic trend, and the expression level of the experimental group was lower than that of the control group. After the injection of growth hormone (GH), the expression of the GHR gene in the liver of both sexes showed a significant increase in the first week, decreasing to the control group level in the second week, and then gradually increasing. Finally, a significant level of difference in the expression of the GHR gene was reached at 3 and 4 weeks. In terms of the IGF-I gene, the changing trend of the expression level in the liver was the same as that of the GHR gene. After the injection of exogenous growth hormone, although the expression of the SS gene increased the inhibition of the secretion of the GHR gene by the Reeves' turtle, exogenous growth hormone could replace the synthesis of GH and GHR, accelerating the growth of the turtle. The experiments showed that the injection of recombinant human growth hormone affects the expression of SS, GHR, and IGF-1 genes, and promotes the growth of the Reeves' turtle.

Genome-wide identification of NF-Y gene family in maize (Zea mays L.) and the positive role of ZmNF-YC12 in drought resistance and recovery ability.

Nuclear factor Y (NF-Y) genes play important roles in many biological processes, such as leaf growth, nitrogen nutrition, and drought resistance. However, the biological functions of these transcription factor family members have not been systematically analyzed in maize. In the present study, a total of 52 ZmNF-Y genes were identified and classified into three groups in the maize genome. An analysis of the evolutionary relationship, gene structure, and conserved motifs of these genes supports the evolutionary conservation of NF-Y family genes in maize. The tissue expression profiles based on RNA-seq data showed that all genes apart from ZmNF-Y16, ZmNF-YC15, and ZmNF-YC17 were expressed in different maize tissues. A weighted correlation network analysis was conducted and a gene co expression network method was used to analyze the transcriptome sequencing results; six core genes responding to drought and rewatering were identified. A real time fluorescence quantitative analysis showed that these six genes responded to high temperature, drought, high salt, and abscisic acid (ABA) treatments, and subsequent restoration to normal levels. ZmNF-YC12 was highly induced by drought and rewatering treatments. The ZmNF-YC12 protein was localized in the nucleus, and the Gal4-LexA/UAS system and a transactivation analysis demonstrated that ZmNF-YC12 in maize (Zea mays L.) is a transcriptional activator that regulates drought resistance and recovery ability. Silencing ZmNF-YC12 reduced net photosynthesis, chlorophyll content, antioxidant (superoxide dismutase, catalase, peroxidase and ascorbate peroxidase) system activation, and soluble protein and proline contents; it increased the malondialdehyde content, the relative water content, and the water loss rate, which weakened drought resistance and the recoverability of maize. These results provide insights into understanding the evolution of ZmNF-Y family genes in maize and their potential roles in genetic improvement. Our work provides a foundation for subsequent functional studies of the NF-Y gene family and provides deep insights into the role of the ZmNF-YC12 regulatory network in controlling drought resistance and the recoverability of maize.

Genome-Wide Identification and Expression Analysis of the Phytocyanin Gene Family in Nicotiana tabacum

Phytocyanin (PC) is a class of plant-specific blue copper proteins involved in electron transport, plant growth, development, and stress resistance. However, PC proteins have not been systematically evaluated in tobacco plants. We determined the whole-genome sequences of the PC family in the tobacco cultivar ‘K326.’ The transcriptome data were used to analyze the expression of the NtPC family at different development stages and tissue-specific genes. Real-time fluorescence quantitative analysis was used to analyze the expression of the NtPC gene family under low temperature and methyl jasmonate stress. The tobacco NtPC family contained 110 members and was divided into four subfamilies: early nodulin-like protein (NtENODL), uclacyanin-like protein, stellacyanin1-like protein, and plantacyanin-like protein. According to phylogenetic and structural analyses, the NtPC family could be divided into eight structural types. Fifty-three NtPCs were randomly distributed on 22 of 24 tobacco chromosomes. Collinearity analysis revealed 33 pairs of genes belonging to the NtPC family. Gene ontology analysis showed that the PC genes are components of the plasma membrane and may participate in plasma membrane-related functions. The NtPC family contained numerous elements related to hormonal and abiotic stress responses and was specifically expressed in the tobacco prosperous, maturation, and budding periods. Tissue-specific expression analysis showed that some genes were tissue specific. The expression of NtENODL58 and other genes was significantly induced by low-temperature and methyl jasmonate stress. Thus, the NtPC gene family plays an important role in plant stress response.

Experimental study of reversal of multidrug resistance in human leukemia K562/DOX cells by toad venom

Acute leukemia is a malignant tumor originating from the hematopoietic system with the highest incidence and mortality. At present, the main clinical treatment of leukemia is still chemotherapy, during the course of which the multidrug resistance (MDR) will significantly reduce remission rate and disease-free survival rate of patients. MDR is the most important factor affecting refractory/recurrent acute leukemia. Therefore, reversing leukemia MDR is one of the best ways to improve the complete remission rate of refractory/recurrent acute leukemia, and the study of drugs and methods to overcome leukemia MDR has received extensive attention in the leukemia research field. This study was to primarily investigate the effects of Liushen pills on leukemia drug-resistant cell line K562/DOX in inhibiting growth, reversing resistance and inducing apoptosis in anticipation of providing useful cytological and molecular biological basis for the treatment of refractory/recurrent acute leukemia. The serum containing toad venom was prepared by means of Chinese drug serum pharmacology. MTT assay was used to detect the inhibitory rates of human leukemia cell line K562/DOX after being treated with the serum containing toad venom as well as daunorubicin, or with the serum containing toad venom alone at different time points. Real-time fluorescent quantitative analysis (RT-PCR) was performed to determine the effects of serum containing toad venom on the expression of BCL-2 mRNA in human leukemia cell line K562/DOX. Compared to the control group, toad venom showed inhibitory effects on K562/DOX cells; the expression level of BCL-2 mRNA in toad venom group were decreased, indicating that toad venom may reverse the resistance of K562/DOX cells by down-regulating the expression level of MDR1.

Cadmium induces endoplasmic reticulum stress-mediated apoptosis in pig pancreas via the increase of Th1 cells

Cadmium (Cd), an environmental pollutant, causes several adverse reactions in animals. High dose of Cd has serious cytotoxicities, including the induction of programmed cell necrosis, autophagy and apoptosis, which has aroused wide public concern. The balance of cytokine network is affected by Th1/Th2 balance which is closely related to immune response and the occurrence, development, treatment and outcome of various diseases. Cd can induce severe apoptosis, but the relationship between Cd induced apoptosis and Th1/Th2 balance has not been clarified. In this study, we established a pig Cd poisoning model, exposing to CdCl2 for 40 days (20 mg Cd/kg diet). Firstly, deviation of Th1/Th2 balance was observed by fluorescence staining, and apoptosis was observed by TUNEL staining. Then, real-time fluorescence quantitative analysis and Western blot were used to detect the expression of related proteins. The results show that Cd can interfere with the balance of Th1/Th2 and shift the balance towards Th1. In addition, through the experiments, we found that Cd exposure can increase the expression of glucose-regulated protein 94 (GRP94) and glucose-regulated protein 78 (GRP78), marker proteins of unfolded protein response (UPR). Cd exposure can increase the expression of pancreatic endoplasmic reticulum kinase (PERK), CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF-6), cysteinyl aspartate specific proteinase (Caspase12), indicating the three branches (ATF6, PERK and IRE-1) of endoplasmic reticulum stress (ER-stress) were activated. Moreover, we found that the expression of pro-apoptosis genes in the downstream pathway of ER-stress increased. In summary, our results indicated that Cd exposure upregulated the expression of pro-apoptosis related genes and caused apoptosis via the activation of the ER-stress signaling pathways in pancreas cells. And these negative effects were correlated with the equilibrium drift of Th1/Th2, increase in the expression and secretion of Th1 cytokines.