Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis / 生物工程学报

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

Could be FOXO3a, miR-96-5p and miR-182-5p useful for Brazilian women with luminal A and triple negative breast cancers prognosis and target therapy?

Abstract FOXO3a dysregulation is frequently implicated in tumorigenesis, and its inhibition can occur by several molecular mechanisms. Among these, post-transcriptional suppression by miRNAs has been associated with various cancers initiation. Here, we assessed the expression profiles of the most relevant miRNAs for breast tumorigenesis, using Luminal A (LA) and Triple-Negative (TN) breast cancer from Brazilian patients, by the quantitative real time-PCR method. Their potential prognostic role for the patients was also evaluated. We identified the miRNAs miR-96-5p and miR-182-5p, de-scribed as negative regulators of FOXO3A, with differential expression both in LA and TN tumors when compared to normal tissue. The miR-96-5p and miR-182-5p miRNAs were upregulated in LA (7.82 times, p < 0.005; 6.12 times, p < 0.005, respectively) and TN breast cancer samples (9.42 times, p < 0.0001; 8.51 times, p < 0.0001) compared to normal tissues. The samples with higher miR-96-5p and miR-182-5p expression (FR ≥ 4) were submitted for FOXO3a immunostaining. Reduced protein detection was observed in all of the tumors compared to normal tissues. The most prominent miRNA expression and FOXO3a protein suppression were observed in TN samples (p < 0.001), indicating the relevant role of these molecules in this tumor biology and clinical behavior. Our results corroborate the literature regarding to the relevance of FOXO3a in the breast cancer, and they open new perspectives for alternative target therapy options for Brazilian patients expressing both FOXO3a and its regulatory miRNAs.

Effects of immune infiltration-related gene CSAG1 on the progression of renal clear cell carcinoma / 现代泌尿外科杂志

【Objective】 To explore the correlation between CSAG1 expression and the prognosis and tumor-infiltrating lymphocytes in renal clear cell carcinoma (RCCC), and to predict the survival and tumor progression. 【Methods】 The gene expression profiles and clinical information of CSAG1 were downloaded from the Cancer Genome Atlas (TCGA). Based on the differential mRNA expression, GO annotation and KEGG pathway analysis were performed. The relationship between CSAG1 and tumor immune infiltration was assessed with Tumor Immunoassay Resource (Timer 2.0) database. The mRNA expression of CSAG1 in human RCCC specimens was validated with qRT-PCR. 【Results】 CSAG1 expression was significantly higher in RCCC tissues than in normal tissues (P<0.05). The qRT-PCR results revealed that the mRNA level of CSAG1 was consistent with that predicted by bioinformatic analysis. The KEGG analysis and GO annotation indicated high GSAG1 expression in RCCC was related to transmembrane transport, tricarboxylic acid cycle and lysosome. CSAG1 expression was positively related to the infiltration of pDC, aDC, CD8+ T cells, cytotoxic cells, TFH, TH1 cells, Tem, NK CD56dm cells, Treg and T cells, but negatively correlated with macrophage infiltration. 【Conclusion】 CSAG1 may be associated with poor prognosis of RCCC and become a potential immunotherapy target.

Comparison of Automated and Manual Viral Nucleic Acid Extraction Kits for Covid-19 Detection Using qRT-PCR

@#Introduction: The emergence of a novel Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a pandemic. Rapid and accurate diagnosis method is crucial to reduce the disease burden and to improve early diagnosis approaches to control of the disease. Real time Reverse transcriptase PCR (qRT-PCR) has been identified by the World Health Organization as the most sensitive and specific method of detection. However, the success of this assay relies on the quantity and quality of the extracted viral RNA. Methods: Various methods have been developed for nucleic acid extraction however, the methods have not been assessed. RNA extraction was performed from 24 nasopharyngeal swab samples using a manual extraction kit (GF-1) and an automated extraction kit (Genolution). The concentration and purity of the extracted RNA samples were measured, and its performance were tested using qRT-PCR. Results: The average concentration and purity of the RNA samples extracted using GF-1 kit was higher compared to Genolution. Similarly, the qRT-PCR assay using the RNA samples extracted using manual extraction was better compared to automated kit. Conclusion: Both the manual and automated extraction kits have its advantages and disadvantages in terms of yield and purity. However, with proper optimization, both methods may be used for routine molecular diagnostic of COVID-19 in laboratories.

Utility of BMI-1 and NANOG expression levels in survival prediction of pediatric acute lymphoblastic leukemia

ABSTRACT Background Acute lymphoblastic leukemia (ALL) is the most common malignancy in children characterized by the overproduction and accumulation of immature lymphoid cells in the bone marrow and peripheral blood. The BMI-1 is an important component of the Polycomb Repressive Complex-1 (PRC1). It is an important molecule for the self-renewal of hematopoietic stem cells (HSCs). The BMI-1 expression is generally high in HSCs and decreases after cell differentiation. The BMI-1 is required for the maintenance of normal and cancer stem cells and has been reported as an oncogene in various tumors. The NANOG is a homeodomain transcription factor responsible for maintaining the stem cell compartment at the blastocyst stage of developing embryos. The NANOG gene has been proven to be transcribed in CD34+ cells and different leukemic cells. Methods The ribonucleic acid (RNA) was extracted from the peripheral blood mononuclear cells (PBMNCs) of 30 pediatric ALL patients (16 B-ALL and 14 T-ALL) and 14 healthy controls. The Bmi-1 and NANOG expression levels were determined using the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results Compared to normal controls, patients with ALL exhibited upregulated levels of Bmi-1 (p = 0.03). Patients who overexpressed Bmi-1 and NANOG displayed a significantly worse survival than low-expressing patients (hazard ratio (HR) 5.74, 95% confidence interval (CI):1.48-22, p = 0.012 and HR 3.8, 95% CI:1.009-14.3, p = 0.048, respectively). Conclusions Taken together, these data suggest that the Bmi-1 and NANOG might serve as a novel survival predictor in ALL patients. Our observation also suggests that the Bmi-1 and NANOG could serve as new therapeutic targets for treatment of pediatric ALL.

Rootstock mediates transcriptional regulation of citrulline metabolism in grafted watermelon / Mediação do porta-enxerto na regulação transcricional do metabolismo da citrulina na melancia enxertada

Abstract Citrulline is a non-essential amino acid, involved in key biological functions in plants and humans. Rootstocks have a major impact on citrulline accumulation in grafted watermelon. Information regarding rootstock induced changes in citrulline metabolism is elusive. To understand the regulatory mechanism, parallel changes in the expression profiles of citrulline metabolic genes and citrulline content of watermelon were monitored during the development of self-rooted watermelon and watermelon grafted onto pumpkin, wild and bottle gourd rootstocks. Results demonstrated that rootstocks regulated the expression profiles in different ways to influence the citrulline content. GAT, NAGPR, ASS3 ASS2 and Asl2 showed the negative correlation with citrulline content in pumpkin grafted watermelon. Pumpkin rootstock promoted the citrulline content by high down-regulation and synergistic effect of ASS2, ASS3, ASL1 and ASl2 genes. In wild grafted watermelon, citrulline was accumulated as a result of down regulation of GAT, NAGS and ASL2 genes, which showed an inverse correlation with citrulline. In gourd grafted watermelon, changes in citrulline content were observed to be linked with lower expressions of GAT, NAGK, ASS2, ASS3, ASL1 and ARG which were negatively correlated with citrulline content. Our study will provide the basis to understand the molecular mechanism of citrulline accumulation in various rootstocks.

Resumo A citrulina é um aminoácido não essencial, envolvida em importantes funções biológicas de plantas e seres humanos. Os porta-enxertos têm um grande impacto no acúmulo de citrulina na melancia enxertada. Informações sobre alterações induzidas por porta-enxertos no metabolismo da citrulina ainda não foram descritas. Para entender o mecanismo regulatório, foram monitoradas mudanças paralelas nos perfis de expressão dos genes metabólicos de citrulina e no teor de citrulina da melancia durante o desenvolvimento da melancia e da melancia enxertada em porta-enxertos de abóbora, silvestre e cabaça. Os resultados demonstraram que o porta-enxerto regulou os perfis de expressão de diferentes maneiras para influenciar no conteúdo de citrulina. GAT, NAGPR, ASS3, ASS2 e ASL2 apresentaram correlação negativa com o teor de citrulina em melancia enxertada de abóbora. O porta-enxerto de abóbora promoveu o conteúdo de citrulina por meio de baixa regulação e efeito sinérgico de duas famílias de genes ASS e ASL. Na melancia enxertada, a acumulação de citrulina resultou na regulação negativa de GAT, NAGS e ASL2, que mostraram uma correlação inversa com a citrulina. Na melancia enxertada, observou-se que as alterações no conteúdo de citrulina foram associadas a menores expressões de GAT, NAGK, ASS2, ASS3, ASL1 e ARG, que foram negativamente correlacionadas com o conteúdo de citrulina. Esses resultados fornecem a base para identificar o mecanismo molecular do acúmulo de citrulina em vários porta-enxertos.

Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox / 中国中药杂志

Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.

Transcriptome analysis and validation of key genes involved in biosynthesis of iridoids in Gentiana lhassica / 中国中药杂志

As the main chemical constituents, iridoids are widely distributed within Gentiana, Gentianaceae, with promising bioactivities. Based on the previous work, the transcriptome of G. lhassica, an original plant of Tibetan herb "Jieji Nabao", was sequenced and analyzed in this study, and the transcriptome databases of roots, stems, leaves, and flowers were constructed so as to explore unigenes that may encode the key enzymes in the biosynthetic pathway of iridoids. Then, qRT-PCR was used to validate the relative expression levels of 11 genes named AACT, DXS, MCS, HDS, IDI, GPPS, GES, G10H, 7-DLNGT, 7-DLGT, and SLS in roots, stems, leaves, and flowers. Also, the total contents of gentiopicroside and loganic acid were determined by HPLC, respectively. The results are as follows:(1)a total of 76 486 unigenes with an average length of 852 bp were obtained;(2)335 unigenes were involved in 19 stan-dard secondary metabolism pathways in KEGG database, with phenylpropanoid biosynthesis having the maximum number(75 unigenes), and no isoflavone biosynthetic pathway was annotated;(3)171 unigenes participatedin 27 key enzymes encoding in the biosynthetic pathway of iridoids, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR) gene was highly expressed;(4)qRT-PCR results were approximately consistent with RNA-Seq data and the relative expression levels of the 11 genes were higher in the aboveground parts(stem, leaf, and flower) than in the underground part(root);(5)the total contents of gentiopicroside and loganic acid were higher in the aboveground parts(stem, leaf, and flower) than in the underground part(root), and the difference was significant. This study provides basic scientific data for accurate species identification, evaluation of germplasm resources, research on secondary pro-duct accumulation of medicinal plants within Gentianaceae, and protection of endangered alpine species.

Selection of q RT-PCR reference genes for Amomum tsaoko seeds during dormancy release / 中国中药杂志

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.

Transcriptome analysis and exploration of genes involved in the biosynthesis of iridoids in Gentiana crassicaulis (Gentianaceae) / 药学学报

italic>Gentiana crassicaulis Duthie ex Burk. in Gentiana (Sect. Cruciata), Gentianaceae, is one of the original plants of both Gentianae Macrophyllae Radix and Tibetan herb Jie-Ji Na-Bao, which contain such bioactive iridoids as gentiopicroside, loganic acid and others. In this study, based on previous work, the transcriptome of G. crassicaulis was sequenced and analyzed to construct transcriptome databases of roots, stems, leaves and flowers. qRT-PCR verification was conducted for parts of unigenes that may be key enzymes in the pathway of iridoid biosynthesis. The results are as follows: ① a total of 159 534 unigenes were obtained, with an average length of 679 bp. According to the functional classification of GO, unigenes can be divided into 3 categories with 67 branches. The unigenes were aligned in the KOG database and were classified into 25 categories according to function. ② In the KEGG database, 215 unigenes were implicated in 20 standard secondary metabolism pathways. The analysis shows that 305 unigenes encoded 28 key enzymes in the pathway of iridoid biosynthesis, and their expression in different organs is different; and ③ qRT-PCR was approximately consistent with RNA-Seq results. The 7 annotated unigenes identified in this study, HMGS, DXS, MCS, GPPS, G10H, 7-DLNGT and STR, all had higher relative expression levels in the above-ground parts (stem, leaf and flower) than in the underground part (root). Iridoids are common active and index components of such traditional Chinese medicines as Qinjiao, Longdan, Dangyao, and Qingyedan, among others. Therefore, this work provides basic scientific data for further development including obtaining active components or intermediates through biotechnology, exploring the accumulation of effective components, evaluating the quality of different ecotype varieties, and identifying authentic biosynthesis pathways of medicinal materials.

Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots / 药学学报

Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.

Silicon Mediated Alleviation of Salinity Stress Regulated by Silicon Transporter Genes (Lsi1 and Lsi2) in Indica Rice

Abstract Silicon accumulation is known to improve tolerance of plants under both biotic and abiotic stress. Salinity stress is an inevitable crisis causing wide spread damage to rice leading to food insecurity. The influence of Si (1mM) on two rice cultivars cv. Ghanteswari (high accumulator) and cv.Badami (low accumulator) which differs in Si uptake potential under saline (10ds/m EC) and non- saline conditions were studied in nutrient culture. The Si transporter genes were isolated and characterized to determine their function in salinity tolerance. Under stress, there was an increase in Si accumulation, Na+/K+ ratio, electrolyte leakage, lipid peroxidation and antioxidant activities. On addition of silicon, the K+ uptake increased, membrane damage reduced and osmolytes balance improve under salinity. But, the level of resurgence was varied in both cultivars, due to their differential Si-accumulation. Molecular characterizations of Lsi1 protein revealed its involvement in the movement of ion and water and therefore prevent osmotic stress. The Lsi2 is responsible for removal of Na+, reducing salt toxicity. Silicon accumulation is responsible for maintenance of cell water status, osmotic balance and Na+ ion exclusion during high salinity. The variable relative expression of Lsi2 provides a possible explanation for differential genotypic uptake of silicon.

Cloning, subcellular location and expression analysis of an acteoside synthase gene from Rehmannia glutinosa / 中草药

Objective: To clone the acteoside synthase gene (RgAcS1) from Rehmannia glutinosa, and analyze its subcellular localization and expression pattern. Methods: The cDNA sequence of RgAcS1 was identified based on the annotation of the transcriptome data of R. glutinosa, and the RgAcS1 gene was cloned by polymerase chain reaction (PCR). Constructing the GFP fusion expression vector and observing the subcellular localization of RgAcS1 mediated by Agrobacterium tumefaciens. The expression pattern of RgAcS1 in different parts of tuberous root of R. glutinosa was detected by real-time fluorescence quantitative PCR (qRT-PCR). Results: A full-length coding sequence of a shikimate-O-hydroxy cinnamoyl transferase from R. glutinosa was obtained and named RgAcS1. The length of the RgAcS1 cDNA was 1659 bp, including an open reading frame (ORF) of 1 296 bp, encoding 431 amino acid residues, the molecular weight of the protein was 475 900, and it has a typical domain of shikimic acid-O-hydroxy cinnamoyl transferase. The result of subcellular localization showed that RgAcS1 was mainly distributed in cytoplasm and also in nucleus. The qRT-PCR analysis showed that the expression levels of RgAcS1 were higher in the periderm and root hair of R. glutinosa tuberous root, but lower in the xylem and phloem. The expression levels of RgAcS1 were higher in non-radial striation than that in radial striation of BJ1, QH1 and 85-5. Conclusion: In this study, we obtained the cDNA sequence of RgAcS1, and analyzed the subcellular location and expression patterns of RgAcS1, which will lay foundations for further study on roles of RgAcS1 gene in the synthesis of acteoside in R. glutinosa.

Cloning and expression stability analysis of Actin, Tubulin and GAPDH genes in Dipsacus asper / 中草药

Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.

Cloning and expression analysis of PlERG24 gene in ergosterol biosynthesis of Phellinus linteus / 中草药

Objective: To clone the gene full length of ergosterol C14 reductase (ERG24) in Phellinus linteus and analyze its bioinformatics and expression pattern. Methods: The primers of PlERG24 were designed according to the transcription sequence of P. linteus, the cDNA full-length sequence of PlERG24 was obtained by PCR, its bioinformatics was analyzed by Ex PASy and other online analysis software, and its expression pattern in mycelia of P. linteus was analyzed by real-time fluorescence quantitative PCR. Results: The full-length cDNA of PlERG24 gene was 1 412 bp, which encoding a protein of 441 amino acids with a predicted molecular weight of 49 358.61 and isoelectric point of 5.28; ERG24 protein was a hydrophobic protein without signal peptide, which was presumably located in the plasma membrane with six phosphorylation sites. Phylogenetic tree analysis indicated that amino acid sequences of ERG24 in P. linteus were genetically closely related to ERG24 in Sanghuangporus baumii. The qRT-PCR results showed that gene expression of PlERG24 reached the highest level of 6.36 at 25d during the growth cycle of mycelia in P. linteus. Conclusion The full length of PlERG24 gene was obtained, which lays a foundation for further studies on gene function and genetic regulatory mechanism of ergosterol biosynthesis.

Cloning and expression analysis of tryptophan synthase gene BcTSB in Baphicacanthus cusia / 中草药

Objective: To clone the tryptophan synthase gene named as BcTSB (GenBank accession number AYM45644.1) involved in the synthesis pathway of indole alkaloids from Baphicacanthus cusia, meanwhile, the bioinformatics analysis and expression analysis were also performed. Method: The open reading frame (ORF) of BcTSB gene was obtained by the database of prophase Baphicacanthus cusia transcriptome. The function of the BcTSB gene was preliminarily predicted by a series of bioinformatics tools. The entire protein-coding cDNA of BcTSB was cloned into the prokaryotic expression vector pET32a, then the recombinant plasmid was transformed into E. coli BL21 (DE3) cells, with IPTG induction. SDS-PAGE was used to investigate the situation of expression. The expression of the gene in root, stem and leaf was determined by using real-time PCR (qRT-PCR). Results: The open reading frame (ORF) of cloned BcTSB gene was 1 452 bp, and encoding 483 amino acids, it was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts. Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52 kDa, because prokaryotic expression vector pET32a contained 18 kDa label, SDS-PAGE results showed that a protein band at 70 000 was in consistent with molecular weight of the predicted protein. The QRT-PCR revealed that BcTSB gene was expressed in different tissues of B. cusia, the expression level of BcTSB in stems was much higher than that in roots and leaves. Conclusion: In this study, BcTSB gene of B. cusia was cloned and its expression was analyzed successfully, which laid an experimental foundation for further study on the function and regulation of the gene.

Application of laboratory diagnostic technologies for SARS-CoV-2: current progress and prospect / 南方医科大学学报

Since the outbreak of COVID-19 pandemic, the detection capability has been improving and the detection techniques have been evolving with innovations. qRT- PCR and mNGS, which represent the current mainstay diagnostic technologies, play key roles in disease diagnosis and monitoring of virus variation. The detection technologies based on serum and plasma IgM and IgG antibodies are important for auxiliary diagnosis. RT-LAMP is highly specific for a diagnostic purpose. Digital PCR could quantitatively detect nucleic acid and SHERLOCK has a higher sensitivity. These techniques all have great potential for future development and application for pathogen detection. In this review the authors summarize the basic rationales, technical characteristics and the current application of the SARS-CoV-2 detection techniques.

Cloning and expression analysis of 2-oxoglutarate-dependent dioxygenase from Schisandra chinensis / 药学学报

The 2-oxoglutarate-dependent dioxygenase (2-ODD) gene is regarded as the key enzyme gene involved with aryl naphthalene lignan-podophyllotoxin synthesis. To study the expression pattern and function of the Sc2-ODD gene, a full-length cDNA of the gene was cloned. Bioinformatic analysis, the expression pattern, and prokaryotic expression and purification were implemented. The open reading frame of Sc2-ODD gene was 1 077 bp and encoded 358 amino acids with a molecular weight of 40.16 kD. The Sc2-ODD protein contained the conserved 2OG-FeII-oxy sequence of the 2-ODD protein. The results of phylogenetic analysis revealed that Sc2-ODD is most closely related to Corchorus olitorius 2-ODD. qRT-PCR results showed that Sc2-ODD expression displayed obvious up-regulation at the fruit-swelling stage, then down-regulation in the fruit-coloring period. The Sc2-ODD gene was cloned into the bacterial expression vector pGS21T, the recombinant Sc2-ODD protein was expressed in Escherichia coli Rosetta (DE3) cells and the fusion protein was obtained and purified by GST fusion protein purification technology. This study will lay a foundation for further research on the function and expressional regulation of the Sc2-ODD gene in the aryl naphthalene lignans biosynthesis pathway, and also provides a scientific basis for improving the lignan content and the medicinal quality of Schisandra chinensis using plant genetic engineering.

Expression Analysis of Pyrenophora teres f. maculata-Responsive Loci in Hordeum vulgare

Abstract Pyrenophora teres f. maculata is the causal agent of barley spot form net blotch (SFNB), a major stubble-borne disease in many barley-growing areas worldwide. In plants, the Nucleotide-Binding Site-Leucine-Rich Repeat (NBS-LRR) gene family functions in immunity against a variety of pathogens and pests. From a pre-established set of NBS-type resistance gene candidates, we have selected three candidate genes, HvNBS10, HvNBS72 and HvNBS85, to analyze their possible involvement in P. teres f. maculata resistance. The studied genes were mapped on chromosomes 5H and 7H. Expression profiles using qRT-PCR, 48 hours after infection by P. teres. f. maculata, revealed that the transcription of all genes acted in the same direction (down-regulation) in both resistant and susceptible cultivars, although they showed a variation in transcript dosage. This result suggests that coordinated transcriptional responses of multiple barley NBS genes would be required to an efficient response against P. teres f. maculata. Moreover, the phylogenetic analysis revealed that the studied barley candidate R genes were characterized by a high homology with the barley Nbs2-Rdg2a gene conferring resistance to the fungus Pyrenophora graminea, suggesting a common origin of P. graminea and P. teres resistance genes in barley, following pathogens evolution. The genes characterized in the present study hold potential in elucidating the molecular pathways and developing novel markers associated with SFNB resistance in barley.

Cloning and expressive characteristics of GPS gene and its correlation with saponins content in Eleutherococcus senticosus / 中草药

Objective To clone cDNA sequence of geranyl pyrophosphate synthase (GPS) gene from Eleutherococcus senticosus and analyze genetic characteristics, gene expression level in different organs and the correlation between GPS gene expression and saponins content. Methods RNA was extracted from E. senticosus and reverse transcribed into cDNA. Gene specific primers were designed according to the unigene (c37362.graph_c0) of GPS from transcriptome sequencing data. The full length of the GPS gene cDNA was amplified by PCR. The expression level of GPS gene in different organs was analyzed by qRT-PCR. The content of E. senticosus saponins was detected by spectrophotometry method. Results GPS gene cDNA was cloned from E. senticosus and encodes 419 amino acids with full length of 1 260 bp. GPS protein located in mitochondria does not have transmembrane region. The GPS gene was expressed in each organ and had the highest expression in blade, which is 5.26 times in root. The relative expression of GPS gene and content of saponin showed the same trend and significant positive correlation (r = 0.851, P < 0.05). Conclusion The whole length of cDNA sequence of GPS gene is cloned for the first time, and there is a positive correlation between the expression level of GPS gene and the saponin content of E. senticosus.