Synergistic effect on biosynthesis of Panax notoginseng saponins by overexpressing a transcription factor PnbHLH and RNA interference of cycloartenol synthase gene / 中国中药杂志

This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.

A key for hypoxia genetic adaptation in alpaca could be a HIF1A truncated bHLH protein domain / Una clave para la adaptacion genética de la hypoxia en las alpacas podria ser un dominio de proteina bHLH truncado con HIF1A

Animals exposed to hypobaric hypoxia triggers a physiological hypoxia response via Hypoxia Inducible Factor (HIF) proteins that functions as transcriptional complexes. As the South American camelids inhabit at high Andean altitudes we have asked if they have developed genetic adaptations to live at high altitudes. In the present study we investigate genetic structures of the HIF1A proteins carried by members of the superorder Cetartiodactyla. During our investigation we discovered the existence of a genetic event that caused the loss of most of the bHLH domain in the proteins borne by the Alpaca and other members of the Cetartiodactyla superorder; we designate them as bHLH short sequences. Further analysis at the nucleotide level revealed in the 12 short sequences included in the study the presence at the 5´end of the bHLH domains stop codons. Seven out of the 12 short HIF1A proteins, have an identical or almost identical nucleotide sequence at their 5´end with a same TAA stop codon and at the same position. As the mutations affects to both the Artiodactyls and Cetaceans, we postulate that the mutation(s) occurred before their divergence about 55 million years ago. The relevance of these findings for genetic adaptation of Alpacas to hypobaric hypoxia of high altitude conditions is discussed.

Los animales expuestos a hypoxia hipobárica generan una respuesta hipóxica fisiológica debido a unas proteinas de Factor-Hipoxia Inducible (HIF) que funcionan como complejos transcripcionales. Debido a que los camelidos Americanos habitan en las grandes alturas andinas, nos hemos preguntado si han desarrollado una adaptación genética para vivir a grandes alturas. Eneste estudio hemos investigado la estructura genética de las proteinas HIF1A que llevan consigo los miembros de la superorden de los cetartiodáctilos. Durante nuestra investigación, descubrimos la existencia de un evento genético que causó la perdida de la mayoría del dominio bHLH en las proteinas transmitidas por la alpaca y otros miembros de la superorden de los cetartiodáctilos; las hemos designado como secuencias cortas de bHLH. Análisis posteriores a nivel nucleótido revelaron que en la doceava secuencia corta incluida en el studio, hubo presencia de codones de terminación en el extreme 5' del dominio de bHLH. Siete de las doce proteinas cortas HIF1A, tiene una secuencia idéntica o casi idéntica de nucleotidos en su extremo 5', con el mismo codón de terminación TAA y en la misma posición. Debido a que la mutación afecta tanto a Artiodáctilos como Cetáceos, proponemos que la mutación(es) ocurrió antes de su divergencia hace unos 55 millones de años. Analizamos la relevancia de estos descubrimientos sobre la adaptación genética de las alpacas a la hipoxia hipobárica en condiciones de grandes alturas.

Identification and bioinformatics analysis of bHLH transcription factor gene family in Lycium ruthenicum Murr under NaCl stress / 中草药

Objective: Based on the transcriptome sequencing results of Lycium barbarum under different concentrations of NaCl stress, the bHLH transcription factor family members of Lycium barbarum were identified by bioinformatics method. Methods: The bHLH family genes were screened by transcriptome sequencing of leaf and root samples of Lycium ruthenicum Murr. under NaCl stress. Results: The physicochemical properties, conserved domain, gene structure, cell location and phylogenetic development of these genes were analyzed by bioinformatics method. The results showed there were 89 bHLH family of Lycium ruthenicum. under NaCl stress. Their physicochemical properties were different, of which 71.90% proteins were weakly acidic and they were hydrophilic protein. The bHLH family of Lycium ruthenicum contained two conserved domains, which were located in the alkaline amino acid region of N and the helical ring and spiral region of C, respectively. The subcellular localization prediction of these bHLHs were mainly in the nucleus and extracellular. Phylogenetic analysis showed that 89 bHLH genes were divided into 20 subgroups, among which the 3 subgroup contained the most abundant bHLH members,including 11 bHLH proteins; the 7, 11, 13, 22 subgroups had only 1 member, respectively. In the other subgroups, the range was between 2 and 8. Conclusion: There are 89 members of bHLH transcription factor family in Lycium barbarum under NaCl stress, and most of the bHLH family proteins are weakly acidic, belonging to hydrophilic proteins, which can be divided into 20 subgroups.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Cloning and expression analysis of CtbHLH transcription factor gene from Carthamus tinctorius and construction of plant expression vector / 中国中药杂志

To clone bHLH( basic helix-loop-helix) gene from Carthamus tinctorius,analyze the expression level in different plant tissues and construct the plant expression vector. The bHLH1 gene was cloned by RT-PCR techniques,and the protein characteristics were analyzed by bioinformatics,and phylogenetic tree was constructed. The expression of bHLH1 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR,and the plant expression vector p BASTA-bHLH1 was constructed. The obtained ORF sequence of bHLH1 gene was 897 bp,encoded a protein of 298 amino acids. Sequence alignment and phylogenetic tree analyses showed that C. tinctorius bHLH1 had a certain homology with other species of amino acids,and was the most similar to the amino acid sequence of tobacco. Real-time PCR results showed significant differences,CtbHLH1 gene in red flower petals in different tissues and different flowering period had remarkable difference in expression level,its high amount expressed in petals,flowers third day after blossom expressed the highest quantity,at the end of the flowering the expression quantity is low. In addition,it is expressed in the root,and the expression in the stem and leaves is extremely low. The bHLH1 gene of C. tinctorius is successfully cloned,and the expression is analyzed. The plant expression vector p BASTA-bHLH is constructed.

Progress in bHLH transcription factors regulating the response to iron deficiency in plants / 生物工程学报

Iron is one of the essential mineral micronutrients for plants. Low concentrations of effective iron in soil can easily increase risk of plant iron deficiency. Several members of bHLH transcription factors family participate in the response to iron deficiency and play an important role in iron regulation of plants. In order to better understand the mechanism of iron deficiency response, an overview of the structure, classification, function and regulatory mechanism of bHLH transcription factors was given in this review as well as signaling pathway triggered by iron deficiency. It will provide theoretical basis and design strategies for cultivating iron deficiency tolerant or iron-rich crops using bHLH transcription factors.

Genome-wide characterization and analysis of bHLH transcription factors in

Ginseng ( C.A. Meyer) is one of the best-selling herbal medicines, with ginsenosides as its main pharmacologically active constituents. Although extensive chemical and pharmaceutical studies of these compounds have been performed, genome-wide studies of the basic helix-loop-helix (bHLH) transcription factors of ginseng are still limited. The bHLH transcription factor family is one of the largest transcription factor families found in eukaryotic organisms, and these proteins are involved in a myriad of regulatory processes. In our study, 169 bHLH transcription factor genes were identified in the genome of , and phylogenetic analysis indicated that these PGbHLHs could be classified into 24 subfamilies. A total of 21 RNA-seq data sets, including two sequencing libraries for jasmonate (JA)-responsive and 19 reported libraries for organ-specific expression analyses were constructed. Through a combination of gene-specific expression patterns and chemical contents, 6 PGbHLH genes from 4 subfamilies were revealed to be potentially involved in the regulation of ginsenoside biosynthesis. These 6 PGbHLHs, which had distinct target genes, were further divided into two groups depending on the absence of MYC-N structure. Our results would provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factor action in .

Analysis of bioinformatics and expression level of bHLH transcription factors in Scutellaria baicalensis / 中草药

Objective The information of bHLH transcription factor genes from transcriptome dataset of Scutellaria baicalensis was predicted by bioinformatics methods and the gene expression analysis was used to deduce its probable function. Methods The bHLH genes were screened from the transcriptome dataset of S. baicalensis by using BLAST comparison software. Then the open reading frames (ORFs) from the full-length of cDNA of bHLH genes were predicted by ORF Finder online tool, and its protein characteristics were analyzed using bioinformatic method. The expression of bHLH genes was detected by qPCR in different organs and treatments stimulated by Gibberellin A3 (GA3). Results Six genes of bHLH transcription factors were obtained, which belonged to six subfamilies of bHLHs of A. thaliana, two of which had completed ORFs. The results of gene expression showed that: The expression of bHLH2 and bHLH3 increased after 100 μmol/L GA3 treatment, and the expression of bHLH1, bHLH5, bHLH6 and bHLH7 decreased. The bHLH gene had the highest expression level in roots and flowers of S. baicalensis of them, among which bHLH1, bHLH2, bHLH5 and bHLH7 had highest expression in flowers and bHLH3 had highest expression in root. There was a correlation between bHLH gene and expression of biosynthesis and regulation genes of flavonoid. Conclusion These results provided the basis for further improving the molecular regulation network of flavonoids of bHLH genes in S. baicalensis.

Changes of differentiation- and proliferation-related proteins in neural stem cells in rats treated with L-monosodium glutamate / 第三军医大学学报

Objective To investigate the roles of differentiation and proliferation related proteins in neural stem cells (NSCs) in rats treated with L monosodium glutamate (MSG). Methods Tissue proteins were extracted from the rat forebrain. Western blotting was used to investigate the changes of differentiation and proliferation related proteins in NSCs in rats treated with MSG. Results Compared with those in rats of the normal control group, the related proteins (Notch1, hes5, Mash1, and NeuroD) in NSCs were significantly decreased in MSG rats at 30 and 60 d, especially for Notch1 and hes5 ( P

Inhibition of BETA2/NeuroD by Id2

Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.

Changes of related proteins of neural stem cell differentiation and proliferation in rats treated with corticosterone / 中国病理生理杂志

AIM: To explore the changes of related proteins of neural stem cell differentiation and proliferation in rats treated with corticosterone (CORT). METHODS: Western blot method was used to investigate the changes of related protein of neural stem cell differentiation and proliferation. RESULTS: Compared with the normal control group, related proteins (Notch1, hes5, Mash1 and NeuroD) of neural stem cell differentiation and proliferation were significantly decreased in the CORT rats on the day 30 and 60, especially for Mash1 and NeuroD (P