Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.     

Constraint line methods and the applications in ecology北大核心CSCD

With increasing data availability in the big data era, many traditional statistical analyses based on the mean or median are insufficient or inappropriate to elucidate the complex patterns of variation. This is particularly the case when multiple factors are involved and the bivariate scatter occurs as scatter clouds. In such circumstances, constraint line (or envelope) method could be an alternative and effective tool to extract the data boundaries, thus improves our understanding of the complex relationships between limiting factor and response factor. Here, we synthesize the major findings and achievements in the field of applying the constraint line method in ecology. Specifically, we first describe the history and development of the constraint line method. We then discuss the techniques to establish the constraint lines with examples, and discuss the applications and implications of the constraint lines in species distribution, population performance, and optimization problem. We suggest simultaneously application of both constraint lines and regression techniques to the same datasets to achieve a comprehensive understanding of ecological process and underlying mechanisms. Such combined methods should be used with special attention to the role of spatial heterogeneity and scale dependency. We also discuss in detail the potential applicability of the constraint line method in studying the linkages between ecosystem services, and land system design.     

Isolation a P450 gene in <Emphasis Type="Italic">Pinus armandi</Emphasis> and its expression after inoculation of <Emphasis Type="Italic">Leptographium qinlingensis</Emphasis> and treatment with methyl jasmonate

In conifers, the cytochrome P450 monooxygenase of the CYP720B family plays an important role in the synthesis of diterpene resin acids, which are specialised metabolites of the oleoresin defence. We isolated CYP720B19 from Pinus armandi and found that it belongs to the CYP720B family. The CYP720B19 open reading frame of 1467 bp encodes a protein of 488 amino acid residues with high similarity to abietadienol/abietadienal oxidase. Differential expression of the CYP720B19 gene significantly changed when plants were inoculated with the fungus Leptographium qinlingensis, mechanically wounded, or treated with methyl jasmonate (MeJa) and Tween at 4 days and 8 days. Increased expression of the CYP720B19 gene after these treatments suggested that the gene was involved in pine defence against inoculation by L. qinlingensis, MeJa treatment and mechanical injury.     

Chalcone synthase homologous genes cloning and expression pattern in flowering <Emphasis Type="Italic">Fagopyrum tataricum</Emphasis> Gaertn

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is highly nutritious and an excellent dietary source of flavonoid compounds. Chalcone synthase (CHS) is the first key enzyme involved in flavonoid biosynthesis. Here, three putative CHS genes (designated as FtCHS1 (GU172165), FtCHS2 (KT284884), and FtCHS3 (KT284885) were isolated from tartary buckwheat. Nucleotide sequence analysis indicated that FtCHS1 and FtCHS2 each contained one intron of 444 bp and 157 bp, respectively. FtCHS3 included two introns, one of 86 bp and another of 73 bp. The results of quantitative real-time PCR (qRT-PCR) showed the FtCHSs expression presented the same pattern in the stems and flowers, with FtCHS1>FtCHS3>FtCHS2. A different tendency was found in leaves, with FtCHS3>FtCHS2>FtCHS1. However, there was no direct correlation between the three CHS expression and total flavonoids. Furthermore, high-performance liquid chromatography (HPLC) performance reveals rutin is the most abundant flavonoid in all tissues, leaves should be the main location for quercetin storage in tartary buckwheat.     

Physiological and proteomic analysis of mycorrhizal <Emphasis Type="Italic">Pinus massoniana</Emphasis> inoculated with <Emphasis Type="Italic">Lactarius insulsus</Emphasis> under drought stress

This study aimed to investigate physiological and protein expression alterations of mycorrhizal Pinus massoniana Lamb. inoculated with Lactarius insulsus in response to drought stress. The P. massoniana seedlings were inoculated with L. insulsus (Li group) and ectomycorrhized fungal-free filtrate (control, CK group), respectively. After two and a half years, all the plants were exposed to a simulate drought condition without water for 21 days. The soil relative water content (SRWC), wilting degree (WD) and wilting rate (WR) of the plants were measured and root proteome was analyzed based on two-dimensional gel electrophoresis (2-DE), respectively at four time points as 0, 7, 14 and 21 days during the whole drought period. Finally, the electrospray ionization mass spectrometry (ESI-MS) was used to identify the differentially expressed proteins (DEPs) between Li and CK groups. The SRWC was higher, while WR and WD were lower in Li group, compared with that in CK group. Based on 2-DE and ESI-MS, 22 DEPs were identified between Li and CK groups during drought stress. Among them, four proteins had the annotated information in relevant databases, including 1,4-benzoquinone reductase, PSCHI4, ribosomal protein L16 (RPL16) and AINTEGUMENTA-like (AIL) protein. Mycorrhizal P. massoniana inoculated with L. insulsus achieved an enhanced drought resistance as compared to the non-mycorrhizal, and the altered protein expressions such as 1,4-benzoquinone reductase, PSCHI4, RPL16, and AIL might contribute to the improved resistance under drought stress.     

Production of proinsulin in marker-free transgenic tobacco plants using <Emphasis Type="Italic">CRE</Emphasis>/loxP system

The demand for INSULIN is increasing rapidly along with the increased number of diabetic patients. Using the CRE/loxP system, we developed a selective marker-free system without crossing to produce PROINSULIN in transgenic plant. In frame of this approach, the induced promoter pRD29A was isolated from Arabidopsis. The CRE recombinase gene was placed under the control of pRD29A between two loxP recombination sites together with the selective NPTII gene. Furthermore, the binary vector with CRE recombinase and PROINSULIN was constructed and introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Gene excision was used to remove the sequence between the two loxP sites at the presence of 200 mM NaCl. PCR analysis showed that self-excision occurred in several T₀ transgenic plants. Transgenic plants without any marker gene successfully expressed PROINSULIN. This auto-excision strategy provides efficient means of removing the selectable marker gene from transgenic plants. It is an efficient method for producing bio-safe recombinant protein and other valuable substances in plants.     

Effects of exogenous spermidine on antioxidant system of tomato seedlings exposed to high temperature stress

The effects of foliar spraying with spermidine (Spd) on antioxidant system in tomato (Lycopersicon esculentum Mill.) seedlings were investigated under high temperature stress. The high temperature stress significantly inhibited plant growth and reduced chlorophyll (Chl) content. Application of exogenous 1 mM Spd alleviated the inhibition of growth induced by the high temperature stress. Malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content and superoxide anion (O₂) generation rate were significantly increased by the high temperature stress, but Spd significantly reduced the accumulation of reactive oxygen species (ROS) and MDA content under the stress. The high temperature stress significantly decreased glutathione (GSH) content and activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), but increased contents of dehydroascorbic acid (DHA), ascorbic acid (AsA), and oxidized glutathione (GSSG) in tomato leaves. However, Spd significantly increased the activities of antioxidant enzymes, levels of antioxidants and endogenous polyamines in tomato leaves under the high temperature stress. In addition, to varying degrees, Spd regulated expression of MnSOD, POD, APX2, APX6, GR, MDHAR, DHAR1, and DHAR2 genes in tomato leaves exposed to the high temperature stress. These results suggest that Spd could change endogenous polyamine levels and alleviate the damage by oxidative stress enhancing the non-enzymatic and enzymatic antioxidant system and the related gene expression.