寄主凤凰柚对柑橘衰退病病毒甜橙分离株构成的影响

 为研究不同柑橘类型对柑橘衰退病病毒构成的影响，将9个保存在新会橙上的引起甜橙茎陷点症状的柑橘衰退病毒强毒分离株嫁接接种到凤凰柚后，比较了接种前后p25基因的HinfⅠRFLP 和SSCP变化情况。分离株 CT90与CT20接种前后的RFLP组群没有发生变化；RFLP组群发生变化的分离株中有6个由混合组群变为单一组群。此外，除分离株CT90接种前后的SSCP谱带保持不变外，其余8个分离株从新会橙接种到凤凰柚后，SSCP谱带数均呈减少趋势。试验结果倾向于表明某些能在甜橙上增殖的CTV株系在凤凰柚上的增殖受到了抑制。     

Development of hypo-allergenic apples: silencing of the major allergen Mal d 1 gene in ‘Elstar’ apple and the effect of grafting

Summary

Many people who are allergic to birch pollen are also allergic to apple fruit, due to cross- allergenicity. Since apples are the most extensively consumed fruit in Europe, it is highly relevant to develop a hypo-allergenic apple.Apples with significantly reduced levels of the allergen, Mal d 1, may allow many apple allergics to eat them without an allergic reaction. We are currently collaborating to develop a hypo-allergenic apple within the European Integrated Research Project, ISAFRUIT (www.isafruit.org). Hypo-allergenic apple plants (Malus × domestica Borkh., ‘Elstar’) with decreased levels of Mal d 1 mRNA were produced by RNA interference (RNAi) technology. Ten genetically modified (GM) apple lines were selected. In vitro plantlets were first transferred to a greenhouse, then grafted onto wild-type M.9 rootstock to promote the development of fruit-producing trees. Levels of Mal d 1 gene silencing were measured repeatedly by quantitative real-time PCR. Compared to leaf samples from wild-type ‘Elstar’, two GM lines showed modest levels of gene silencing (up to 250-fold), whereas the other eight GM lines were significantly silenced (up to10,000-fold) in Mal d 1 gene expression. These levels of silencing were unaffected by grafting, and have been stable over more than 3 years, and throughout all developmental stages.     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.     

柑橘黄龙病和衰退病的同步PCR和RT-PCR检测

柑橘黄龙病(HLB)和衰退病(CTV)是2种危害严重的世界性柑橘病害,国内外对其病原检测的研究相当多。研究建立了同时检测HLB和CTV的多重RT-PCR体系,从影响多重RT-PCR扩增的引物浓度、Mg2+浓度、Taq酶浓度、dNTPs浓度、退火温度等方面进行体系优化。结果表明,多重PCR反应的退火温度应优先考虑退火温度高的引物,退火温度高的引物所需浓度要大于相应退火温度低的引物。该体系实现了DNA病原和RNA病原的统一检测,进一步体现了多重RT-PCR的优越性。     

Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

AIM: To evaluate the expression of c-Met in papillary thyroid cancer (PTC) by constructing lentiviral vectors for RNA interference (RNAi) of c-met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS: Immunohistochemical assay was performed to detect the expression of c-Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c-met gene was constructed and the silencing effect was detected by RT-PCR and Western blotting. The colony-forming ability, cell cycle, migration and invasion of K1 cells were measured by colony-forming assay, flow cytometry, wound-healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS: The expression of c-Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c-met gene were successfully constructed, and they efficiently inhibited the expression of c-met at mRNA and protein levels. Transfection of c-met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION: Lentivirus-mediated c-met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.     

Assessing the potential impacts of climate change on the alpine habitat suitability of Japanese stone pine (Pinus pumila)

To assess the potential distribution of Pinus pumila, a dominant species of the Japanese alpine zone, and areas of its habitats vulnerable to global warming, we predicted potential habitats under the current climate and two climate change scenarios (RCM20 and MIROC) for 2081–2100 using the classification tree (CT) model. The presence/absence records of P. pumila were extracted from the Phytosociological Relevé Database as response variables, and five climatic variables (warmth index, WI; minimum temperature for the coldest month, TMC; summer precipitation, PRS; maximum snow water equivalent, MSW; winter rainfall, WR) were used as predictor variables. Prediction accuracy of the CT evaluated by ROC analysis showed an AUC value of 0.97, being categorized as “excellent”. We designated Third Mesh cells with an occurrence probability of 0.01 or greater as potential habitats and further divided them into suitable and marginal habitats based on the optimum threshold probability value (0.06) in ROC analysis. Deviance weighted scores revealed that WI was the largest contributing factor followed by MSW. Changes in habitat types from the current climate to the two scenarios were depicted within an observed distribution (Hayashi’s distribution data). The area of suitable habitats under the current climate decreased to 25.0% and to 14.7% under the RCM20 and MIROC scenarios, respectively. Suitable habitats were predicted to remain on high mountains of two unconnected regions, central Honshu and Hokkaido, while they were predicted to vanish in Tohoku and southwestern Hokkaido. Thus P. pumila populations in these regions are vulnerable to climate change.     

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells

AIM:To investigate the inhibitory effect of vector-based RNA interference ( RNAi) on the expression of melanoma associated antigen A3 (MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells. METHODS:A vector for transcribing specific small hairpin RNA ( shRNA) targeting MAGEA3 gene was constructed ,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000. The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively. The cell apoptosis was studied by DNA fragmentation, electron microscopy ，TUNEL assay, and annexin V/PI staining. RESULTS:The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells. After the shRNA expression vector was transfected into the MEL-ED1 cells, the expression of MAGEA3 gene was inhibited significantly ( by 90% ). DNA fragmentation，electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ±1.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group (both P<0.01). CONCLUSION:The specific small hairpin RNA targeting MAGEA3 mRNA can inhibit the expression of MAGEA3 and cause apoptosis of hepatocellular carcinoma cells , which suggests inhibitory effect of MAGEA3 on apoptosis in cancer and provides an experimental basis for treating human tumors with RNAi.     

Evaluation of an alternative D-amino acid/DAAO selection system for transformation in apple (Malus × domestica Borkh.)

Summary

The conventional selection system for apple transformation is based on the selectable marker gene, nptII, encoding antibiotic resistance against kanamycin. We tested an alternative selection system based on the use of D-amino acids using the gene, D-amino acid oxidase 1 (dao1) as the selectable marker, in order to avoid the presence of antibiotic resistance genes in the resulting transgenic apple plants. In addition, dao1 allowed the selection as well as the elimination of dao1-transgenic plants, based on differences in the toxicity of different D-amino acids. Regeneration experiments using apple leaf explants revealed that 2 mM D-serine or D-alanine inhibited shoot regeneration. We performed transformation experiments using the apple cultivars ‘Gala’, ‘Holsteiner Cox’, and a progeny of the apple cultivar ‘Pinova’, and the vector p35S::dao1-intron, containing the dao1 and nptII selectable marker genes. Several shoots regenerated successfully on selection media containing various concentrations of D-serine or D-alanine, but transgenic shoots were not obtained. However, three dao1/nptII transgenic apple lines were obtained after selection with kanamycin, indicating that the vector was functional. Furthermore, we showed that 20 mM D-serine could be used to select dao1-transgenic shoots from non-transgenic in vitro shoots, whereas 13 mM D-isoleucine had the opposite effect.     

Function and anatomy of plant siRNA pools derived from hairpin transgenes

Background  

RNA interference results in specific gene silencing by small-interfering RNAs (siRNAs). Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs will retain cross-species function and that transgenic plants expressing heterologous siRNAs might serve as useful siRNA bioreactors. Here we report a detailed evaluation of the above proposition and present evidence regarding structural features of siRNAs extracted from plants.     

Effects of shRNA-mediated hWAPL silencing on proliferation and apoptosis of human cervical cancer CaSki cells

AIM：To investigate the role of human wings apart-like (hWAPL) protein in proliferation and apoptosis of human cervical cancer CaSki cells through hWAPL gene silencing by specific short hairpin RNA (shRNA) duplexes. METHODS：The relative hWAPL mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. Cell proliferation was detected by MTT assay, and the apoptosis was determined by Annexin V-PE and Hoechst 33258 staining. Western blotting was used to analyze the expression of cleaved caspase-3, p21 and p27. The effect of hWAPL gene silencing on the in vivo tumorigenic capacity of CaSki cells was investigated in a tumor-bearing nude mouse model. RESULTS：Real-time fluorescence quantitative PCR and Western blotting showed that hWAPL mRNA and protein expression in CaSki cells was efficiently inhibited by hWAPL shRNA. The shRNA-mediated hWAPL silencing inhibited the proliferation and induced the apoptosis of CaSki cells. Additionally, the expression levels of cleaved caspase-3, p21 and p27 were up-regulated in hWAPL knockdown cells. Knockdown of hWAPL also inhibited the in vivo tumorigenic capacity of CaSki cells. CONCLUSION：hWAPL is involved in the regulation of the proliferation and apoptosis of CaSki cells in vitro and in vivo, and might serve as a therapeutic target in cancer treatment.     

Construction of a lentiviral vector for RNA interference of human EGFL7 gene and its stable expression in human laryngeal carcinoma cells

AIM：To investigate the role of epidermal growth factor-like domain 7 (EGFL7) in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference (RNAi) of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma (HEp-2) cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs (shRNAs) against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.     

Production of transgenic sweetpotato plants resistant to stem nematodes using oryzacystatin-I gene

Stem nematode (Ditylenchus destructor Thorne) is one of most serious diseases limiting sweetpotato (Ipomoea batatas (L.) Lam.) production, and it is urgent to develop sweetpotato varieties resistant to this disease. In present study, we have developed transgenic sweetpotato (cv. Xushu 18) plants resistant to stem nematodes using oryzacystatin-I (OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, uidA gene and hptII gene. Selection culture was conducted using 7 mg/l hygromycin. A total of 2119 plants were produced from the inoculated 1710 cell aggregates of Xushu 18 via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 92.8% of the regenerated plants were transgenic plants. Transgenic plants exhibited enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation and the inoculation test with stem nematodes and stem nematode-resistant plants were selected from the transgenic plants. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 3. High level of OCI gene expression in the resistant transgenic plants was demonstrated by real-time quantitative PCR analysis. This study provides a way for improving stem nematode resistance of sweetpotato.     

Effects of HIF-1α silencing on expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919

AIM: To study the effect of hypoxia-inducible factor 1α(HIF-1α) silencing on the expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919 under hypoxia. METHODS: Hypoxic condition was induced by CoCl₂ and the expression of HIF-1α was silenced by small interference RNA. HIF-1α-specific RNAi lentiviral vector was constructed. Real-time RT-PCR and Western blotting were used to detect the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells under hypoxia. The expression of p27 and Ki67 was observed by Western blotting after HIF-1α silencing was performed. The cell cycle of hepatoma cells was detected by flow cytometry. RESULTS: Under hypoxic condition, the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells increased significantly (P<0.05). HIF-1α silencing significantly reduced the expression of Ki67 but increased the expression of p27 (P<0.05) in CBRH-7919 cells. In transfected cells, the number of cells in G₀/G₁ phase was much higher and that in S phase was much lower than those in the control cells. CONCLUSION: Hypoxia induces the expression of HIF-1α. HIF-1α silencing can regulate the proliferation of hepatoma cells through reducing the expression of Ki67 and increasing the expression of p27.     

Development of a gene silencing DNA vector derived from a broad host range geminivirus

Background  

Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.