Pineapple mealybug wilt associated virus-2 （PMWaV-2） is an important pathogen causing Mealybug wilt of pineapple （MWP）. We established a realtime quantitative RT-PCR （RT-qPCR） method with TaqMan probe based on s...Pineapple mealybug wilt associated virus-2 （PMWaV-2） is an important pathogen causing Mealybug wilt of pineapple （MWP）. We established a realtime quantitative RT-PCR （RT-qPCR） method with TaqMan probe based on specific primers of conserved nucleotide sequence of PMWaV-2 coat protein gene. The results showed that the method was higldy sensitive to positive sample, but had no fluorescence signal to health sample and blank control. The sensitivity of RTqPCR was about 100 times higher than general PCR. Reproducibihty test revealed that the coefficients of variation between intra-and inter-assay were beth within 1.85%, indicating it was a quantitative detection method for PMWaV-2 with simple operation, strong specificity, high sensitivity, and reliable reproducibility.展开更多
The present study was conducted to investigate the effect of agitation rate on the increase in fresh weight of MD2 pineapple protocorm-like bodies (PLBs) and shoots cultured in liquid medium. PLBs were cultured in 250...The present study was conducted to investigate the effect of agitation rate on the increase in fresh weight of MD2 pineapple protocorm-like bodies (PLBs) and shoots cultured in liquid medium. PLBs were cultured in 250 ml Erlenmeyer flasks (7 g per flask) containing MS medium and plant growth regulators (1.5 mg/L 6-Benzylaminopurine, BAP and 0.2 mg/L 1-Naphthaleneacetic acid, NAA). The orbital shaker was set at speeds of 50, 80, 100, 120, and 150 rpm. After 40 days, the cultures shaken at 80 rpm showed the highest fresh weight and the highest number of shoots at 76 g and 41 shoots, respectively. A comparative study of agitation found that 80 rpm was the best speed which enhanced both PLB and shoot formation. The findings in the present study would be helpful in setting up large-scale in vitro mass propagation of MD2 pineapple.展开更多
AIM: To investigate the expression of toll-like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells (KCs)and their role in ischemia-reperfusion (IR) injury of ratliver graft.METHODS: KCs were isolated at 0 (co...AIM: To investigate the expression of toll-like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells (KCs)and their role in ischemia-reperfusion (IR) injury of ratliver graft.METHODS: KCs were isolated at 0 (control group), 2, 12,24 h (IR group) following IR in rat liver graft, mRNA expressionof TLR4 and MD-2 was detected by RT-PCR analysis, proteinexpression of TLR4/MD-2 was detected by flow cytometric(FCM) analysis, and tumor necrosis factor-(~ (TNF-(~) levelin supernatant was measured by ELISA. Then isolated KCswere incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-(~ level was measured again.RESULTS: The mRNA and protein expression of TLR4/MD-2 and the level of TNF-(~ in IR group increased significantlyat 2 h following IR, and reached the maximum at 12 h, andslightly decreased at 24 h, but were still significantly higherthan those in the control group (P<O.01). The expression ofthese factors was markedly decreased after anti-TLR4antibody treatment as compared with the IR group (P<O.01).CONCLUSION: Lipopolysaccharide (LPS) following IR canup-regulate TLR4/MD-2 gene and protein expression inKCs, and synthesize cytokine TNF-(~. Anti TLR4 antibodycan inhibit the production of TNF-(~ induced by LPS. TLR4and its partner molecule MD-2 may play an important rolein Kupffer cell activation and IR injury.展开更多
基金Supported by Applied Research and Industrialization Projects of Key Science and Technology Plan of Hainan Province(ZDXM20130031)Special Fund for Agro-scientific Research in the Public Interest(201203021)+3 种基金Scientific Operation Fund of Hainan Province(QCY[2013]131)Natural Science Foundation of Hainan Province(QK[2013]32)Key Research and Development Project of Hainan Province(ZDYF2016035)Special Program for Agricultural Science and Technology Innovation of Hainan Academy of Agricultural Sciences(CXZX201410)
文摘Pineapple mealybug wilt associated virus-2 （PMWaV-2） is an important pathogen causing Mealybug wilt of pineapple （MWP）. We established a realtime quantitative RT-PCR （RT-qPCR） method with TaqMan probe based on specific primers of conserved nucleotide sequence of PMWaV-2 coat protein gene. The results showed that the method was higldy sensitive to positive sample, but had no fluorescence signal to health sample and blank control. The sensitivity of RTqPCR was about 100 times higher than general PCR. Reproducibihty test revealed that the coefficients of variation between intra-and inter-assay were beth within 1.85%, indicating it was a quantitative detection method for PMWaV-2 with simple operation, strong specificity, high sensitivity, and reliable reproducibility.
文摘The present study was conducted to investigate the effect of agitation rate on the increase in fresh weight of MD2 pineapple protocorm-like bodies (PLBs) and shoots cultured in liquid medium. PLBs were cultured in 250 ml Erlenmeyer flasks (7 g per flask) containing MS medium and plant growth regulators (1.5 mg/L 6-Benzylaminopurine, BAP and 0.2 mg/L 1-Naphthaleneacetic acid, NAA). The orbital shaker was set at speeds of 50, 80, 100, 120, and 150 rpm. After 40 days, the cultures shaken at 80 rpm showed the highest fresh weight and the highest number of shoots at 76 g and 41 shoots, respectively. A comparative study of agitation found that 80 rpm was the best speed which enhanced both PLB and shoot formation. The findings in the present study would be helpful in setting up large-scale in vitro mass propagation of MD2 pineapple.
文摘目的探讨脂多糖作用小鼠RAW264.7巨噬细胞24 h内细胞表面TLR4/CD14/MD-2受体复合物表达的变化特点。方法分别用低剂量(100 ng/ml)和高剂量(1 000 ng/ml)脂多糖刺激RAW264.7,应用RT-PCR检测TLR4、CD14、MD-2m RNA水平的变化,ELISA检测细胞上清TNF-α含量的变化。结果低剂量脂多糖下调TLR4 m RNA表达,14 h降至最低,24 h维持在低水平表达;CD14和MD-2 m RNA表达增加。高剂量脂多糖上调TLR4 m RNA表达,1 h上升至高峰,此后维持在高水平表达;CD14和MD-2 m RNA表达也明显增强。高剂量脂多糖作用后TNF-α含量显著高于低剂量脂多糖(P<0.05)。结论高剂量脂多糖逆转低剂量脂多糖下调的TLR4表达,提高TLR4/CD14/MD-2受体复合物的表达水平,放大下游信号转导通路的炎症效应。
基金Supported by the National Natural Science Foundation of China,No.30300337,30200278,30170919
文摘AIM: To investigate the expression of toll-like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells (KCs)and their role in ischemia-reperfusion (IR) injury of ratliver graft.METHODS: KCs were isolated at 0 (control group), 2, 12,24 h (IR group) following IR in rat liver graft, mRNA expressionof TLR4 and MD-2 was detected by RT-PCR analysis, proteinexpression of TLR4/MD-2 was detected by flow cytometric(FCM) analysis, and tumor necrosis factor-(~ (TNF-(~) levelin supernatant was measured by ELISA. Then isolated KCswere incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-(~ level was measured again.RESULTS: The mRNA and protein expression of TLR4/MD-2 and the level of TNF-(~ in IR group increased significantlyat 2 h following IR, and reached the maximum at 12 h, andslightly decreased at 24 h, but were still significantly higherthan those in the control group (P<O.01). The expression ofthese factors was markedly decreased after anti-TLR4antibody treatment as compared with the IR group (P<O.01).CONCLUSION: Lipopolysaccharide (LPS) following IR canup-regulate TLR4/MD-2 gene and protein expression inKCs, and synthesize cytokine TNF-(~. Anti TLR4 antibodycan inhibit the production of TNF-(~ induced by LPS. TLR4and its partner molecule MD-2 may play an important rolein Kupffer cell activation and IR injury.
