根结线虫活体染色条件的优化
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摘要
【目的】为了提高根结线虫的成活率。【方法】以番茄和南方根结线虫为试验材料,对根结线虫二龄幼虫(J2)进行荧光标记,改进标记溶液浓度,进行染液浓度梯度和染色时间的筛选,确定最佳的染液浓度和染色时间,在保证线虫有效标记的前提下,不影响线虫的活力和致病性,深入了解线虫的行为。【结果】FITC:0.025mg/mL、0.25%间苯二酚、15mol/L章鱼胺盐酸盐缓冲液浓度下标记3h,线虫可以保持活力,入侵植株。【结论】该方法降低了染液浓度,缩短了染色时间,减少试剂用量,增加了线虫的存活率。
【Objective】In order to increase the survival rate of root knot nematodes.【Methods】The tomato and Mi.ncognita were used as test materials to identify the nematode fluorescence of the second instar larvae(J2)of root-knot nematode,improve the solution concentration of labeled nematodes,and screen the stain concentration gradient and staining time to determine.Under the premise of effective labeling of nematodes,it doesnot affect the vitality and pathogenicity of the nematodes to determine the optimal staining concentration and time,learning more about the behavior of nematodes.【Results】When FITC:0.025 mg/mL,0.25% resorcinol,15 mol/L octopamine are marked for 3 hat the hydrochloride buffer concentration,the nematode can remain viable and invade the plants.【Conclusion】This method reduces the concentration of dye solution,shortens the dyeing time,reduces reagent usage and increases the survival rate of nematodes.
【Objective】In order to increase the survival rate of root knot nematodes.【Methods】The tomato and Mi.ncognita were used as test materials to identify the nematode fluorescence of the second instar larvae(J2)of root-knot nematode,improve the solution concentration of labeled nematodes,and screen the stain concentration gradient and staining time to determine.Under the premise of effective labeling of nematodes,it doesnot affect the vitality and pathogenicity of the nematodes to determine the optimal staining concentration and time,learning more about the behavior of nematodes.【Results】When FITC:0.025 mg/mL,0.25% resorcinol,15 mol/L octopamine are marked for 3 hat the hydrochloride buffer concentration,the nematode can remain viable and invade the plants.【Conclusion】This method reduces the concentration of dye solution,shortens the dyeing time,reduces reagent usage and increases the survival rate of nematodes.
引文
[1]Hussey R S,Janssen G J W.Root-knot nematodes:Meloidogyne species[A].In Starr J L,Cook R,BridgeJ editors.Plant resistance to parasitic nematodes[C].Wallingford:CABInterntional,2002:43-70
[2]刘国坤,肖顺,张绍升,等.拟禾本科根结线虫对水稻根系的侵入特性及其生活史[J].热带作物学报,2011,32(4):743-748
[3]Li X,Zhao W,Gao Y,et al.The use of toluidine blue staining combined with paraffin sectioning and the optimization of freeze-thaw counting methods foranalysing root-knot nematodes in tomato[J].Horticulture,Environment and Biotechnology,2017,58(6):620-626
[4]刘奇志,李娜,王丽,等.南方根结线虫侵入黄瓜幼根组织病理学观察[J].中国农业大学学报,2008,13(4):51-56
[5]Rosso M N,Dubrana M P,Cimbolini N,et al.Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins[J].Molecular Plant-Microbe Interactions,2005,18(7):615-620
[6]王佳佳,周桦,张进,等.土壤样品中微生物活性的荧光分析方法[J].环境化学,2012,31(10):1637-1643
[7]Linlin Dong,Xiaolin Li,Li Huang,et al.Lauric acid in crown daisy root exudate potently regulates root-knot nematode chemotaxis and disrupts Mi-flp-18expression to block infection[J].Journal of Experimental Botany,2014,65(1):131-141
[8]逯晓青,赵量,马昕,等.异硫氰酸荧光素标记膀胱癌靶向肽NYZL1的体外特异性鉴定[J].中国药物临床,2018,18(9):1478-1481
[9]Jo-Anne CHTan,Michael GKJones,John Fosu-Nyarko.Gene silencing in root lesion nematodes(Pratylenchus spp.)significantlyreduces reproduction in a plant host[J].Experimental Parasitology,2012,13(2013):166-178
[2]刘国坤,肖顺,张绍升,等.拟禾本科根结线虫对水稻根系的侵入特性及其生活史[J].热带作物学报,2011,32(4):743-748
[3]Li X,Zhao W,Gao Y,et al.The use of toluidine blue staining combined with paraffin sectioning and the optimization of freeze-thaw counting methods foranalysing root-knot nematodes in tomato[J].Horticulture,Environment and Biotechnology,2017,58(6):620-626
[4]刘奇志,李娜,王丽,等.南方根结线虫侵入黄瓜幼根组织病理学观察[J].中国农业大学学报,2008,13(4):51-56
[5]Rosso M N,Dubrana M P,Cimbolini N,et al.Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins[J].Molecular Plant-Microbe Interactions,2005,18(7):615-620
[6]王佳佳,周桦,张进,等.土壤样品中微生物活性的荧光分析方法[J].环境化学,2012,31(10):1637-1643
[7]Linlin Dong,Xiaolin Li,Li Huang,et al.Lauric acid in crown daisy root exudate potently regulates root-knot nematode chemotaxis and disrupts Mi-flp-18expression to block infection[J].Journal of Experimental Botany,2014,65(1):131-141
[8]逯晓青,赵量,马昕,等.异硫氰酸荧光素标记膀胱癌靶向肽NYZL1的体外特异性鉴定[J].中国药物临床,2018,18(9):1478-1481
[9]Jo-Anne CHTan,Michael GKJones,John Fosu-Nyarko.Gene silencing in root lesion nematodes(Pratylenchus spp.)significantlyreduces reproduction in a plant host[J].Experimental Parasitology,2012,13(2013):166-178