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Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica

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Horticulture Research 2020年第1期7卷 1561-1571页

作者： Lina Qiu Yongzhang Wang haiyong qu College of Horticulture Qingdao Agricultural UniversityQingdao CityChina

Cytosolic Ca^(2+)plays a key role in signal transduction in *** imaging is the most common approach to studying dynamic changes in the cytoplasmic Ca^(2+)***,we used mature‘Fuji’apples(Malus pumila Mill.)to obtain viable protoplasts from flesh tissue cells by enzymatic hydrolysis;then,three small-molecule fluorescent probes(fluo-8/AM,fluo-4/AM,and rhod-2/AM)were loaded into the *** three Ca^(2+)fluorescent probes successfully entered the cytoplasm but did not enter the *** the Ca^(2+)chelator(EGTA)and Ca^(2+)channel blocker(La3+)reduced the fluorescence intensity in the *** calcium ionophore A23187 increased the fluorescence intensity in the cytoplasm at 5μmol/L but decreased it at 50μmol/***,A23187 reversed the fluorescence intensity in the cytoplasm,which was decreased by La3+.Ionomycin is also a calcium ionophore that can increase the fluorescence intensity of calcium in the *** results suggest that small-molecule Ca^(2+)fluorescent probes can be used to detect changes in cytosolic calcium levels in the cells of fruit flesh *** addition,the optimum concentration of fluo-8/AM was determined to be 5μmol/*** was the first time that protoplasts have been isolated from apple flesh tissue cells and small-molecule fluorescent probes have been used to detect calcium in the cytoplasm of flesh tissue *** study provides a new method to study calcium signal transduction in fruit flesh tissue.

关键词： reversed probes hydrolysis

A Rapid Method for Isolating Single Cells from Apple Flesh

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Horticultural Plant Journal 2017年第2期3卷 47-52页

作者： GUAN Yaqin qu haiyong College of Horticulture Qingdao Agricultural University

The identification,separation and analysis of individual living cells can be used to analyze the heterogeneity and operation mechanism of living *** study of fruit development is based on the extraction of active single *** this study,we investigated the effects of different enzymes and enzymatic hydrolysis times on the extraction of single cells from the‘Fuji’apple(Malus×domestica Borkh.‘Fuji’).The results showed that the extraction of single cells in apple flesh was a suitable method when 0.1%macerozyme was used and the enzymolysis time was 0.5 *** brightening agent VBL staining showed that the cell wall was intact,while fluorescein diacetate FDA and azo dye Evans blue staining indicated that the extracted single cells were *** extracted single cells could be further used as materials for protoplast extraction.

关键词： apple flesh single cells enzyme liquid concentration enzymolysis time

Calcium channel identification,modulation and function in the pollen tube of Pyrus pyrifolia

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Calcium channel identification,modulation and function in th...

2013全国植物生物学大会

作者： WU Juyou qu haiyong WU Jun TAO Shutian HUANG Xiaoshan ZHANG Shaoling College of Horticulture State Key Laboratory of Crop Genetics and Germplasm EnhancementPear Engineering Research CentreNanjing Agricultural University

The pollen tube has been widely usedas a model to study signal transduction in *** tip-high free cytosolic calcium([Ca])gradient is essential for pollen tube *** Ca influx mediated by plasma membrane Ca channels is involved in maintain pollen tube[Ca] *** investigate the molecular mechanism of pear pollen tube plasma membrane Ca channels,we have developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus *** using patch clamp technique,we have further identified a hyperpolarization activated calcium channel in the *** calcium channel displayed a strong selectivity for divalent cations,with a relative permeability sequence of Ba≈Ca>*** also showed that the channel was inhibited by the Ca channel blockers ***,channel activity depended on extracellular pH and pollen viability. In the next step,we tested how to modulate this channel by chemicals such as spermidine (Spd).Spd has been correlated with pollen tube *** our testing,Spd induces an increase in the[Ca] in the pollen ***,exogenous Spd induces the hyperpolarization activated Ca current:the addition of Spd cannot induce the channel open probability increase in excised outside-out patches,indicating that the effect of Spd in the induction of Ca currents is exerted via a second *** our further study,we found this messenger is hydrogen peroxide(HO),and is generated during Spd oxidation,a reaction mediated by polyamine oxidase(PAO).These reactive oxygen species directly trigger the opening of the hyperpolarization activated Ca channels in *** provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca channels,two Arabidopsis mutants lacking AtPAO3 gene were isolated and *** from these mutants was unable to induce the opening of the Ca channels in the presence of Spd,resulting in reduced pollen tube growth and seed ***,a high Spd concentr

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负压渗透处理对花粉管加载荧光染料的影响

西北植物学报 2018年第11期38卷 2138-2147页

作者：李坤王永章屈海泳青岛农业大学园艺学院山东青岛266109

该研究采用负压渗透技术,以正常培养2h的丰水梨花粉为实验材料,探索负压渗透条件下,花粉管中加载Ca^(2+)荧光探针(Fluo-4/AM)的方法。结果显示:(1)将花粉及花粉管进行负压处理2h,花粉萌发率及花粉管的活性没有受到影响。(2)对培养2h后... 详细信息

该研究采用负压渗透技术,以正常培养2h的丰水梨花粉为实验材料,探索负压渗透条件下,花粉管中加载Ca^(2+)荧光探针(Fluo-4/AM)的方法。结果显示:(1)将花粉及花粉管进行负压处理2h,花粉萌发率及花粉管的活性没有受到影响。(2)对培养2h后的花粉管进行不同条件下的负压渗透处理,辅助荧光探针Fluo-4/AM进入花粉管;激光共聚焦显微镜观察发现,在低温(4℃)条件下,负压(-80kPa)渗透加载荧光探针30min,花粉管尖端可以观察到明显的Ca^(2+)梯度。(3)抑制花粉管外Ca^(2+)内流或降低花粉管外Ca^(2+)浓度,花粉管中荧光密度也显著降低。研究认为,负压渗透辅助加载的方法可以有效促进荧光探针进入花粉管细胞内与Ca^(2+)结合。

关键词： Ca^2+荧光探针 Ca^2+梯度花粉管激光共聚焦负压渗透

不同质地的‘金帅’无锈芽变果肉细胞活性染色观察比较

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果树学报 2018年第6期35卷 702-710页

作者：黄译瑾屈海泳王永章杨绍兰原永兵青岛农业大学园艺学院山东青岛266109

【目的】比较不同质地的‘金帅’无锈芽变果实徒手切片、染色差异和荧光活性,以期寻找区分不同质地芽变果实新方法。【方法】利用直接对比、显微观察、荧光染色等方法对不同质地果实进行区分。【结果】贮藏后的‘金帅’无锈芽变果实可... 详细信息

【目的】比较不同质地的‘金帅’无锈芽变果实徒手切片、染色差异和荧光活性,以期寻找区分不同质地芽变果实新方法。【方法】利用直接对比、显微观察、荧光染色等方法对不同质地果实进行区分。【结果】贮藏后的‘金帅’无锈芽变果实可通过感官评测分为硬、软、绵不同质地;随质地变软,观察单一层次细胞排列越易,细胞变形程度越大,细胞层堆叠程度越小;染色后不同质地果实具有不同表现,其中荧光素二乙酸(FDA)区分不同质地果实效果最佳,质地硬、软、绵、无活性的细胞荧光值差异极显著。【结论】活性染色可以用于区分不同质地的‘金帅’无锈芽变果实,FDA染色后质地硬的果肉细胞活性最强,质地软的果肉细胞活性中等,质地绵的最弱。

关键词： '金帅’无锈芽变质地果肉细胞染色活性

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冷藏温度对蓝莓果实品质的影响

安徽农业大学学报 2014年第5期41卷 871-874页

作者：屈海泳刘连妹张旻倩叶正文青岛农业大学园艺学院青岛266109 淮阴工学院生命科学与化学工程学院淮安223003 上海农业科学院上海201403

采用不同的冷藏温度贮藏蓝莓,以蓝莓的失重率、腐烂率、可溶性固形物的含量、果皮的色差及维生素C的含量等为参考指标,研究冷藏下蓝莓品质的变化。结果显示1℃-3℃低温冷藏蓝莓,经60 d长期检测,低温显著抑制了果实的失重率,腐烂率和Vc... 详细信息

采用不同的冷藏温度贮藏蓝莓,以蓝莓的失重率、腐烂率、可溶性固形物的含量、果皮的色差及维生素C的含量等为参考指标,研究冷藏下蓝莓品质的变化。结果显示1℃-3℃低温冷藏蓝莓,经60 d长期检测,低温显著抑制了果实的失重率,腐烂率和Vc含量的下降,较好的保持了果实原有色泽,显著抑制了果实可滴定酸含量的下降,维持了果实原有风味,保持了果实的营养价值。

关键词：蓝莓冷藏温度品质

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桃花粉活性检测和离体萌发特性的研究

中国农学通报 2010年第21期26卷 207-212页

作者：屈海泳刘连妹孔文涛叶正文淮阴工学院园艺与景观工程系江苏淮安223001 上海农业科学院林木果树研究所上海市设施园艺技术重点试验室上海201106

果树花粉活力直接影响授粉、受精乃至坐果。为了解果树品种的花粉生活力,给引种栽培和杂交育种工作创造有利条件,以桃花粉为试验材料,采用染色方法检测花粉的活性。试验结果表明,含有蓝墨水的花粉培养基,花粉萌发率可以达到64.67%,与对... 详细信息

果树花粉活力直接影响授粉、受精乃至坐果。为了解果树品种的花粉生活力,给引种栽培和杂交育种工作创造有利条件,以桃花粉为试验材料,采用染色方法检测花粉的活性。试验结果表明,含有蓝墨水的花粉培养基,花粉萌发率可以达到64.67%,与对照相比差异不明显,因此该方法比较适合于快速检测花粉活性;固体培养基培养2个品种桃花粉,萌发率比液体培养基培养分别高25%和20%。另外温度、花粉密度、培养基中蔗糖及硼酸浓度都对花粉萌发率和花粉管长度有影响,在温度为24℃、1粒花药/100μL、10%的蔗糖或100mg/L的硼酸的条件下,均有利于花粉萌发和花粉管生长。

关键词：桃花粉活力染色方法萌发率花粉管长度

木霉菌对苹果主要真菌病害的抑制及其在土壤中的定殖作用

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北方园艺 2021年第6期 15-22页

作者：张莉莉刘美媛王永章原永兵屈海泳青岛农业大学园艺学院山东青岛266109

以胶东半岛老的果园中土壤为试材,采用逐步稀释法,涂平板于孟加拉红培养基中,根据形态和分子方法鉴定分离木霉菌。分离出的木霉菌与苹果主要病害对峙培养在不同pH的培养基上,观察不同pH条件下,对真菌病害的抑制效果。土壤有机质和pH是... 详细信息

以胶东半岛老的果园中土壤为试材,采用逐步稀释法,涂平板于孟加拉红培养基中,根据形态和分子方法鉴定分离木霉菌。分离出的木霉菌与苹果主要病害对峙培养在不同pH的培养基上,观察不同pH条件下,对真菌病害的抑制效果。土壤有机质和pH是目前胶东果园的主要问题,采用正交法设计,研究了不同的土壤条件对木霉菌生长的影响。结果表明:在不同pH条件下,木霉菌抑制苹果病原真菌的效果不同,木霉菌在不同的pH条件下对腐烂病(Valsa mali)和炭疽病(Colletotrichum gloeosporioides)均可以抑制,但对轮纹病(Physalospora piricola Nose)只有在pH 6.0时没有效果外,其它pH时都对轮纹病有抑制作用。在土壤pH 6.5和土壤有机质含量为75 g·kg^(-1)时,最有利于木霉菌定殖。同时还发现木霉菌对土壤的pH具有调节作用,无论在碱性条件下,还是酸性条件下,均可以使土壤pH趋于中性,另外木霉菌可以增加土壤有机质含量,特别是在高土壤有机质含量的情况下,增加更加显著。总之,分离出的木霉菌对苹果腐烂病、炭疽病和轮纹病抑制效果好。土壤pH和有机质含量对木霉菌的生长有影响,高土壤有机质含量和中性土壤有利于木霉菌定殖。另外,木霉菌能有效地改良土壤有机质含量和土壤pH。

关键词：木霉菌苹果轮纹病腐烂病炭疽病抑制

高校以农业开发项目为载体在“三农”发展中的作用研究

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科教导刊 2016年第2期 20-21页

作者：马春晖刘连妹屈海泳青岛农业大学园艺学院山东青岛266109 淮阴工学院科技处江苏淮安223001

本文论述了地方性高校在地方"三农"发展中所起的作用,以农业开发项目为依托,产学研结合,推动地方农业的可持续发展,完善了地方农业技术推广体系,并且锻炼了高校教师队伍,从客观上又增强了科研团队建设。

关键词：高校农业开发项目三农