CN107271351A - 一种花瓣细胞中钙离子测定方法 - Google Patents
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 229910001424 calcium ion Inorganic materials 0.000 title claims abstract description 36
- 238000003556 assay Methods 0.000 title claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 26
- 238000005286 illumination Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 8
- 210000001339 epidermal cell Anatomy 0.000 claims abstract description 6
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- 210000002615 epidermis Anatomy 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 4
- 238000006073 displacement reaction Methods 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000004575 stone Substances 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims 1
- 239000011575 calcium Substances 0.000 abstract description 10
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 9
- 229910052791 calcium Inorganic materials 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 230000010355 oscillation Effects 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241000234435 Lilium Species 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000009131 signaling function Effects 0.000 description 2
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
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Abstract
本发明公开了一种花瓣细胞中钙离子测定方法,该方法通过非损伤微测技术测定不同光强下花瓣细胞中钙离子流,钙离子流速均为负值,表明花瓣表皮细胞中钙离子体表现为内流。在黑暗环境中,钙离子流均处于较平稳状态,施加不同强度的光强,钙离子的内流表现出不同程度增大的趋势,且光照越强,内流的速度越大。通过该方法解决了现有技术中无法获得花瓣细胞中钙信号的作用的问题,为花的培养及繁殖提供了新思路。
Description
技术领域
本发明涉及一种花瓣细胞中钙离子测定方法。
背景技术
钙离子作为一种非常重要的信号分子,不仅调节植物细胞内的多种生理活动,还参与细胞对外界环境的响应过程。钙离子信号主要通过细胞质和细胞器中钙离子分布以及浓度的变化而产生。植物中,多种生物或非生物胁迫作用都会导致细胞质钙离子浓度的瞬间变化,触发钙响应的下游反应。
目前,对于花瓣中钙信号的研究较少,对于其信号作用还不清楚。花瓣作为植物重要的器官,对于植物的繁殖以及观赏价值具有重要意义;因此,研究花瓣细胞中的钙信号具有重要意义。
发明内容
针对现有技术中存在的问题,本发明的目的在于提供一种花瓣细胞中钙离子测定方法,其有效解决了现有技术中存在的问题。
为实现上述目的,本发明采用以下技术方案:
一种花瓣细胞中钙离子测定方法,所述方法包括如下步骤:
1)选择盛开期花朵的花瓣,用镊子轻轻撕取1cm2表皮,迅速浸泡在测试缓冲液中,用彩色石粒轻压,浸泡30min,使表皮细胞离子跨膜交换达到平衡;
2)将浸泡后的表皮放入新的测试缓冲液中,然后置于非损伤微测系统的显微镜下;在显微镜下观察表皮边缘,寻找细胞结构完整的区域,选择形态良好的细胞作为测点;
3)将电极对准测点,距离细胞2μm,电极移动距离为30μm,5s记录一个数据;
4)将冷光源放在屏蔽罩中,用可调光强的软管LED冷光源控制光强,照射测点,用照度计检测光强,首先在黑暗条件下测定3min,黑暗条件下测试完成后打开冷光源设备开关,进行光照条件下的测定,设置100μmol/m2·s、300μmol/m2·s、600μmol/m2·s、1000μmol/m2·s、1500μmol/m2·s 5个光强梯度,每个梯度分别测定7min,重复5次,并分别记录不同光照强度下每次测定的测点内钙离子流速。
进一步,所述测试缓冲液的pH为5.8,其含有0.1mM的KCl,0.1mM的CaCl2,0.3mM的MES。
本发明具有以下有益技术效果:
本发明通过非损伤微测技术测定不同光强下花瓣细胞中钙离子流,钙离子流速均为负值,表明花瓣表皮细胞中钙离子体表现为内流。在黑暗环境中,钙离子流均处于较平稳状态,施加不同强度的光强,钙离子的内流表现出不同程度增大的趋势,且光照越强,内流的速度越大。通过该方法解决了现有技术中无法获得花瓣细胞中钙信号的作用的问题,为花的培养及繁殖提供了新思路。
附图说明
图1为花瓣细胞中钙离子对光照强度的响应图。
具体实施方式
下面,参考附图,对本发明进行更全面的说明,附图中示出了本发明的示例性实施例。然而,本发明可以体现为多种不同形式,并不应理解为局限于这里叙述的示例性实施例。而是,提供这些实施例,从而使本发明全面和完整,并将本发明的范围完全地传达给本领域的普通技术人员。
本发明提供了一种花瓣细胞中钙离子测定方法,该方法包括如下步骤:
1)选择盛开期花朵的花瓣,用镊子轻轻撕取1cm2表皮,迅速浸泡在测试缓冲液中,用彩色石粒轻压,浸泡30min,使表皮细胞离子跨膜交换达到平衡;
2)将浸泡后的表皮放入新的测试缓冲液中,然后置于非损伤微测系统的显微镜下;在显微镜下观察表皮边缘,寻找细胞结构完整的区域,选择形态良好的细胞作为测点;
3)将电极对准测点,距离细胞2μm,电极移动距离为30μm,5s记录一个数据;电极用来测定离花瓣不同距离两个点的钙离子浓度,最后转化为钙离子流动速度;
4)将冷光源放在屏蔽罩中,用可调光强的软管LED冷光源控制光强,照射测点,用照度计检测光强,首先在黑暗条件下测定3min,黑暗条件下测试完成后打开冷光源设备开关,进行光照条件下的测定,设置100μmol/m2·s、300μmol/m2·s、600μmol/m2·s、1000μmol/m2·s、1500μmol/m2·s 5个光强梯度,每个梯度分别测定7min,重复5次,并分别记录不同光照强度下每次测定的测点内钙离子流速。
测试缓冲液的pH为5.8,其含有0.1mM的KCl,0.1mM的CaCl2,0.3mM的MES。
如图1所示,通过非损伤微测技术测定不同光强下百合花瓣细胞中钙离子流,Ca2+流速均为负值,表明百合花瓣表皮细胞Ca2+总体表现为内流。在黑暗环境中,Ca2+流均处于较平稳状态(图中阴影部分),施加不同强度的光强,Ca2+的内流表现出不同程度增大的趋势,且光照越强,内流的速度越大。100μmol/m2·s光照下,Ca2+平均内流速度较黑暗条件下增幅在100pmol/cm2·s以内,变化不明显。而光强从0μmol/m2·s增加到300μmol/m2·s时,Ca2+平均流速由增加了近一倍。当分别施加600、1000、1500μmol/m2·s照度时,内流均迅速增加。当受到600μmol/m2·s光强照射后,与黑暗处理相比,Ca2+内流速度增加了一倍。但随着光强增加到1000和1500μmol/m2·s,Ca2+内流速度虽然也呈现增加的趋势,但是与600μmol/m2·s光强处理没有明显差异。并且在600、1000、1500μmol/m2·s照度下,Ca2+内流的速度达到最大后又逐渐回落。
本申请中的屏蔽罩是非损伤微测系统的一部分,用于屏蔽外界电流的影响。非损伤微测系统的用来测定离子流的成熟系统;本申请的主要目的是解决了在研究光对花瓣组织生理生化的影响中,作为早期信号的钙离子流的测定方法。本发明通过非损伤微测技术为研究花瓣中钙离子对光信号的响应规律,及其信号作用提供了新思路。
上面所述只是为了说明本发明,应该理解为本发明并不局限于以上实施例,符合本发明思想的各种变通形式均在本发明的保护范围之内。
Claims (2)
1.一种花瓣细胞中钙离子测定方法,其特征在于,所述方法包括如下步骤:
1)选择盛开期花朵的花瓣,用镊子轻轻撕取1cm2表皮,迅速浸泡在测试缓冲液中,用彩色石粒轻压,浸泡30min,使表皮细胞离子跨膜交换达到平衡;
2)将浸泡后的表皮放入新的测试缓冲液中,然后置于非损伤微测系统的显微镜下;在显微镜下观察表皮边缘,寻找细胞结构完整的区域,选择形态良好的细胞作为测点;
3)将电极对准测点,距离细胞2μm,电极移动距离为30μm,5s记录一个数据;
4)将冷光源放在屏蔽罩中,用可调光强的软管LED冷光源控制光强,照射测点,用照度计检测光强,首先在黑暗条件下测定3min,黑暗条件下测试完成后打开冷光源设备开关,进行光照条件下的测定,设置100μmol/m2·s、300μmol/m2·s、600μmol/m2·s、1000μmol/m2·s、1500μmol/m2·s 5个光强梯度,每个梯度分别测定7min,重复5次,并分别记录不同光照强度下每次测定的测点内钙离子流速。
2.根据权利要求1所述的花瓣细胞中钙离子测定方法,其特征在于,所述测试缓冲液的pH为5.8,其含有0.1mM的KCl,0.1mM的CaCl2,0.3mM的MES。
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CN203908989U (zh) * | 2014-05-09 | 2014-10-29 | 中南大学 | 一种钙离子动态监测实验装置 |
CN106171579A (zh) * | 2016-07-18 | 2016-12-07 | 甘肃农业大学 | 外源油菜素内酯处理缓解玉米幼苗镉逆境胁迫伤害的方法 |
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CN203908989U (zh) * | 2014-05-09 | 2014-10-29 | 中南大学 | 一种钙离子动态监测实验装置 |
CN106171579A (zh) * | 2016-07-18 | 2016-12-07 | 甘肃农业大学 | 外源油菜素内酯处理缓解玉米幼苗镉逆境胁迫伤害的方法 |
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ZENGHUI HU等: "Ca2+ signal contribution to the synthesis and emission of monoterpenes regulated by light intensity in lilium‘siberia’", 《PLANTS PHYSIOLOGY AND BIOCHEMISTRY》 * |
尹丽: "动态离子流检测小麦耐盐性的研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
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