CN117821659A - 一种油松基因表达与定位检测方法 - Google Patents
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Abstract
本发明公开了一种油松基因表达与定位检测方法，包括以下步骤：S1.将油松下胚轴组织侵染；S2.将步骤S1所得下胚轴组织进行水凝胶切片；S3.使用共聚焦显微镜对步骤S2所得切片进行层扫拍摄；S4.利用Imaris软件对侵染的油松下胚轴组织进行基因的三维空间定位解析与表达量化分析。具有稳定、易于检测、灵敏度高、具有广谱性等优势，激光扫描共聚焦显微镜可以提供精确的三维成像、准确的细胞结构及动态过程。
Description
技术领域
本发明涉及植物基因工程技术领域，尤其涉及一种高分辨、多维度的油松基因表达与定位检测方法。
背景技术
油松(Pinus tabuliformis Carr.)属于松科松属，是我国重要的本土针叶树种，广泛分布于我国西北、华北和东北部分地区。油松童期相对较短、适生区辽阔，是进行针叶树生长发育关键基因筛选、功能验证及调控机制研究的理想材料。此外，油松是重要的经济林木之一，在木材生产和森林保护方面具有重要价值。
油松等针叶树具有复杂的遗传背景、庞大的基因组和较长的营养生长期，严重阻碍了利用遗传学方法进行功能基因组研究。随着高通量测序技术的革新和生物信息学的飞速发展，2022年油松完成全基因组测序，为完成油松生长发育、代谢调控、胁迫响应等生物学研究提供了宝贵的资源。植物遗传转化是开展基因工程育种和鉴定基因功能的重要途径，种子植物尤其是针叶树受限于遗传转化技术，稳定的遗传转化植物很难获得，而农杆菌介导的瞬时表达系统操作简单、转化效率高，被广泛应用于植物功能基因的研究。
在油松下胚轴通过农杆菌侵染进行基因瞬时表达的方法已经被报道，通过携带GFP标签，具有稳定、易于检测、灵敏度高、具有广谱性等优势。
激光扫描共聚焦显微镜可以提供精确的三维成像、准确的细胞结构及动态过程。
因此本领域技术人员致力于开发一种高分辨、多维度的油松基因表达与定位检测方法。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是提供一种高分辨、多维度的油松基因表达与定位检测方法。
为实现上述目的，本发明提供一种油松基因表达与定位检测方法，包括以下步骤：
S1.将油松下胚轴组织侵染；
S2.将步骤S1所得下胚轴组织进行水凝胶切片；
S3.使用共聚焦显微镜对步骤S2所得切片进行层扫拍摄；
S4.利用Imaris软件对侵染的油松下胚轴组织进行基因的三维空间定位解析与表达量化分析。
进一步的，步骤S1中，油松下胚轴组织侵染具体为：将萌发一周，子叶还未完全展开的油松幼苗切根，将含有目的基因的融合荧光蛋白载体转入农杆菌感受态，将得到的下胚轴组织在农杆菌感受态菌液中浸泡侵染30-40min，侵染后移至土中，黑暗条件下培养1天，然后转入23℃、16小时光照、8小时黑暗条件继续培养2-4天。
进一步的，步骤S2中，取距离侵染位置1cm的油松下胚轴切下，用预冷的固定液置于冰上抽真空进行固定，将缓冲液稀释10倍成1×PBS溶液，用预冷的1×PBS溶液冲洗3次，每次10min，将低熔点琼脂糖调制成质量浓度为5％的低熔点琼脂糖悬浊液，用5％的低熔点琼脂糖悬浊液进行包埋，用切片机进行切片，切片厚度为50μm。
进一步的，步骤S3中，使用共聚焦激光扫描显微镜对油松下胚轴切片进行层扫拍摄，使用488nm进行GFP荧光蛋白成像，接收光谱范围控制为510-533nm。
进一步的，使用Gating门控调整显微镜高速相机光子寿命接收阈值，以避开叶绿体自发荧光，GFP光子接收时间为0.3ns至2.6ns。
进一步的，使用荧光光谱拆分模块对样品荧光光谱进行扫描，对GFP与叶绿体等自发光进行拆分。
进一步的，步骤S4中，利用Surface模块进行细胞膜和基因表达信号的三维渲染，获取基因三维空间定位。
进一步的，计算渲染后基因表达各部分的各自体积和表面积，以及荧光的Max、Mean、Min和StdDev值，确定最后基因表达水平。
本发明的有益效果是：本发明的油松基因表达与定位检测方法，包括以下步骤：S1.油松下胚轴组织侵染；S2.下胚轴组织进行水凝胶切片；S3.使用共聚焦显微镜对切片进行层扫拍摄；S4.利用Imaris软件进行基因的三维空间定位解析与表达量化分析。具有稳定、易于检测、灵敏度高、具有广谱性等优势，激光扫描共聚焦显微镜可以提供精确的三维成像、准确的细胞结构及动态过程。
附图说明
图1是正常油松488nm激光激发后下胚轴发射光谱与GFP荧光蛋白发射光谱对比图；
图2是正常油松在488nm激光下胚轴发射荧光光谱拆分图；
图3是油松下胚轴PtPR5-GFP共聚焦成像和荧光光谱拆分结果图；
图4是油松下胚轴PtPR5-GFP荧光定位分析图；
图5是PtPR5-GFP荧光定量分析结果示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本说明书中公开的所有特征，或公开的所有方法或过程中的步骤，除了相互排斥的特质和/或步骤以外，均可以以任何方式组合，除非特别叙述，均可被其他等效或具有类似目的的替代特征加以替换，即，除非特别叙述，每个特征之一系列等效或类似特征中的一个实施例而已。
本发明所采用的主要试剂：
固定液：品牌：coolaber，品名：4％组织细胞固定液/PFA固定液(RNase Free)，货号：SL18301；
缓冲液：品牌：biosharp，品名：10×PBS缓冲液，货号：BL551A；
低熔点琼脂糖:品牌：coolaber，品名：LM Agarose，货号：CA1351；
农杆菌感受态：品牌：唯地生物，品名：GV3101，货号：AC1001。
本发明所采用的主要仪器：切片机为徕卡震荡切片机VT1200S，共聚焦激光扫描显微镜为徕卡共聚焦激光扫描显微镜SP8。
实施例1：一种油松基因表达与定位检测方法，包括以下步骤：
S1.将油松下胚轴组织侵染：
将萌发一周，子叶还未完全展开的油松幼苗切根，将含有PtPR5基因的融合GFP荧光蛋白载体转入农杆菌感受态，将得到的下胚轴组织在农杆菌感受态菌液中浸泡侵染30-40min，侵染后移至土中，黑暗条件下培养1天，然后转入23℃、16小时光照、8小时黑暗条件继续培养2-4天。
S2.下胚轴组织进行水凝胶切片：
取距离侵染位置约1cm的油松下胚轴切下，用预冷的固定液置于冰上抽真空进行固定，将缓冲液稀释10倍成1×PBS溶液，用预冷的1×PBS溶液冲洗3次，每次10min，将低熔点琼脂糖调制成质量浓度为5％的低熔点琼脂糖悬浊液，用5％的低熔点琼脂糖悬浊液进行包埋，用徕卡震荡切片机VT1200S进行切片，切片厚度为50μm。
S3.使用共聚焦显微镜对步骤S2所得切片进行层扫拍摄：
使用488nm进行GFP荧光蛋白成像，接收光谱范围控制为510-533nm。使用Gating门控调整显微镜高速相机光子寿命接收阈值，以避开叶绿体自发荧光，GFP光子接收时间为0.3ns至2.6ns。使用荧光光谱拆分功能模块对样品荧光光谱进行扫描，对GFP与叶绿体等自发光进行拆分。
S4.利用Imaris软件对侵染的油松下胚轴组织进行基因的三维空间定位解析与表达量化分析：
利用surface功能模块进行细胞膜和基因定位的三维渲染，获取基因三维空间定位。计算渲染后基因表达各部分的各自体积和表面积，以及荧光的Max、Mean、Min和StdDev值，确定最后PtPR5基因的表达水平。
结果如附图1至附图5所示，其中附图1表示正常油松488nm激光激发后下胚轴发射光谱与GFP发射光谱对比图，这是油松下胚轴在莱卡共聚焦显微镜下的成像，使用488nm激光进行激发。上图红色虚线是利用莱卡相机的光谱扫描功能，将488nm激光激发后，产生胚轴自发荧光所有光谱进行统计图。横坐标代表发射光的波长(nm)，纵坐标代表百分比。绿色图案代表由于488nm激发GFP产生的发射峰。对比两个发射峰图可知，自发荧光存在525/600/675nm三个荧光峰，其中525nm和GFP荧光发射峰重合，严重影响GFP荧光成像。因此，需要把在525nm发射出的自发光荧光拆分。
附图2是正常油松在488nm激光下胚轴发射荧光拆分图，同图1，自发荧光存在525/600/675nm三个荧光峰，图像中荧光较强。由于GFP接受范围设置在505-530nm之间，因此后面的荧光不做考虑，仅拆分525nm附近的荧光。
附图3是油松下胚轴PtPR5-GFP共聚焦成像和荧光拆分结果图，图片从左到右依次为融合、GFP和明场图。
附图4是油松下胚轴PtPR5-GFP荧光定位分析图，图3放大后，PtPR5-GFP亚细胞定位图，由图可知GFP在细胞内荧光分布在细胞壁、液泡和细胞外，且荧光强度为细胞外大于液泡大于细胞壁。
附图5是PtPR5-GFP荧光定量分析结果示意图，利用Imaris软件对原始荧光信号进行surface表面识别和重建，然后对细胞壁，细胞间隙，液泡分别对荧光最大值Max、最小值Min、平均值Mean、中位数Median、标准偏差StdDev和总量值Sum进行量化分析。
本发明提供了以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思做出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
Claims (8)
1.一种油松基因表达与定位检测方法，其特征在于：包括以下步骤：
S1.将油松下胚轴组织侵染；
S2.将步骤S1所得下胚轴组织进行水凝胶切片；
S3.使用共聚焦显微镜对步骤S2所得切片进行层扫拍摄；
S4.利用Imaris软件对侵染的油松下胚轴组织进行基因的三维空间定位解析与表达量化分析。
2.如权利要求1所述的油松基因表达与定位检测方法，其特征在于：步骤S1中，油松下胚轴组织侵染具体为：将萌发一周，子叶还未完全展开的油松幼苗切根，将含有目的基因的融合荧光蛋白载体转入农杆菌感受态，将得到的下胚轴组织在农杆菌感受态菌液中浸泡侵染30-40min，侵染后移至土中，黑暗条件下培养1天，然后转入23℃、16小时光照、8小时黑暗条件继续培养2-4天。
3.如权利要求1所述的油松基因表达与定位检测方法，其特征在于：步骤S2中，取距离侵染位置1cm的油松下胚轴切下，用预冷的固定液置于冰上抽真空进行固定，将缓冲液稀释10倍成1×PBS溶液，用预冷的1×PBS溶液冲洗3次，每次10min；将低熔点琼脂糖调制成质量浓度为5％的低熔点琼脂糖悬浊液，用5％的低熔点琼脂糖悬浊液进行包埋，用切片机进行切片，切片厚度为50μm。
4.如权利要求1所述的油松基因表达与定位检测方法，其特征在于：步骤S3中，使用共聚焦激光扫描显微镜对油松下胚轴切片进行层扫拍摄，使用488nm进行GFP荧光蛋白成像，接收光谱范围控制为510-533nm。
5.如权利要求4所述的油松基因表达与定位检测方法，其特征在于：使用Gating门控调整显微镜高速相机光子寿命接收阈值，以避开叶绿体自发荧光，GFP光子接收时间为0.3ns至2.6ns。
6.如权利要求4所述的油松基因表达与定位检测方法，其特征在于：使用荧光光谱拆分模块对样品荧光光谱进行扫描，对GFP与叶绿体等自发光进行拆分。
7.如权利要求1所述的油松基因表达与定位检测方法，其特征在于：步骤S4中，利用Surface模块进行细胞膜和基因表达信号的三维渲染，获取基因三维空间定位。
8.如权利要求7所述的油松基因表达与定位检测方法，其特征在于：计算渲染后基因表达各部分的各自体积和表面积，以及荧光的Max、Mean、Min和StdDev值，确定最后基因表达水平。
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