枣果实维管组织中荧光示踪剂引入方法的应用

doi:10.13560/j.cnki.biotech.bull.1985.2020-1502

摘要/Abstract

摘要：

本研究以不同发育时期的“灵武长枣”果实为实验材料,采用石蜡切片技术明确不同发育时期果实维管束结构特征和变化规律,在此基础上,通过枣果实维管组织中荧光示踪剂引入方法的应用,采用荧光染料活细胞示踪技术,探讨枣果实维管束韧皮部同化物卸载路径的研究方法。结果表明:(1)膨大前期果实的维管束较多,尤其是内果皮附近的维管束数量较多、分布集中,此时有单个维管束进行发育,也有两个维管束背靠背进行发育。快速膨大期果实中果皮内薄壁细胞发育迅速,由外向内体积逐渐增大,排列松散,有些甚至排列成网状结构,围成一个个较大的空腔,相同视野内维管束数量下降,维管束多为两两背靠背式、3个或4个聚在一起发育。着色期中果皮的细胞空腔进一步增大、数量增多,相比前两个时期相同视野内的维管束数量更少,由多个维管束形成了多分支结构。与着色期相比,完熟期果实薄壁细胞形成的空腔空间继续增大,维管束数量无明显变化,多维管束分支结构略有差异。(2)采用荧光染料活细胞示踪技术,在不同发育时期枣果实维管束韧皮部中引入了荧光示踪剂羧基荧光素酯CFDA、质膜通透剂洋地黄皂苷,在维管束木质部中引入了荧光示踪剂Texas-Red,通过激光共聚焦显微镜CLSM观察荧光特征,获得了枣果实中韧皮部同化物卸载路径的研究方法,为研究枣果实同化物积累和品质调控奠定了基础。

关键词: 灵武长枣, 果实, 维管组织, 荧光示踪剂, 引入

Abstract:

The fruits of Ziziphus jujuba Mill cv. Lingwuchangzao in different developmental stages were used as experimental materials,the microstructure and change laws of vascular bundles in the fruits of different developmental stages can be clarified using paraffin section method. Based on these results and applying methods of leading fluorescent tracers into vascular tissues of Z. jujuba Mill cv. Lingwuchangzao fruits,the techniques of fluorescent dye tracer in living cell were used to explore the research methods of the unloading path of photosynthates in vascular bundles phloem in the Z. jujuba Mill cv. Lingwuchangzao fruit timely. The results showed that:1)The vascular bundles(especially the vascular bundles nearby endocarp)in the fruit was concentrated and abundant in the early bulking period,the vascular bundles were single or two back-to-back gathered together to be developing. In the rapid enlargement period fruit,the parenchyma cells in the mesocarp developed rapidly and arranged loosely,they gradually increased from the outer to the inner volume,some even arranged into a reticular structure,surrounded by a larger cavity,the number of vascular bundles decreased in the same visual field,and the vascular bundles were mostly two back-to-back,three or four gathered together to be developing. The cell cavities were further enlarged and the number increased in the pericarp in the coloring period,compared with the previous two stages,the number of vascular bundles was fewer in the same visual field,and formed multi-branch structures by multiple vascular bundles. Compared with the coloring period,the cavity space formed by parenchyma cells continued to increase,and the number of vascular bundles did not change significantly,multi-branch structures of multiple vascular bundles were slightly different in the maturation period fruit. 2)By using the techniques of fluorescent dye tracer in living cell,the fluorescent tracer carboxyfluorescein diacetate and plasma membrane penetrating reagent digitonin were led into the phloem of vascular bundles,and the fluorescent tracer Texas-Red were led into the vascular bundles xylem of Z. jujuba Mill cv. Lingwuchangzao fruit in different developmental stages,the fluorescence characteristics were observed by CLSM,the research methods of the phloem unloading path of photosynthates in Z. jujuba Mill cv. Lingwuchangzao fruit were gained,which provides scientific bases for photosynthates accumulation and quality regulation for Z. jujuba Mill cv. Lingwuchangzao fruit.

Key words: Ziziphus jujuba Mill cv. Lingwuchangzao, fruit, vascular tissues, fluorescent tracers, leading into

章英才, 黄月, 海源, 张媛, 扈亚杰, 赵梦怡. 枣果实维管组织中荧光示踪剂引入方法的应用[J]. 生物技术通报, 2021, 37(6): 295-304.

ZHANG Ying-cai, HUANG Yue, HAI Yuan, ZHANG Yuan, HU Ya-jie, ZHAO Meng-yi. Method Application of Leading Fluorescent Tracers into the Vascular Tissues of Ziziphus jujuba Mill cv. Lingwuchangzao Fruits[J]. Biotechnology Bulletin, 2021, 37(6): 295-304.

图/表 9

图1 CFDA的引入方法1图示 A:用一细针将棉线从上向下穿过短枝韧皮部;B:用细铁丝将Eppendorf管固定并保持竖直;C:将棉线缠绕于短枝四周进行固定,并用微量注射器向Eppendorf管中注入CFDA稀释液;D:Eppendorf管盖好并用锡箔纸包好

Fig.1 Photos showing the introduction method 1 of CFDA A:A cotton thread passes through the short branch phloem from up to down with a fine needle. B:Fix the Eppendorf tube with fine iron wire and keep it upright. C:Fix the cotton thread around the short branch and inject CFDA diluent into the Eppendorf tube with microinjector. D:The Eppendorf tube is closed with the lid and wrapped in silver paper

图2 CFDA的引入方法2图示 A:砂布磨擦后将棉花团缠绕在靠近果柄的短枝;B:用封口膜将棉花团部分密封后微量注射器注入CFDA溶液;C:用封口膜将棉花团完全密封,两端涂抹凡士林;D:用锡箔纸将密封的棉花团完全覆盖

Fig.2 Photos showing the introduction method 2 of CFDA A:Intertwine the cotton dough around short branch near fruit stalk after rubbing with sandcloth. B:Inject CFDA into the cotton dough with a microinjector after partly sealing up the cotton dough with sealing film. C:Completely seal up the cotton dough with sealing film and applying vaseline on both ends. D:Completely wrap the sealed cotton dough with silver paper

图3 CFDA和洋地黄皂苷同时引入的方法图示 A:棉线穿过短枝韧皮部,在针穿进和穿出的部位滴加适量EDTA;B:用细铁丝将Eppendorf管固定并保持竖直,将棉线缠绕于短枝四周进行固定,在Eppendorf管附近短枝砂布磨擦后用棉花团缠绕;C:将棉花团用封口膜部分密封后用微量注射器注入洋地黄皂苷,然后全部密封;D:在Eppendorf管中用微量注射器注入CFDA稀释液;E:Eppendorf管中注入CFDA稀释液后迅速盖好盖子;F:用锡箔纸将密封的棉花团和Eppendorf管完全包好

Fig.3 Photos showing the simultaneous introduction meth-ods of CFDA and digitonin A:A cotton thread is passed through the phloem of short branch and the appropriate amount of EDTA into and out of the needle is dripped. B:Fix the Eppendorf tube with a fine iron wire and keep it upright,fix the cotton thread around the short branch,intertwine the cotton dough around the short branch near the Eppendorf tube after rubbing with sandcloth. C:Inject digitonin into the cotton dough with a microinjector after partly sealing up the cotton dough with sealing film,then completely sealing up. D:Inject CFDA diluent into the Eppendorf tube with a microinjector. E:The Eppendorf tube is closed with a lid rapidly after injecting CFDA diluent. F:Seal the cotton dough and completely wrap the Eppendorf tube with silver paper

图4 Texas-Red标记的方法图示 A:快速膨大期果实果柄向下引入Texas-Red图示;B:完熟期果实果柄向下引入Texas-Red图示

Fig.4 Photos showing the introduction method of Texas-Red A:Photo showing the introduction of fruit stalk downward Texas-Red during the rapid enlargement period. B:Photo showing the introduction of fruit stalk downward Texas-Red during the maturation period

图5 膨大前期果实维管束特征 A:中果皮中的维管束;B:中果皮中的维管束及其他结构;C:内果皮及外部的维管束;D:果实中维管束的结构 X:木质部;P:韧皮部;VC:维管形成层;VB:维管束;MC:黏液腔;CA:空腔;ME:中果皮;EN:内果皮;SE:内表皮。Bar=50 μm。下同

Fig.5 Vascular bundles characteristic of fruit during the early bulking period A:Vascular bundles of mesocarp. B:Vascular bundles and other structures of mesocarp. C:Endocarp and external vascular bundles. D:Vascular bundle structures of fruit X:Xylem. P:Phloem. VC:Vascular bundle cambium. VB:Vascular bundle. MC:Mucous cavity. CA:Cavity. ME:Mesocarp. EN:Endocarp. SE:Inner epidermis. Bar=50 μm. The same below

图6 快速膨大期果实维管束特征 A:中果皮中的维管束及其他结构;B:果实中维管束的结构;C-D:果实中维管束的结构及其分支

Fig.6 Vascular bundles characteristic of fruit during the rapid enlargement period A:Vascular bundles and other structures of mesocarp. B:Vascular bundle structures of fruit. C-D:Vascular bundle structures and branches of fruit

图7 着色期果实维管束特征 A:果实中果皮结构及其维管束的结构;B:中果皮2个维管束的分支结构;C:中果皮多维管束的分支结构;D:多个维管束彼此相连的结构

Fig.7 Vascular bundles characteristic of fruit during the coloring period A:Structures of mesocarp and vascular bundles in fruit. B:Branch structures of two vascular bundles of mesocarp. C:Branch structures of multiple vascular bundles of mesocarp. D:Structures connected by multiple vascular bundles

图8 完熟期果实维管束特征 A:果实中果皮结构及其维管束的结构;B:中果皮中维管束的结构;C:多维管束的分支结构;D:维管束的分支及同时出现的纵横切结构

Fig.8 Vascular bundles characteristic of fruit during the maturation period A:Structures of mesocarp and vascular bundles in fruit. B:Vascular bundles structures of mesocarp. C:Branch structures of multiple vascular bundles. D:Branches of vascular bundles and length cutting and cross section structures

图9 枣果实维管束荧光示踪图 A1、A2、A3分别为膨大前期果实引入CFDA 48 h后维管束横切荧光图、对应的透射图、对应的荧光图和明视野的叠加图;B1、B2分别为快速膨大期果实只引入CFDA、同时引入CFDA和洋地黄皂苷48 h后维管束纵切荧光图;C1、C2分别为着色期果实只引入CFDA、同时引入CFDA和洋地黄皂苷48 h后维管束纵切荧光图;D1、D2分别为完熟期果实只引入CFDA、同时引入CFDA和Texas-Red后荧光在维管束中的分布(韧皮部为CF标记的绿色荧光,木质部为Texas-Red标记的红色荧光)。X:木质部;P:韧皮部。A1-A3,Bar=100 μm;B1、B2、C1、C2、D1、D2,Bar=250 μm

Fig.9 Fluorescence tracing map of vascular bundles in jujube fruit A1,A2,and A3 shows vascular bundles fluorography,the corresponding bright field,the overlaid pictures from fluorescence and bright field,respectively after CFDA was introduced into the phloem of the early bulking period fruit for 48 h. B1 andB2 shows vascular bundles fluorography when only CFDA and CFDA and digitonin introduced simultaneously into the phloem of the rapid enlargement period fruit after treatment with 48 h.C1 and C2 shows vascular bundles fluorography when only CFDA and CFDA and digitonin introduced simultaneously into the phloem of the coloring period fruit after treatment with 48 h. D1 and D2 shows vascular bundles fluorography when only CFDA and CFDA and Texas-Red were introduced simultaneously into the maturation period fruit(CF green fluorescence shows the phloem and Texas-Red red fluorescence shows the xylem). X:Xylem. P:Phloem. A1-A3,Bar=100 μm. B1,B2,C1,C2,D1,D2,Bar=250 μm

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