论文总字数：22856字

摘 要

本论文拟建立油菜细胞悬浮体系，并通过荧光示踪技术，探究聚谷氨酸（ γ-PGA ）在油菜细胞中的作用位点。在实验过程中通过控制变量的方法，对无菌苗获取时采用的两种消毒剂 3%(v/v)NaClO 和 0.1%HgCl₂进行了比较，对愈伤组织诱导培养基中的激素配比进行了优化，同时不确立了最佳的悬浮细胞液体培养基，最后利用培养的悬浮细胞，进行了γ-PGA荧光示踪实验。实验结果表明：经 0.1%HgCl₂ 处理的油菜种子，实验中染菌率较低，无菌苗长势较好；当愈伤组织诱导培养基中，选择 2,4-二氯苯氧乙酸 （ 2,4-D ）和 6-苄氨基腺嘌呤 （ 6-BA ）浓度分别为 2.5mg/L 和 0.5mg/L 时，愈伤组织的出愈率最高；采用 BA 培养基培养的悬浮细胞的生长相较于 B5 和 AA 培养基更快而且不易褐化，所以选择 BA 培养基进行油菜悬浮细胞的培养；所得悬浮细胞用荧光标记 γ-PGA进行处理，在激光扫描共聚焦显微镜下观察， 发现 γ-PGA 在植物细胞中的作用位点是细胞膜。

关键词：无菌苗；愈伤组织；悬浮细胞； γ-PGA ，荧光示踪

Establishment of Suspension System of rapeseed cell and Exploration of γ-PGA’s acting site

Abstract

This paper intends to establish rapeseed cell suspension system, and by a fluorescent tracer technique, explore polyglutamate (γ-PGA)'s acting site in rapeseed cell.During the experiment, by the process control variables,two kinds of disinfectants were compared in the acquirement of no vaccine .The combination of different hormones in callus induction was optimized, meanwhile ,we established the best cell suspension liquid medium. Finally, the γ-PGA fluorescent tracer experiments were carried on by suspension cells. The results showed that: rape seeds treated by 0.1% HgCl2 have a low rate in experiments contamination , no vaccine grew better; when select the 2,4-dichlorophenoxyacetic acid (2, 4-D) and 6-benzylamino adenine (6-BA) concentrations of 2.5mg / L and 0.5mg / L.We got the highest rate of healing callus; The suspension cell cultured in BA Culture medium growed faster than that in B5 and AA Culture medium,so the BA Culture medium is the best choice.The protoplast samples were stained with FITC (fluorescein isothiocynate),and were photographed with laser scanning confocal microscope.And we can ensure that γ-PGA acts in cell membrane.

Keywords::Tissue culture; The callus; Tissue culture conditions; γ-PGA tracing

目 录

摘要.............................................................................................................................Ⅰ

ABSTRACT.............................................................................................................Ⅱ

第一章 文献综述.....................................................................................................1

1.1植物细胞悬浮培养研究概况..............................................................................1

1.1.1植物细胞悬浮培养的概念及特点...............................................................1

1.2影响悬浮细胞体系建立和再生的因素..............................................................2

1.2.1愈伤组织和细胞悬浮体系的建立...............................................................2

1.2.2 细胞悬浮培养的条件..................................................................................2

1.3 γ-PGA 在农业应用中的研究发展.....................................................................4

1.3.1 γ-PGA 的结构和性质...............................................................................4

1.3.2 γ-PGA 在作物种子萌发及苗期生长中的应用.......................................4

1.3 .3 γ-PGA 作为肥料增效剂在农业中的应用..............................................4

1.4本课题研究的内容和意义..................................................................................6

第二章 实验材料和方法.......................................................................................7

2.1 实验材料.............................................................................................................7

2.2 实验方法...........................................................................................................12

2.2.1 无菌苗的获取..........................................................................................13

2.2.2 愈伤组织的诱导......................................................................................13

2.2.3 悬浮细胞体系的建立..............................................................................13

2.2.4 γ-PGA示踪实验....................................................................................14

第三章 结果分析与讨论.....................................................................................16

3.1 无菌苗的培养...................................................................................................16

3.2 愈伤组织的诱导...............................................................................................17

3.3 悬浮细胞体系的建立.......................................................................................18

3.3.1 培养基的选择............................................................................................18

3.3.2 悬浮细胞生长曲线绘制............................................................................19

3.3.3不同接种量对悬浮细胞系生长的影响.....................................................20

3.3.4 油菜悬浮细胞生长周期中的糖消耗........................................................22

3.4 γ-PGA 示踪实验................................................................................................23

3.4.1 荧光标记γ-PGA的核磁表征.........................................................................23

3.4.2 γ-PGA在油菜悬浮细胞内的荧光示踪..........................................................24

第四章 结论和展望..............................................................................................26

4.1 结论....................................................................................................................26

4.2 展望....................................................................................................................26

参考文献...................................................................................................................27

致谢.............................................................................................................................30

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