【作者】 杨旸；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 园林植物与观赏园艺， 2007， 硕士

【摘要】 本试验以金花茶(Camellia nitidissima Chi.)成年材料离体再生体系为材料来源，进行了体胚发生调控、发育解剖学观察，并对金花茶正常体胚发育和花状多子叶形胚体胚发育途径不同阶段的离体培养材料进行了蛋白质表达的双向电泳分析。金花茶花状多子叶形胚这一异常发育途径，是植物离体形态发生的一种特殊现象，在其他植物上未见报道。对该现象的研究，有助于了解植物离体形态过程的一些特殊机制，对完善发育生物学理论有重要意义。本试验主要研究结果如下：1、比较了不同激素组合对金花茶愈伤组织诱导的影响。发现将本实验室原有的金花茶体胚增殖再生系统中获得的子叶胚切块后接种在附加2mg／L 2，4-D和1mg／L KT的培养基上每7d继代一次，诱导出的部分愈伤组织，在不添加激素的分化培养基上分化出体胚，并且相当数量都是花状多子叶形胚类型。2、建立了金花茶花状多子叶形胚发育途径的体胚循环再生系统。通过逐步提高培养基渗透压，经过逐代筛选，在附加0.22mol／L甘露醇和0.12mol／L蔗糖的MS培养基上获得基本一致的花状多子叶胚。将体胚转入附加0.2mol／L NAA和2mol／L BA的1／2 MS培养基中，体胚变红甚至褐变，最后在褐变的胚体表面又生长出较同步的同一类型的次生胚。将其重新接种到原培养基上，又可重复这一发育途径。3、将花状多子叶形胚不同发育阶段的离体培养材料制作石蜡切片，并与正常发育的材料进行对比，发现了其组织形态上具有特殊性。同时发现子叶向真叶的转化是从两端叶缘向主脉方向进行的。与拟南芥lec突变体比较，发现二者不是同一类型的突变。4、用双向电泳的分析方法，得到了金花茶正常体胚发育途径和花状多子叶形胚体胚发育途径球形胚、子叶胚、成熟胚和幼苗4个阶段的离体培养材料以及2mg／L 2，4-D和1mg／L KT的激素组合诱导的愈伤组织的总蛋白的双向电泳图谱。金花茶正常体胚发育途径，从球形胚阶段发育至子叶胚阶段，明显消失或减弱的蛋白有4个：CnPG1(63.9kD)、CnPG2(29.9kD)、CnPG3(30.6kD)、CnPG4(29.1kD)，其中CnPG2(29.9kD)、CnPG3(30.6kD)和CnPG4(29.1kD)从子叶胚阶段开始明显减弱；而在子叶胚阶段明显新增的蛋白，有9个：CnPC1(73.3kD)、CnPC2(70.5kD)、CnPC3(35.4kD)、CnPC4(44.1kD)、CnPC5(28.4kD)、CnPC6(20.1kD)、CnPC7(19.1kD)、CnPC8(17.4kD)、CnPC9(19.0kD)，其中CnPC1(73.3kD)和CnPC2(70.5kD)在随后的发育进程中逐渐减弱，在幼苗阶段完全消失。从子叶胚发育至成熟胚阶段，明显消失或减弱的蛋白有25个CnPC10(58.4kD)、CnPC4(44.1kD)、CnPC11(48.1kD)、CnPC12(45.0kD)、CnPC13(40.0kD)、CnPC14(19.0kD)、CnPC15(42.1kD)、CnPC6(20.1kD)，CnPG4(29.1kD)，CnPC7(19.1kD)，CnPC16(23.0kD)、CnPC17(18.8kD)、CnPC9(19.0kD)，CnPC8(17.4kD)，CnPC18(21.3kD)、CnPC19(35.1kD)、CnPC20(29.2kD)、CnPC21(30.4kD)、CnPC22(20.7kD)、CnPC23(19.0kD)、CnPC24(18.9kD)、CnPC25(16.0kD)、CnPC26(32.6kD)、CnPC27(32.3kD)、CnPC28(28.4kD)。其中CnPC4(44.1kD)、CnPC6(20.1kD)、CnPC7(19.1kD)、CnPC8(17.4kD)和CnPC9(19.0kD)仅在子叶胚阶段出现，CnPG4(29.1kD)在球形胚阶段开始明显减弱，至子叶胚阶段完全消失。而在成熟胚阶段新出现的蛋白有3个CnPM1(21.0kD)、CnPM2(18.5kD)、CnPM4(15.3kD)，CnPM3(16.3kD)明显增强。成熟胚发育到幼苗阶段，明显消失的蛋白有16个：CnPM5(72.5kD)、CnPM6(73.1kD)、CnPM7(60.6kD)、CnPMS(71.1kD)、CnPG2(29.9kD)、CnPG3(30.6kD)、CnPM9(27.6kD)、CnPM1(21.0kD)、CnPM10(21.5kD)、CnPM11(21.0kD)、CnPM12(20.0kD)、CnPM3(16.3kD)、CnPM13(15.7kD)、CnPM14(14.1kD)、CnPM15(13.1kD)、CnPM16(12.2kD)。其中CnPM1(21.0kD)仅在成熟胚阶段出现，CnPG2(29.9kD)和CnPG3(30.6kD)在球形胚阶段开始明显减弱，至成熟胚阶段则完全消失。而在幼苗阶段新增的蛋白有5个：CnPP1(55.7kD)、CnPP3(57.2kD)、CnPP4(55.3kD)、CnPP5(55.3kD)、CnPP6(47.2kD)，CnPP2(57.5kD)虽然存在于正常体胚所有的发育阶段，但在幼苗阶段的表达量特别大。而金花茶花状多子叶形胚发育途径蛋白表达变化的差异有：①CnPG5(31.4kD)在正常体胚发育过程中的球形胚阶段就已经出现；而在花状多子叶形胚体胚发育过程中，则要到子叶胚阶段才出现。②CnPC4(44.1kD)和CnPC5(28.4kD)在正常体胚发育过程中的子叶胚阶段才出现；而在花状多子叶形胚体胚发育过程中，在球形胚阶段就已经出现。③CnPG4(29.1kD)在正常体胚发育过程中的球形胚阶段开始减弱，在进入成熟胚阶段前消失；而CnPG 4(29.1kD)在花状多子叶形胚体胚发育过程中的球形胚阶段的表达量就明显低于正常发育的体胚，并且在于叶胚阶段就已消失。④花状多子形叶胚体胚发育过程的子叶胚至成熟胚阶段，CnPP2(57.5kD)和CnPM17(46.4kD)在此期间消失；而正常体胚发育过程中这一时期没有这一变化。⑤CnFM1(21.0kD)在花状多子叶形胚体胚发育过程中未见表达。⑥CnPD(14.9kD)是花状多子叶形胚体胚发育过程中特有的蛋白，从球形胚至成熟胚阶段一直稳定存在；而正常体胚发育过程中，没有见到该蛋白的表达。两种发育途径最终获得的幼苗的蛋白表达差异分析发现，正常体胚比花状多子叶形胚多出5个蛋白：CnPP1(55.7kD)、CnPP5(55.3kD)、CnPP6(47.2kD)、CnPP7(16.1kD)、CnPM4(15.3kD)；而在花状多子叶形胚幼苗阶段，CnPC7(19.1kD)仍未消失。金花茶愈伤组织与花状多子叶形胚的蛋白表达差异分析发现，花状多子叶形胚发育途径的特有的CnPD(14.9kD)和提早出现的CnPC5(28.4kD)在愈伤组织阶段也有表达。更多还原

【Abstract】 In this experiment the repetitive in vitro regenerating system originated from the adult stem-tips was used as the material resource for the studies on the regulation of somatic embryogenesis and the related developmental anatomy in Camellia nitidissima Chi.(yellow camellia; besides, 2-Dimensional Electrophoresis was applied to analyze protein expression of different developmental phases during somatic embryogenesis. The abnormal developing pathway in Camellia nitidissima Chi., through which somatic embryos formed multiple cotyledons and flower-like shape, (named as MCFS pathway) was a special phenomenon in plant development, which had not been reported. The studies on the phenomenon was conducive to fathom the mechanism of some specific processes of plant in vitro morphogenesis and important to consummate the theory of the developmental biology. The obtained results showed as follows:1．The effects of different combinations of plant growth regulators on the callus induction were compared. The results indicated that the pieces cut from somatic cotyledonary embryo derived from the repetitive somatic embryogenesis system established in our laboratory were cultured on the medium adding 2mg/L 2,4-D and 1mg/L KT and subcultured every 7 days, the calli formed after 3～4 times, and then somatic embryogenesis occurred on the differentiation medium without growth regulators, and many somatic embryos with multiple cotyledons and flower-like shape (named as MCFS embryo) were observed..2．The repetitive MCFS embryogenesis system was established. Almost the same type of MCFS embryos clould be obtained on MS medium adding 0．22mol/L mannitol and 0．12 mol/L sucrose after augmenting osmotic pressure of the medium step by step and selecting typical materials every generation. After being transferred onto the 1/2 MS medium adding 0．2mol/L NAA and 2mol/L BA, the abnormal somatic embryos turned magenta or even became brown; finally, the browned body generated nearly synchronized and identical secondary MCFS embryos on the surface. Going back to the first step with the secondary MCFS embryos could repeat the process.3．The anatomic observations on the materials of different developmental phases in the MCFS pathway and normal materials by paraffin sections were compared. The materials from MCFS embryos had unique morphologic features, and the transforming order from cotyledon to leaf was from leaf margins of both sides to main vein. The MCFS embryo was distinct from lee mutant of Arabidopsis by comparison.4．The 2-DE analyses of the expression of the total proteins of the materials in the different phases during the MCFS pathway and normal developmental pathway, including globular embryos, cotyledon embryos, mature embryos, plantlets, and the calli induced by adding 2mg/L 2,4-D and 1mg/L KT, was carried out..During the normal developmental pathway, from the globular embryo phase to the cotyledon embryo phase, there were 4 protein spots disappeared or weakened obviously, i.e., CnPGl(63．9KD), CnPG2(29．9kD), CnPG3(30．6kD) and CnPG4(29．1 kD), among which CnPG2(29．9kD), CnPG3(30．6kD) and CnPG4(29．1kD) weakened obviously since the cotyledon embryo phase; and there were 9 new protein spots in the cotyledon embryo phase, i.e., CnPCl(73．3kD), CnPC2(70．5kD), CnPC3(35．4kD), CnPC4(44．1 kD), CnPC5(28．4kD), CnPC6 (20．1kD), CnPC7(19．1kD), CnPC8( 17．4kD), CnPC9(19．0kD), among which CnPC1 (73．3kD) and CnPC2(70．5kD) weakened gradually later and completely disappeared in the plantlet phase. From the cotyledon embryo phase to the mature embryo phase, there were 25 protein spots disappeared or weakened obviously, i.e.,CnPC10(58．4kD), CnPC4(44．1 kD), CnPC11(48．1kD), CnPC12 (45．0kD), CnPC13(40．0kD), CnPC14(19．0kD), CnPC 15(42．1 kD), CnPC6(20．1 kD), CnPG4 (29．1kD), CnPC7(19．1kD), CnPC 16(23．0kD), CnPC17(18．8kD), CnPC9(19．0kD), CnPC8 (17．4kD), CnPC 18(21．3kD), CnPC 19(35．1kD), CnPC20(29．2kD), CnPC21(30．4kD), CnPC22 (20．7kD), CnPC23(19．0kD), CnPC24(18．9kD), CnPC25(16．0kD), CnPC26(32．6kD), CnPC27 (32．3kD), CnPC28(28．4kD). among which CnPC4(44．1 kD), CnPC6(20．1 kD), CnPC7(19．1kD), CnPC8(17．4kD), CnPC9(l9．0kD) appeared in the cotyledonary embryo phase only and CnPG4 (29．1kD) completely disappeared in the cotyledonary embryo phase. There were 3 new protein spots in the mature embryo phase, i.e., CnPM1(21．0kD), CnPM2(18．5kD), and CnPM4(15．3kD). CnPM3(16．3kD) strengthened obviously. From the mature embryo phase to the plantlet phase, there were 16 protein spots disappeared, i.e., CnPM5(72．5kD), CnPM6(73．1kD), CnPM7(60．6kD), CnPM8(71．1kD), CnPG2(29．9kD), CnPG3(30．6kD), CnPM9(27．6kD), CnPM 1(21．0kD), CnPM10 (21．5kD), CnPM 11(21．0kD), CnPM12(20．0kD), CnPM3(16．3kD), CnPM13(15．7kD), CnPM14 (14．1kD), CnPM15(13．1kD), CnPM16(12．2kD), among which CnPM 1(21．0kD) only appeared in the mature embryo phase, CnPG2(29．9kD) and CnPG3(30．6kD) weakened since the globular embryo phase, then disappeared in the mature embryo phase. There were 5 new protein spots in the plantlet phase, i.e., CnPP1(55．7kD), CnPP3(57．2kD), CnPP4(55．3kD), CnPP5(55．3kD), CnPP6 (47．2kD). CnPP2(57．5kD) existed in all the phases of the normal developmental pathway, and dramatically increased in expression level in the plantlet phase.The discrepancy of protein expression in MCFS pathway was as follows:①CnPG5(31．4kD) had already appeared in the globular embryo phase of the normal developmental pathway, but had not appeared until the cotyledonary embryo phase of MCFS pathway.②CnPC4(44．1kD) and CnPC5(28．4kD) had not appeared until the cotyledonary embryo phase of the normal developmental pathway, but had already appeared in the globular embryo phase of MCFS pathway.③CnPG4(29．1kD) weakened since the globular embryo phase and disappeared before the mature embryo phase during the normal developmental pathway. In contrast, during MCFS pathway, its expression amount was obviously less in the globular embryo phase than that of the normal developmental pathway, and the protein spot disappeared in the cotyledonary embryo phase.④From the cotyledonary embryo phase to the mature embryo phase during MCFS pathway, CnPP2(57．5kD) and CnPM17(46．4kD) disappeared, which had not yet happened during the normal developmental pathway.⑤CnPM 1(21．0kD) had never appeared during the MCFS pathway.⑥CnPD(14．9kD) which existed from the globular embryo phase to the mature embryo phase was exclusive for the MCFS pathway, but it never appeared in any phase during the normal developmental pathway.In the plantlet phase, there were 5 more protein spots during the normal developmental pathway than that during the MCFS pathway; besides, in the plantlet phase during the MCFS pathway, CnPC7(19．1kD) still existed.CnPD(14．9kD) and CnPC5(28．4kD) were also observed in the callus phase by comparing with the globular embryo phase during the MCFS pathway.更多还原