【作者】 王凤华；

【导师】 吕柳新； 郑金贵；

【作者基本信息】 福建农林大学 ， 果树学， 2003， 博士

【摘要】 龙眼胚性愈伤组织的体细胞胚胎发生与合子胚的发育过程基本相同，依次经过愈伤组织、球形胚、心形胚、鱼雷胚、子叶胚以及成熟胚等阶段。通过对2，4-D浓度及生长发育时间的控制获得了高度同步化的各发育阶段体胚材料。 采用SDS法、CTAB法、LiCl沉淀法均可从龙眼胚性培养物提取高质量的DNA。LiCl沉淀法简便、快捷、经济且提取的DNA较前两者质量更高，是适合用于胚性培养物DNA提取的一种方法。采用RNA抽提试剂盒提取的RNA适合mRNA差别显示分析。 本试验首次建立了龙眼胚性培养物的mRNA差别显示技术，并利用此技术对龙眼体细胞胚胎发生整个过程中的mRNA变化进行分析，结果获得了30条差异cDNA片段，经Northern和反Northern杂交获得阳性片段5条(longan1、longan2、longan3、longan4、longan4’)，其中longan1在龙眼体胚的胚性愈伤组织和球形胚阶段表达；longan2为组成型表达，即存在于整个体胚发生过程中；longan3在球形胚和鱼雷形胚阶段特异表达；longan4、longan4’在子叶形胚阶段特异表达。 碱基序列分析表明，longan1在3’端存在一个开放阅读框(ORF)，longan2、longan3分别在5’端存在一个开放阅读框，longan1在3’方向，longan2、longan3在5’方向均没有终止密码子，所以可能往相应的方向延伸仍为各自的ORF，因此可以通过RACE或者筛选cDNA文库的办法来获得它们的全长cDNA。登陆Genbank进行同源性比较发现longan2、longan3、longan4、longan4’均没有同源基因，而longan1与番茄apx基因具有较高的同源性(92％)，推测longan1可能是龙眼体细胞胚胎中存在的apx基因。 龙眼体细胞胚胎发生过程中不仅mRNA的种类和数量存在明显变化，而且蛋白质的种类和数量也存在显著变化。采用蛋白质水平双向电泳技术研究龙眼体胚发生过程中的蛋白质变化，获得13个特异蛋白；分析发现这些特异蛋白的等电点与龙眼体细胞胚胎发育进程相关，随着发育进程的推进，特异蛋白的等电点呈降低趋势。推测龙眼体胚的发育与特异蛋白的酸碱性相关。 分析mRNA差别显示的结果和蛋白质双向电泳结果，发现龙眼在胚性愈伤组织和球形胚阶段，差异表达的mRNA多于后期阶段(子叶形胚和成熟胚阶段)，但蛋白质却较后期少。推测胚性愈伤组织阶段和球形胚阶段某些基因正处于转录mRNA或以mRNP的形式存在，为以后体胚发育作准备，此后由于mRNA翻译产生大量特异蛋白，促使了特定阶段体细胞胚胎的发生与发育。更多还原

【Abstract】 Longan somatic embryogenesis occurred through the following stages- embryogenic callus, globular embryoid, heart-shaped embryoid, torpedo-shaped embryoid, cotyledonary embryoid and matured cotyledonary embryoid, which was similar to that of zygotic embryo. Synchronized embryogenic cultures at different developmental stages were obtained by controlling 2,4-D concentration and developmental time. High-quality DNA of longan embryogenic cultures were extracted by SDS ,CTAB and LiCl methods. DNA extracted by LiCl was better with less contamination and high-quality than that by the other 2 methods. Furthermore, it was cheaper and took less time, which was suitable for the DNA extraction of in vitro materials. High-quality RNA of longan embryogenic cultures for mRNA differential display were also extracted by the Sangon RNA extraction kit.The mRNA differential display system for longan embryogenic cultures was first established, and 30 specific expression cDNA fragments were obtained. Among them, 5 cDNAs(longan1, longan2, longan3, longan4, Iongan4’) were proved to be specific expression cDNAs by northern blot and reverse northern blot. Longanl expressed from the embryogenic callus stage to the globular embryoid stages. Longan2 expressed in the whole process of longan somatic embryogenesis, which was probably a housekeeping gene. Longan3 expressed from the globular embryoid stage to the torpedo-shapedembryoid stage. Longan4, longan4’ expressed only at the cotyledonary embryoid stage.Sequence analysis showed that an open reading frame(ORF) without the termination code existed at the 3’ end of longanl and at the 5’ end of longanl and longanl, which suggested that they should be the part of ORF at their 3’ or 5’ extension end, respectively. The whole length cDNA of these three genes could obtain by RACE or screening cDNA library. Blast results in Genbank showed that there were no homologous genes to longan2, longan3, longan4, longan4’ However longanl was highly homology to apx gene(92%) from tomato. It was predicted that longanl was an apx gene in longan somatic embryoids.Proteins in longan somatic embryogenesis changed greatly as the mRNAs did. 13 specific proteins were obtained with horizontal two-dimensional electrophoresis. The PIs of these specific proteins were decreased as somatic embryogenesis occurred and developed, which suggested that the PIs of the specific proteins should be negative relative to the developmental satages of somatic embryoids.The obtained results of the mRNA differential display and two-dimensional electrophoresis of the proteins showed that the cDNAs of embryogenic calli and globular embryoids were more than those of cotyledonary embryoids and matured-cotyledonary embryoids, While the proteins were in turn. It was predicted that some genes existed in transcripting mRNAs or exiting in mRNPs, which would be translated into proteins which were important to somatic embryogenesis at the later stages.更多还原