【作者】 彭华正；

【导师】 朱睦元；

【作者基本信息】 浙江大学 ， 遗传学， 2005， 博士

【摘要】 植物分生组织在植物生活周期完成和形态建成中扮演十分重要的角色,是了解植物生长发育基本特点和基本规律的一个重要切入点。竹子是颇具特色的禾本科植物,与水稻、小麦等其它禾本科植物有着显著的不同,如竹子的快速生长能力和较高的木质化程度,不同竹种和同一竹种有散生和丛生的性状差异,竹子开花和繁殖规律的独特性等,竹子的这些特点与竹子分生组织发育特别是竹子地下茎(竹鞭)芽的发育紧密相关。因此研究竹子分生组织的发育特别是侧枝的形态发生规律对于丰富植物分生组织发育的认识非常有益。本研究筛选出一批与早竹分生组织发育过程相关的基因,克隆部分调控基因并初步揭示其功能和表达规律,为丌展竹子发育调控提供理论依据。1.竹子分生组织发育特点和基因芯片杂交分析 最近十几年,植物分生组织发育的分子机理研究有了不少重要的发现,这些研究大多建立在拟南芥,水稻等模式植物上。本研究利用竹子这种有着独特发育规律的植物,在其地下茎芽(鞭芽)和笋芽切片分析的基础上,借助于水稻基因表达谱芯片,检测了竹子地下茎鞭侧芽相对于成熟叶片的基因表达,共检测到1329个上调基因,结合拟南芥和水稻等模式植物的相关基因功能,列举了其中31个司一能对竹子发育研究有重要意义的基因,将这些基因归为分生组织发育、细胞壁合成、激素调控和其它等相关功能,并进行了简单分析,为竹子分生组织发育相关基因的克隆提供依据,也为进一步开展竹子发育的分子生物学研究提供思路。2.竹子发育相关基因的克隆和序列分析 根据基因芯片的分析结果,利用RACE技术从竹子鞭侧芽中克隆了6个与已知分生组织发育和激素调控基因相似的序列,序列分析表明它们分别与拟南芥的ARFI,AHKI,CLAVATAI,REVOLUTA,SPINDLY和SLVAT5基因相似;从竹子各组织混合样品中克隆到3个NAC家族的基因(分别命名为PpNACI,PpNAC2和PpNAC3)更多还原

【Abstract】 Meristems provide a reservoir of undifferentiated stem cells as well as a population of proliferating cells that will produce the various tissues of a plant, which is the key to understand the basic characteristic of plant development. Bamboos are a group of special Monocots in the grass family Poaceae, subfamily Bambusoideae, which are quite different in phenotype from rice , wheat and so on. Most cultured bamboo are woody although they do not form true wood from a vascular cambium. Bamboo elongate at meristems intercalated between mature tissues at the bases of their nodes, which can produce high rates of growth. Bamboo is usually propagated by meristem of rhizome underground which has several typical types among bamboo species including monopodial rhizome and sympodial rhizome. Although it grows fast, bamboo seldom flowers and when it does, it usually dies without breeding.These phenomenon are thought to be closely related to the development of bamboo rhizome. So the study of meristem development especially the branching of shoot in bamboo is helpful for us to deeply understand the development of plant meristem. In this project we identified a batch of genes related to meristem development of bamboo, cloned some of them and then explored their function, which offered the important base for further study on bamboo development.The characteristic of meristem development of bamboo and its analysis by gene chipRecent years have seen a tremendous increase in knowledge about the cellular organization and molecular mechanisms underlying a functional meristem. Most researches focus on the model plants like Arabidopsis thaliana and rice or other herbaceous plants which can be easily studied. Based on the analysis of sections from rhizome bud and bamboo shoot, we used rice gene chip to identify the meristem related genes in lateral rhizome bud of bamboo (Phyllostachys praecox). About 1329 up-regulated genes were found , and based on the homologues from Arabidopsis and rice, 31 of them were listed here which were thought to be very significant in the development of bamboo and were mainly classified into meristem related, phytohormone regulated and cell wall formation related genes. The results would undoubtedly brought new ideas on the study of molecular mechanism of development of bamboo.Cloning and analyzing of genes related to bamboo development.Based on the results from gene chip, six genes possibly related to meristem development and phytohormon pathway were cloned from lateral rhizome bud of bamboo, and then analyzed in sequence, which showed they were similar seprately to known six genes ARF1, AHKl, CLAVATAJ, REVOLUTA, SPINDLY and SINAT5 in Arabidopsis. And four genes belonging to MADS-box family(named as PpMADSl, PpMADS2, PpMADS3 and PpMADS4)and three genes belonging to NAC family(named as PpNACl, PpNAC2 and PpNAC3) were cloned from the mixture samples of bamboo and analyzed, which showed PpNACl was similar to CUC2 gene in Arabidopsis and PpMADS2 to RAP1B in rice. We supposed PpMADS2 was related to the initiation of flowering in bamboo since RAP1BB is the homologue of APETALAl(APl) in Arabidopsis. Besides, 14 gene segments very similar to M0C1 in rice were cloned from 12 bamboo species and the analysis of them indicated that three sites were absent in the conserved domain of the genes in bamboo compared to that in rice.Functional studies on genes related to bamboo developmentBased on the cloning of genes, nine genes including PpRLKl, PpHBl, PpSPY, PpSINA, PpMADSl, PpMADS2, PpMADS3, PpMADSi and PpNACl were analyzed in their expression among tissues including apical rhizome bud, lateral rhizome bud, lateral bamboo bud, leaf and young floret by semi-quantitative RT-PCR. PpSINA and PpMADSl were found to be expressed in all tissues but the former showed the highest expression in lateral rhizome bud and latter in young floret. Other gens were not found to be expressed in leaf, among which PpMADS3, PpMADS4 and PpNACl were only found to be expressed in young florest. Furthermore, six expression vector including three genes (PpMADS 1 ,PpMADS2 and PpNACl) and two kind of vector were constructed, which were then used in transformation of rice and Arabidopsis. Although it needed further verification, the primary experiments on the function of PpNACl in rice showed that the gene could promote the differentiation of rice callus.Chromosome walking by Nonspecific Anchoring of Suppression Sequence(NASS) PCRRP-PCR (Random Primer PCR) is obviously the simplest and most popularmethod for identification of T-DNA or transposon insertions because of no need to endonuclease digestion and ligase ligation. Although it has been successfully used with various genomes in several laboratories, it has some problems especially low efficiency and high nonspecific product which can hardly be solved because of its principle. An innovative method in DNA walking with Nonspecific Anchoring of Suppression Sequence PCR (NASS-PCR) was created here to solve the problem. Long primers instead of short ones were used in the method, which changed most programs used in general RP-PCR before and reached excellent results because of PS (PCR suppression)-effect. To further exert the advantages of this method, a series of suppression sequences should be designed in future. Anyway, the method would no doubt greatly facilitate isolation of DNA segments flanking known sequences and increase the efficiency of gene isolation by positional cloning.The method and application on reconstructing gene networksThe complexity of gene networks is embodied due to the interactions of genes and their products. The inner regulation of information in life system is emphasized by gene networks, which is a part of whole molecular networks. The reconstruction of gene networks, which is the prime task of gene networks research and important way to elucidate the genetic regulatory mechanism, can be divided into forward reconstruction based on biology information and reverse construction based on large-scale gene expression according to the type of data resource. Forward reconstruction stressed the analysis of dynamic features and logic structure, while reverse reconstruction emphasizes the probabilistic reasoning. The applications of gene networks reconstruction in the model organisms are beneficial to exploring the mechanism of vital phenomenon in life science. This chapter reviews the techniques of reconstructing gene networks and mimic the characteristic of gene network with software. And the gene network of meristem development was also discussed, which showed the unstability of gene network should be emphasized in further study.更多还原