Studies On The Characteristic Of Meristem Development And Cloning And Functional Analysis Of Related Genes In Bamboo

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.