Study On PeTua3and PeTub3Encoding Micro Tubulin Of Cytoskeleton In Moso Bamboo (Phvllostachys Edulis)

Bamboo forest is one of important forest resources. Bamboo timber is a complexbiological material consisted of various types of cell walls forming special spatial structurewhich has high strength, good toughness and hardness, and it is widely used in handcraft,construction, paper making, etc. The excellent material characteristics of bamboo is closelyrelated to the composition and structure of its cell wall. The development of cell wall playsimportant roles in the growth and development, physiological characteristics, biochemicalmetabolism of bamboo and the defence reactions to environmental stress, and it also decidesthe characteristics of bamboo material. The biosynthesis of cell wall is guided by cytoskeletonwhich is composed of microfilament, microtubule and intemediate filament. As a key part ofcytoskeleton, microtubule has an important regulatory role in the development of cell wall. Inthis study, two genes encoding microtubule proteins were isolated from Moso bamboo(Phyllostachys edulis) and their functions were identified through prokaryotic expression invitro and ectopic expression in model plants. This work will lay the basis for molecularengineering to breed new bamboo varieties. The main results are as follows:1. Two genes encoding microtubule proteins were cloned using RT-PCR from leaves ofMoso bamboo, and named as PeTua3and PeTub3respectively. The open reading frame (ORF)of PeTua3is1353bp encoding451amino acids, while the ORF of PeTub3is1341bpencoding447amino acids.2. Blast analysis showed that PeTua3and PeTub3had a high similarity with tubilins fromother plants and belonged to α-tubilin and β-tubilin family, respectively. Hydropathic analysisshowed that PeTua3and PeTub3were hydrophobic acidic protein. The protein analysis withMOTIFSCAN showed that Petua3and PeTub3all contained N-glycosylation sites, caseinkinase II phosphorylation sites, N-myristoylation sites, phosphorylation protein kinase sites,GTPase domain and C-terminal domain, however, the positions and quantity of these sites were different in the sequences, which means they might have different functions. Phylogenetic treesbased on tubilins were constructed by using MEGA4.0. The reslut showed that PeTua3andPetub3were closed to α-tubilins and β-tubilins from monocotyledon respectively and wereaway from to those of dicotyledon, the nearest one to PeTua3was that of rice and to PeTub3was that of goose grass, which was consistent with the morphological classification ofBambusoideae in Poaceae of monocotyledon plants.3. The prokaryotic expression vectors of PeTua3and Petub3were constructed and theplasmids of pET-32b-PeTua3and pET-30a-PeTub3were transformed into Escherichia coli(DE3). The positive clones were selected for further experiments. By optimizing the expressionconditions of induction, the soluble recombinant protein of PeTua3was effectively expressedat37℃induced by0.4mmol·L^-1IPTG with2h, while that of PeTub3received higherexpression at28℃induced by0.4mmol·L^-1IPTG with4h. The recombinant proteins werepurified by using His·Tag Bind Purification Kit and were used to treat seeds of Arabidopsisthaliana. The result showed that the hypocotyl of the treated seedlings were thicken and thequantity of lateral root increased significantly, which indicated that the recombinant proteinsmight have biological activity for growth stimulation.4. The plant expression vectors of sense/antisense PeTua3/PeTub3were constructed andtransferred into A. thaliana. The sense and antisense transgenic plants were selected withantibiotics and the postive transgenics of T3were used to further studies. The phenotypes ofthe transgenics were observed, the germination was not affected by the overexpression orinhibition of genes. However, the root growth was affected obviously. The root phenotypes ofsense transgenics all grew fast and had more lateral roots, while the antisense transgenics allgrew abnomal and had lots of hair roots. All the roots of sense and antisense transgenics wereembedded in paraffin and sectioned to8^-10m thick for tissue sections making. The roottissue sections of sense transgenics showed that the quantity of epidermal parenchyma cellswas increasing, the volume of the cells was becoming larger, the xylem was significantlythicker and the volume of the vascular cylinder increased about2times than that of wild type. However the quantity of epidermal parenchyma cells in antisense transgenics was significantlyreduced, the volume of the cells became smaller, the xyle was obviously thinner and thevolume of the vascular cylinder became smaller than that of wild type. These results suggestedthat PeTua3and PeTub3could promote cell growth and division to accelerate the growth ofroots.5. Tobacco (Nicotiana tabacum) BY-2suspension cells were used to study the tubilingenes' influence on cell division. PeTua3and PeTub3were overexpressed respectively in BY-2suspension cells. The result of microscope observation showed that the mitotic index oftransgenic BY-2cells was increased and the shape of the cells was also changed. Comparedwith BY-2cells, the time of G2and S phase for the transgenic BY-2cells was shortenedsignificantly, which accelerated the cell division.The recombinant proteins of expression in vitro have biological activity for promoting cellgrowth, which further confirmed in transgenic A. thaliana. PeTua3and PeTub3promotegrowth by reducing the cell division cycle through transgenic BY-2. Thus prove the PeTua3and PeTub3functions to promote cell development has been proved, which means that PeTua3and PeTub3for molecular breeding have important potential applications.