Study On Mechanism Of Populus Simonii×P. Nigra PsnCYCD1;1 Gene Involved In Cell Division And Secondary Growth

Cell division is a basic characteristic for all organisms, which is playing an important role in the growth and development of plants. Cell cycle and growth regulation depend on a class of important cell division control factors--Cyclins. Studies of plant cyclins are mainly focused on Arabidopsis thaliana, tobacco, there are few reports on the forest trees. In view of the huge difference of genome size and the secondary growth between herbaceous plants and woody plants, the results of studies on the cyclins from herbaceous plants can not reflect the real function of the woody plants. In order to reveal the molecular mechanism of growth and development of woody plants, it is imperative to explore the biological function of woody plant cyclins. In this paper, the expression pattern and function of PsnCYCD1;1 gene from Populus were studied, the main conclusions are as follows:(1) The results of prediction of biological information, tissue-specific expression, induced expression of exogenous substance, function verification of promoter, subcellular localization and other tests showed that PsnCYCD1;1 gene was a typical D-type cyclins, not only containing cyclin-box, LxCxE, PEST and other conserved domains, but also a large number of O- glycosylation sites and N- glycosylation sites; PsnCYCD1;1 was a nuclear localization protein confirmed by in vivo, which is also similar with the prediction of nuclear localization signal site; Moreover, PsnCYCD1; 1 was highly expressed in the meristem or growing tissues, such as buds, xylem, young leaves, and lateral root.Transcription level of PsnCYCD1;1 gene was up-regulated after were treated by sucrose and plant hormones, such as NAA,6-BA, GA3 and ethylene. As above, PsnCYCD1;1 gene was working in the nucleus and responsed to the external growth signal to participate in the process of plant cell division and development.(2) Analysis of ploidy levels by flow cytometry showed that the number of cells with double DNA content in the transgenic lines was far more than that of wild type cells, suggesting that PsnCYCD1;1 gene accelerates cell replication and promotes the change of GO-G1 period. Although PsnCYCD1 ;1 gene can promote cell division, generate small cells, but not participate in the growth of cell in enlargement stage.(3) Morphological traits of transgenic Arabidopsis and P. simonu×P. nigra plants were changed due to the overexpression of PsnCYCD1; 1 gene. Same point:in the macro structure, transgenic Arabidopsis and P.simonu×P. nigra plants showed short root length, curled leaves, curved stems, growth retardation and respose to cytokinin etc.. when compared with wild type plants:In microcosmic structure, transgenic plants also showed the traits of small cell size, abnormal differentiation and so on. Different point:transgenic Arabidopsis showed the phenotype of longer and curved hypocotyl, delayed blossom and low seed-set rate; Transgenic P. simonii ×P. nigra not only showed the abnormal development of meristem and chloroplast, separated wood fiber, morphological changes of wood fiber cell, thickened cell wall. In addition, differentiation of secondary xylem was different on both sides of the buds, resulting in stem eccentric growth.(4) The transcription levels of genes related to CYCDs, CDKs, ICKs, E2F/Rb, and vascular bundle development were varied in transgenic Arabidopsis and P. simonii ×P. nigra plants. The results of yeast two hybrid and bimolecular fluorescence complementation (BiFC) showed that PsnCYCD1;1 could combine with PsnCDKA1, PsnICK3 and PsnICK5, demonstrated that there were interaction relationships between them and they could coordinatedly regulate the cell cycle in an orderly manner.In summary, this study was firstly based on the systematic study of expression patterns of PsnCYCD1;1 gene, then confirmed the morphological changes and interaction proteins of transgenic plants to deeply analyze the biological function of PsnCYCD1;1 gene. Finally, this study will contribute to new findings for the regulation network of plant cycle protein, and provide a theoretical basis and related genes for genetic improvement of forest trees.