Morphological Observation And Preliminary Study Of Several Genes Of Helwingia Willd

The genus Helwingia Willd is a dioecious shrub belonging to Helwingiaceae under the order of Aquifoliales. Until now eight species were documented and five were discovered to distribute in China. The five species include to H. japonica, H. chinensis, H.omeiensis, H. zhejiangensis and H. himalaica. The whole plant can be used as medicine. Many reports illustrate it possesses high medical value, such as antibacterial, anti-inflammatory, and reducing blood lipid. In addition, the flowers and the fruits forming on the midvein of leaf of the Helwingia species makes it special to all other plant species. In our present study, a morphological observation was undertaken by paraffin section and the non-normalized cDNA of leaves and flowers was sequenced using Illumina paired-end sequencing technology. By analysis the data of transcriptome, we made a preliminary study of the possible new genes of Helwingia Wild.(1) Like other common plants, the transverse sections of the male flower of H. Chinese showed that it has butterfly-like anthers. Its clinandrium have three layers, including to the tapetum, mesoderm and epidermis, arranging from inside to outside. The ovary is inferior and unilocular with one pendant ovule, which is axile placenta. The stigma divide and the pollen tube grow along the guided tissue. The longitudinal section of its’oval-shaped ovule indicated that the ovule had thick integument and an oval-shaped nucellar.(2) Paraffin section of the terminal bud of H. Chinensis var.crenata showed that there was a gap on top of the growing tip. According to the rib meristem along the gap, there might be another growing tip. This might indicate that structure may relate to the epiphyllous flowering of Helwingia Willd. Compared with the terminal bud of Swida wilsoniana, there was no axillary bud primordium can be seen besides the terminal bud of H. chinensis Batal.var.crenata. From the observation of the lateral bud of H. chinensis Batal.var.crenata, the rib meristem was not quite clear and no axillary bud primordium can be seen.(3) The young leaf of H. chinensis var.crenata has two layers of vascular tissues.The one of the leaf abdomen side had clear phloem and xylem, while the one of the blade back side had not well developed. The sprouting flower bud could also be seen. In the well developed leaves of Helwingia chinensis var.crenata, two vascular bundles were observed in the base sections of leaf. The vascular of the blade back side (with clear phloem and xylem) was much more developed than that of the leaf abdomen side. While only one vascular was seen on the leaf top part. The transverse section of the eustipes also showed only one vascular. It is assumed that the epiphyllous flowering of H. Willd might caused by the symphysis of the flower stalk and leafstalk.(4) Through the analysis of the "non-blastable"genes of H. japonica, five target genes were chose to isolated.We used the gDNA in H. Japonica to construct the pMOG22-Hj101315oversexpression vector, the pMOG22-Hj10076oversexpression vector and the pMOG22-Hj20587oversexpression vector. We used the cDNA template in H. Japonica to construct the pCAMBIA2300s-Hj40642oversexpression vector and the pCAMBIA2300s-Hj98105oversexpression vector. pMOG22-Hj101315, pMOG22-Hj10076and pMOG22-Hj20587were transformed into agrobacterium GV3101while the pCAMBIA2300s-Hj40642and pCAMBIA2300s-Hj98105were transformed into EHA105respectively.(6) The oversexpression vectors pMOG22-Hj101315and pMOG22-Hj10076was transformed into early flowering Nicotiana tabacum (transformed by PMV-PmCFT).All12pMOG22-Hj101315transformed plants lived after growing in the natural environment and among them66.7%of the transformed plant was positive.16of17pMOG22-Hj10076transformed plant lived after growing in the natural environment and87.5%showed positive.In a word, this study made a relatively deep morphological observation to Helwingia plant, through the vascular anatomy of Helwingia Willd, its odd floral architecture might caused by the fusion of flower stalk to petiole.These results supply the theoretical basis for further study. In addition, the transcriptome data will rich the genomic resources for Helwingia and in the order Asterids. Our preliminary research of the several genes would be a reference thought of further study of Helwingia.