Cloning And Expression Analysis Branching And Development Gene LATERAL SUPPRESSOR (NfLs) In Nervilia Fordii

Branching is a biological trait of plants to adapt to the environment, which directly affect the amount of biomass and production. During plant development, the lateral bud growth is inhibited because of the apical dominance. Research shows that branching and development of plants are subject to a variety of plant hormone, regulation and genes involved in plant branching conserved in different species, which makes them possible to be characterised in medicinal plants. For example, the DOH1gene has been cloned and characterised from Dendrobium nobile, which inhibits the growth of meristems, promotes the formation of apical meristem and precocious flowering. Hence characterizing genes involved in plant branching from medicinal plants will not only further elucidate the plant development mechanism, but also provide theoretical basis for obtaining multi-branching plants to increase production and ease the shortage of certain medicinal plant resources, such as Nervilia fordii, one of the endangered authentic herbs from Lingnan area.Qingtiankui (Herba seu Rhizoma Nervilia fordii) comes from Nervilia fordii (Hance) Schltr. of Orchidaceae which has been included in "Guangdong Traditional Chinese Medicinal Materials Standards". The medicinal part is leaves or the whole plant. It mainly distributes in Guangdong, Guangxi and Hainan province. Qingtiankui has the efficacy of detoxification, relieving pain, lungs and cough, etc. and has been mainly used for pediatric respiratory disease, infantile malnutrition and bruises. Since the low breeding rate and highmarket demand, coupled with excessive excavation, Nervilia fordii has long been in endanger. However the artificial cultivation can not meet the growing market demand. Increase Nervilia fordii medicinal resources become a problem to be urgently solved.In this thesis, one of the branching related genes was found from transcriptome database of Nervilia fordii and preliminarily studied, expecting to lay the foundation of breaking the status quo that only one bulb grows one leaf in Nervilia fordii under the normal circumstances. The main content in the current thesis includes:(1) Selecting suitable total RNA extraction method from Nervilia fordiileaves. Five methods of CTAB, SDS, RNeasy Plant Mini Kit, RNAiso Plus, RNAiso for Polysaccharide-rich Plant Tissue, combined with four extraction reagents, were used to extract the total RNA from Nervilia fordii. We had acquired the best extraction method, which ensured the smoothly carrying out of the subsequent experiments.(2) Through screening branching related Unigenes in the transcriptome database of Nervilia fordii, the Unigene37948, which is homologous to tomato LATERAL SUPPRESSOR gene is obtained. RACE technique was used to get full-length cDNA sequence of Nervilia fordii lateral suppressor gene (NfLs). The NfLs cDNA sequence contains a1191bp Open Reading Frame that encodes396amino acids, which belongs to GRAS family predicted by NCBI BLASTP. The sequence of NfLs has been submitted to GenBank and obtained the accession number of KJ584362.(3) Using β-tubulin as reference gene, real-time quantitative PCR was used to analyse NfLs gene tissue-specific expression pattern in the plant (including leaves, petioles, rhizomes, corms) of Nervilia fordii and expression style in Nervilia fordii leaves under the regulation of circadian clock within24hours. The results revealed that NfLs gene expressed the highest in leaf, however expressed the lowest in bulb, while in the root had more expression than in the petiole. The expression of NfLs gene within24hours showed a light/dark fluctuation, which had a higher expression in the dark cycle than the light cycle.(4) After inserting NfLs gene into the vector by double digestion and ligation, the plant expression vector pRI101AN-DNA-NfLs was constructed and transformed into Agrobacterium EHA105and was then transformed into Arabidopsis col-0using’floral dip’ approach. Further experiments will be carried out to characterize the role of NfLs gene in braching and development of plant.In this paper, we preliminary studied.NfLsgene, which is proposed to be involved in branching and development in Nervilia fordii. It helps setting up a new thought that to solve the problem of endangered resources and to ensure the sustainable use of medicinal resources of Nervilia fordii by increasing the upground production.