Dendrobium Plant Genetic Relationship Research And ACC Oxidase Gene Clone

Dendrobium species are perennial plants of orchidaceae. Dendrobium is one of the largest genus of the orchid, and also an important horticultural plants. Many species of Dendrobium are precious ornamental plants and tranditional Chinese medicine herbals. The traditional classification of the genus Dendrobium relies mainly on morphology and geographical distribution, but the classification accuracy is relatively low, and different researchers have different classification system, thus there exists a lot of difference.In recent years, molecular markers methods have been applied in Dendrobium genetic diversity and relationship analysis.As one kind of Orchidaceae, Dendrobium officinale is also known as Dendrobium candidum, one of the most precious medicinal Dendrobium species. Dendrobium officinale is attached on the wet trunk of trees or in the crack of stones of500-1600meters above sea level. Its habitat conditions is very unique and the ability to reproduce is very weak. In addition,in recent years the exeessive gather and the destruction of natural habitats, Deandidum officinale wildiife resources increasingly decreaseing. To protect the of Dendrobium officinale resources and increase drug source, now there are many scholars do researches on the tissue culture of Dendrobium officinale and the rapid propagation, but there are very few researches on sterile sowing the seeds obtained by artificial pollination and genetic diversity analysis based on molecular markers.ACC oxidase (1-aminocyclopropane-carboxylic oxidase) is a key enzyme in the biosynthesis of ethylene.Ethylene can induce enzyme involved in metabolite biosynthesis such as phenylalanine ammonia-lyase (PAL), peroxidase, polyphenol oxidase and chitinaseo some researchers reported that PAL is the key factor for the synthesis of Dendrobium alkaloidsISSR molecular marker technology was used to analysis the genetic relationship between the26materials of the genus Dendrobium, and do some studies on the conditions of Dendrobium officinale artificial pollination and sterile germination. In addition, we cloned and express the ACC oxidase gene of Dendrobium officinale in E. coli. The main findings are as follows:1.The genetic relationship of the26materials were analyzed by using ISSR Marker.5Primers were chosen from100ISSR-PCR primers for PCR amplification of Dendrobium material DNA,70bands were amplified, and63bands of them were polymorphic(percentage of polymorphic bands was90%) with an average of12.6polymorphic bands per primer.2.Using DPS software to deal with all date. Cluster analysis indicated the genetic distance of26materials was0.0334-0.7314, and all the materials could be divided into six groups with the genetic distance at0.43. I:Den.heterocarpum Lindl.、Den.denneanum Kerr.、 Den.fimbriatum Hook.、Den.chrysanthum Lindl.、Den.crepidatum Lindl.ex Paxt.、 Den.wardianum Warner.、Den.hercoglossum Rchb.f.、Den. aphyllum(Roxb.)C.E.Fisher.、 Den. nobile Lindl.、Den.primulinum Lindl.、Den.devonianum Paxt.、Den.falconeri Hook.、 Den.moniliforme(L.)Sw.、Den.officinale Kimura et Migo.;Ⅱ:Den.loddigesii Rolfe.、Den. Longicornu Lindl.、Den.Capillipes Rchb.f.、Den.lindleyi Stendel.、Den.hancockii Rolfe. Den.salaccens(Bl.)Lindl.;Ⅲ:Den.chrysotoxum Lindl.、Den.densiflorum Lindl.;Ⅳ: Den.williamsonii Day et Rchb.f.、Den.cariniferum Rchb.f.; V:Den.terminale Par.et Rchb.f.; Ⅵ:Den.trigonopus Rchb.f.3. Artificial pollination was performed by using:(1) xenogamy;(2) different inflorescence pollination within one plant;(3) different flwers pollination within one inflorescence;(4) the self pollination;(5) the control (natural pollination after bagging), the results showed that the seeding rate reaches the highest under cross pollination before10o ’clock in the morning after blooming2d to4d.4. Choose healthy seeds obtained by artificial pollination methods (8months old seed) for aseptic seeding, it showed that the optimal medium for seeds germination was MS+NAA0.5mg/L+potato juicel0%+active charcoal0.5%, and cultured for7d in the dark then under light.5. A homolog of ACC oxidase gene (ACO) was isolated from flowers of Dendrobium officinale kimura et migo by PCR-method. The obtained cDNA of Do ACO is970bp long and contains an open reading frame (ORF) encoding a protein with314amino acid residues (The sequence has been submitted to the GenBank data base, the accession number is JX679494)6. The constructed prokaryotic expression vector pET-28a-ACO was transformed into E. coli BL21(DE3) and expressed. One foreign protein, which was the same size as expectation, was obtained. This research layed the foundation for studying ethylene biosynthesis and had an implication to improve the alkaloids content in the future.