Study On Molecular Taxonomy, Physiological Regulation Function Of Nitric Oxide And Gene Cloning Of FPS In Dendrobium Huoshanense

Dendrobium Sw.,perennial herbages in orchidaceae family,normally grow in shadow environment as facultative CAM plants.Many wild species of Dendrobium are rare traditional Chinese medicine.But there is great difference in medicinal components and pharmacological activities between different species of Dendrobium Sw.The original taxonomy and evolution research are based on morphology and geographical distribution, which make the results different.At present,there are several molecular marker methods has been used in the Dendrobium Sw.to study the genetic diversity and relationship. Sequence-related amplified polymorphism(SRAP) is a molecular marker technology, newly developed by Li and Quiros in 2001,however this new technology has not been applied in research of Dendrobium.Dendrobium.huoshanense,a precious wild medicinal plant in China,distributes in Huoshan County,Anhui Province.Because the request for environment comparatively stringent,outgrowth propagate frequency is highly low,adding to lasting collection,the wildness plant is close to annihilation.More attention was paid to its tissue culture and artificial cultivation in order to increase its production.However,there is little research about how to increase its production and the contents of medicinal components by regulating physiological metabolism and molecular regulation.Nitric oxide(NO) is a freely diffusible,gaseous compound and an important signal molecule in living organisms. In plants,NO influences aspects of growth and development,as well as the ability of plant responses to stress.Increasing evidences indicated that NO is a key signal messenger in disease resistance in plants.In this paper,the genetic diversity and kinship of medicinal Dendrobium Sw.were analyzed using RAPD and SRAP markers,and the subordinate classification system was confirmed,which lay a basis for exploitation and utilization of medicinal Dendrobium Sw. On the other hand,the physiological regulation effects of NO on the Dendrobium.huoshanense under stress and non-stress condition were studied to provide a theoretical foundation for action mechanism of NO to facultative cam plant.In addition,a cDNA fragment of farnesyl phosphate synthase genes was amplified by RT-PCR,which provided basic information for regulation of alkaloid synthesis for Dendrobium.huoshanense by using gene engineering technology.The results were as follows:1.This study was carried out in order to analyze genetic diversity and compare the Mark index(MI) between RAPD and SRAP markers in Dendrobium Sw.Using 36 random RAPD primers and 40 SRAP primers combinations,the genetic diversity of 9 species of Dendrobium was studied.A total of 281 loci were generated by 36 RAPD primers,of which 87.09%was polymorphic and the genetic distance ranged from 0.4942 to 0.7731.As for SRAP,1977 bands were obtained and 90.2%of them was polymorphic,and the genetic distance was from 0.3302 to 0.7892.There were some differences existing in the two cluster results revealed by RAPD and SRAP markers,they were as follows:compared to that of RAPD,the result of SRAP could more comfortably manifest the kinship,geographical origin,tree forms and so on,and also was more conformant to the traditional classification.Compared to RAPD,SRAP has higher marker efficiency and diversity detection ability.2.Normal conditions,using the wild seedlings and tube seedlings of D.huoshanense by presoaking in different SNP concentrations,the effects of NO on membrane lipid peroxidation and antioxdant enzyme activities were studied.Accordingly,the 0.1mmol·L^-1 and 0.5mmol·L^-1SNP were selected to treat wild seedlings,and 10,25,50 and 75μmol·L^-1SNP were determined as a appropriate concentration for tube seedlings.In addition,there were some interesting points revealed as follows.(1) Under high light intensity,the capacity of scavenging Active Oxygen for antioxidant system was increased and the level of cell membrane lipid peroxidation was decreased by presoaking with 0.1mmol/L SNP,which alleviated the photoinhibition on leaves of Dendrobium huoshanense significantly.On the contrary,the leaves presoaked in 0.5mmol/LSNP were severely inhibited by the light and the enzyme activities of SOD,POD and CAT were decreased.(2) The increase of the concentration of SNP can further improve chlorophyll content,soluble protein content,PAL activity and alkaloid content of Dendrobium. huoshanense when presoaking within 0～50μmol·L^-1SNP.So,NO can induce the accumulation of secondary metabolic product for Dendrobium.huoshanense.As elicitor, 50μmol·L^-1SNP is a suitable concentration.3.The physiological change law of photoinhibition for Dendrobium.huoshanense at high light intensity were summarized,and the chlorophyll fluorescence parameters were determined under 800μmol·m^-2·s^-1 light intensity based on lake model and puddle model. As a result,the chlorophyll fluorescence parameters Fv/Fm,Φ_psⅡ,qP,qL,NPQ,Φ_NPQ, ETR measured under 800μmol·m^-2·s^-1 light intensity decreased significantly,andΦ_NOrose remarkably,which showed that the Dendrobium.huoshanense suffered photoinhibition and the dissipation of excess light energy was not regulated efficiently by PSⅡreaction center. After dark recovery for 24h,the parameters Fv/Fm,Φ_psⅡ,qP,qL,NPQ,Φ_NPQ,ETR rose back,and the running state of PSⅡreaction center recovered gradually.Comparing with the original florescence parameters based on the puddle model,the new florescence parameters based on the lake model have been found more appropriate for estimating the running state of light energy conversion system of photosystemⅡ.In the experiment,the effects of NO donor sodium nitroprusside(SNP) on the chlorophyll fluorescence under high light stress were studied too.The result showed that the efficiency of light energy conversion and regulated non-photochemical dissipation of excess light energy for PSⅡwere improved by presoaking with 0.1mmol/L SNP,which alleviated the photo inhibition on leaves of Dendrobium huoshanense significantly. Therefore the PSⅡwas protected efficiently and the recovery was faster.On the contrary, the leaves presoaked in 0.5mmol/LSNP were severely inhibited under the strong light.4.Total RNA was extracted from young leaves of D.huoshanense.The degenerate primers were designed and synthesized according to the highly conservative sequences among the known famesyl phosphate synthase genes.A cDNA fragment(sh-fps) of 522bp was amplified by RT-PCR,which was subsequently cloned into pUCm-T Vector for sequencing.The sequence has been submitted to the GenBank data base,the accession number is EU681273.The sequence encoded 174 amino acids.By using the program of Blast on GenBank database,the amino acid sequence deduced from this fragment presented 79%-88%similarity with the FPP synthases from other plants.The amino acid sequence also contains two FPS conservative core fragments.The two areas always appear in isoprenoid transferase,which possibly represent the active center of the sort of enzymes.