| Euodiae Fructus was a commonly used Chinese herbal medicine. Evoida rutaecarpas natural resources were rich and had a wide distribution but genetic variation were complex and genetic background was not clear because these reasons that the eclolgical environment very different,the ecological condition complicated,widely cultivated,for thousands of years all over the frequent exchanges between germplasm resources,drug habits vary greatly and had many species and varieties in Evodia.Now people about the phylogenetic evolution relationship between Evoida rutaecarpa of different species and cultivars and varieties and quality evaluation of genuine medicinal materials of different origin was not clear. In addition,drought stress susceptible to Evodia rutaecarpa during the growth of plants,resulting in excessive accumulation of active oxygen and active oxygen scavenging system disorders,caused serious adverse effects on the growth and quality of medicinal material plant.Therefore, studies on the diversity of germplasm resources of Evodia rutaecarpa and physiological drought has important scientific and practical significance.ObjectiveAlthough molecular marker technique such as AFLP and RAPD on Evodia rutaecarpa germplasm genetic diversity study were used, but because of the number of samples and sample source, germplasm without extensive collection of other Evodia rutaecarpa advocate produce a division, so the genetic analysis had the certain limitation. In this study, Evodia rutaecarpa genuine medicine source plant cultivation and medicinal materials were collected extensively. The genetic germplasm resources and their diversity were analyzed and HPLC fingerprinting was construced. Germplasm resource of Evodia rutaecarpa was evaluated objectively. Its genetic diversity was discussed. These experiments could clarify the phylogenetic relationship of Evodia rutaecarpa cultivation. In addition, Evodia rutaecarpa in the growth process is vulnerable to drought stress, seriously affect the quality and yield of medicinal materials. At present, research on the mechanism of resistance to drought stress mainly concentrated on crops, and little study of medicinal plants, also study on drought resistance of Evodia rutaecarpa not been reported. This experiment using artificial simulated drought stress environment, study of Evodia rutaecarpa cuttings under variation of drought stress and rewatering during various physiological and biochemical indexes and cloned the full-length sequence of key resistance gene Cu/Zn-SOD and Mn/Fe-SOD genes, the next step for the research has laid a solid foundation.Methods1. Literature researchThe ancient books related to this study and the literature home and abroad were retrivealed and summarized. The research trends related to the paper was reviewed. Mainly includes the following four aspects:(1) Herbal research of Evodia rutaecarpa include Survey medical history, resource distribution, function and application, plant characteristics and variety identification.(2) Evodia rutaecarpa germplasm genentic diversity’s research progress about morphological markers, cytological analysis, DNA molecurlar markers and DNA barcoding.(3) Evodia rutaecarpa s cell osmotic adjustment and Active Oxygen Scavenging system(ROS) and plant SOD progress in drought stress environment.(4) Research progress of Evodia rutaecarpa on chemical quality evaluation include chemical fingerprint and chemical composition.2. Experimenal researchDNA molecular marker technology, DNA barcoding and chemical analysis method for the evaluation of germplasm resources and diversity of Evodia rutaecarpa herbs of different source and origin in this paper. The several physiological and biochemical indexes of cutting seedling in artifical simulated drought stress conditions were studied. Finally, Cu/Zn-SOD and Mn/Fe-SOD gene of Evodia rutaecarpa were cloned. The research contents of this paper include:(1) Seventy-one cultivars of Evodia on originates from Guizhou province, Jiangxi province, Hunan province, Hubei province and Guangdong province of plant germplasm resources were analyzed to study the genetic diversity by ISSR molecular markers.(2) The Evoida genus18samples of different varieties and different sources were analyzed using ITS2DNA barcoding technology.(3) The contents of six kinds of chlorogenic acid, isorhamnetin-3-O-galactoside,quercetin, evodiamine, rutaecarpine and evocarpine were determined in37batches of material medicines used HPLC method. HPLC chemical fingerpring on37batches of material medicines of Evodia rutaecarpa were constructed using HPLC method.(4) The Evodia rutaecarpa cutting seedlings were treated using different concentration of PEG-6000solution and different time. The physiological and biochemical indexes such as soluble protein, soluble sugar, praline, malondialdehyde(MDA), catalase(CAT), peroxidase(POD) and superoxide dismutase(SOD) were determinated.(5) By homology cloning method for cloning the core fragments of Evodia rutaecarpa’s Cu/Zn-SOD and Mn/Fe-SOD genes, specific primers were designed according to the sequences.3,5-Rapid Amplification of cDNA Ends(RACE) technology was used to clone the full-length cDNA sequence of the two genes.ResultsThrough the experimental study, this paper has made the following achievements:(1)ISSR genetic diversity of germplasm resources of Evodia rutaecarpa experiment has obtained the following results. The genetic divertiy of Wuzhuyu populations in Guizhou province than Shihu groups of Jiangxi province. The diversity of Shihu populations was higher. The genetic diversity of the populations of different regions are arranged from high to low is:7,10,1,11,9,3,6,2and4. Three genetic base original species of Evodia rutaecarpa medicine diversity from high to low for:Wuzhuyu>Shihu>Shumaowuzhuyu. Different areas and different varieties of groups differentiation are larger, especially the Gst reached0.8164that from different pophlations and different varieties of group Gst was0.6898, but gene flow calculation result was just the opposite. Different populations of Nm less than cultivars in different groups, but the gene flow were weak. The reasons are which reproduction of Evodia rutaecarpa are cutting propagation and growth environments conditions also had certain influence. UPGMA clustering showed that ISSR genetic diversity analysis can not only separate the genuine Evodia rutaecarpa original plant with fake-Evodia glabrifolia and Evodia rutaecarpa also would be different samples of different varieties or even different areas of original samples are effective resolution and shows the relationship between the species, the genetic distance and cluster analysis. ISSR genetic diversity analysis is a reliable means to study the Evodia rutaecarpa genus relationship and cultivar identification.(2) Evodia rutaecarpa germplasm resources ITS2DNA barcoding experiment has obtained the following experimental results. ITS2sequences length are220bp, yielding a total of17single nucleotide(SNP) sequence variation sites, accounting for7.73%of the total length, in which specific stie are seven. The interspecific genetic distance Shihu and Wuzhuyu recently0.036, interspecific genetic distance Shumaowuzhuyu and Shihu up to0.039. The K2-P distance distribution of base the original species and Evodia glabrifolia in the0.042to0.067. Evodia rutaecarpa and Evodia glabrifolia nearest genetic distance. Neighbor-joining(NJ) to construct that Evodia rutaecarpa genus system clustering tree display that18samples are divided into three branches which Evodia glabrifolia to form a single branch,phylogenetic relationships and Evodia rutaecarpa three base stock plant far.(3) The following experimental results of drug content determination were obtained that wuzhuyu and shumaowuzhuyu’s contents of evodiamine, rutaecarpine and evocarpine were higher than shihu’s. These alkaloids contents had the tendency that correlated with altitude while the contents of chlorogenic acid, isorhamnetin-3-O-galactoside and quercetin did not show the above rule.HPLC fingerprint experiment has obtained the following experimental results. Ten batches samples of Evodia rutaecarpa in Jiangxi has19common peaks and the retention time of RSD is less than0.4%and the peak area of RSD is large, which show that Jiangxi province medicinal Evodia rutaecarpa is basically the same as in the chemical composition of the components but the amount of different Guizhou province Yuqing country dragon town, Guizhou Shiqian baisha town, Guizhou Shiqian baisha town, Guizhou province Songtao country zhengda town, Hubei, Hunan and Jiangxi control sample fingerprint similarity was0.952,0.969,0.965,0.874,0.973. Therefore, two different varieties of Evodia rutaecarpa and Shihu similarity, two species of medicinal Evodia rutaecarpa quality more consistent. Study on HPLC fingerprint of the Euclidean distance martrix and ISSR molecular marker genetic similarity coefficient matrix of related coefficient was0.469that the correlation was not significant.(4) Drought stress experiment of Evoida rutaecarpa cutting seeding has obtained the following experimental results. Changes in the trend of the overall content of soluble protein in leaves is that increased first and then decreased with the increasing of stress intensity and time, the complex process of water, soluble protein content decreased slowly but higher than the content of stress state. Changes in the trend of overall soluble sugar content is that an upward trend with the stress intensity and time, the complex process of water, soluble sugar content was significantly decreased but higher than the CK can not be restored to the level of CK. Changes in the trend of overallproline content is that has been an upward trend with the stress intensity and time and at rehydration process that its content is stress showed the downtrend but with the rehydration time prolonging the content and slowly rising. Leaf blade MDA change the general trend is that has been an upward trend with the stress intensity and time and rehydration process, except in severe stress, seven days stress after rewatering conditions the content of MDA continues to rise the content of MDA is relatively stress showed decline. Change tendency of SOD activity in leaf is that has been an upward trend with the stress intensity and time and rewatering process its activity continues to rise and the late activity decreased slowly. Change tendency of CAT activity in leaf is that increased first and then decreased with increasing stress intensity and at rewatering process with the extension of time and slow rise. Change tendency of POD activity in leaf is that has been an upward trend with the stress intensity and time and at rewatering process decreased rapiadly in short time with the time prolonging fell down but could not recover to the level of CK.(5) Evoida rutaecarpa’ s Cu/Zn-SOD and Mn/Fe-SOD gene cloning experiment has obtained the following experimental results. The full-length cDNA sequence of Cu/Zn-SOD gene is717bp and the open reading frame is459bp that encoding152amino acids. Its Genbank accession number is JQ285851This amino acid molecular weight is15142.7Da and isoelectric point is5.47, belonging to the hydrophilic acidic amion acid and stable protein. The full-length cDNA sequence of Mn/Fe-SOD gene is1048bp and the open reading frame is687bp that endoding229amino acids. Its Genbank accession number is JQ285852. This amino acid molecular weight is25434.1Da and isoelectric point is7.16, belonging to the hydrophilic basic protein and stable protein.ConslusionAnalysis of senenty-one samples of germplasm of genus Evoida rutaecarpa in different varieties and different source of the genetic diversity of ISSR analysis and DNA barcoding region ITS2experiments proved that the genetic relationship of the original plants is closer but still affected areas, environment and reproduction method but has the differentiation. HPLC chemical fingerprint proved that Shihu and Shumuwuzhuyu contained in the chemical composition and content. The chemical composition of both confirmed the close genetic relationship. This result is common that the experimental result of molecular marker.But HPLC fingerprinting culster analysis and ISSR clustering and ITS2culstering results were not entirely consistent that showed themedicinal plant’s secondary metabolite accumualtion may be affected by the objective factors such as environmental conditions and culture level.HPLC determination results showed that Evodia rutaecarpa’s alkaloid content had the tendency that related to altitude and cultivation.The change of cell osmotic substances and ROS system in cutting seedling at drought stress and rewatering time proves that the permeability of the somotic and ROS system is not synchronous operation under adverse conditions and the changing law is complicated. Soluble protein, free proline content and POD activity were increased firstly and then decreased. MDA content and SOD activity were increased. CAT activity showed "N"type curve change, namely the first increase then decrease and increase trend.SOD gene cloning experiments proved that Cu/Zn-SOD and Mn/Fe-SOD gene have high similarity with other plants of similar genes. This experiment lays a foundation for breeding of drought resistance and SOD gene regulation. |
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