【作者】 李欣；

【导师】 刘迪秋；

【作者基本信息】 昆明理工大学 ， 药物化学， 2020， 硕士

【摘要】 三七[Panax notoginseng(Burk.).F.H.Chen]是我国一种珍贵的中草药,长期以来被用于促进血液循环和治疗瘀伤。大量研究表明三七具有抗血小板聚集、溶栓、降低血压、抗炎等多方面的药理作用,且临床应用疗效显著。但三七较长的生长周期和独特的生长环境容易诱发病害发生。三七根腐病是一种严重的土传病害,全年均可发生,常年的发病率保持在5%～20%,严重时可达到70%。由三七根腐病引发的多种连作障碍更是使得三七种植成本增加,土壤利用率降低,对三七产业化发展相当不利。三七黑斑病是三七种植园中普遍发生的一类叶部病害,是继三七根腐病之后的一类为害较为严重的三七病害,常年的发病率保持在10%左右,严重的时候可占总发病植株60%以上。这类真菌病害的病原真菌以菌丝或厚垣孢子在三七种子、种苗、病根、落叶、残枝和土壤上越冬,并以它们作为传播媒介进行扩散。因此,任何快速、准确的检测病原真菌的方法对防止三七根腐病和三七黑斑病的发生以及流行都具有重要意义。本论文的研究工作及得到的主要结果如下:1.为了建立一种准确、快速的检测三七根腐病的分子检测体系,本研究针对引发三七根腐病的几种镰刀属真菌开发了一套实时荧光定量PCR快速检测方法。通过基因筛选得到了四种镰刀属真菌的基因位点,它们分别是:茄腐镰刀菌的氨基己二酸还原酶Lys2基因、尖孢镰刀菌的脂肪酸ω-羟化酶CYP505基因、轮枝镰刀菌的博来霉素水解酶Bleo基因和禾谷镰刀菌的Aba A基因。基于这些特异的基因位点设计了它们的实时荧光定量PCR引物。并采用实时荧光定量PCR技术构建了它们的定量检测标准曲线,最终建立了三七根腐病中镰刀属致病真菌的实时荧光定量PCR检测方法。对采集到的三七病害样品进行了检测,结果表明,根腐病植株中镰刀属真菌含量显著高于健康植株。表明镰刀属真菌是引起三七根腐病的一类重要致病菌。同时也证明本研究建立的快速分子检测技术灵敏度高,快速,可以为田间三七根腐病、带菌土壤以及种子种苗的快速分子检测提供理论依据和技术支持。2.为了使诊断技术更加简便易行,本研究以茄腐镰刀菌氨基己二酸还原酶Lys2基因为靶点,设计了实时荧光环介导等温扩增引物。运用实时荧光环介导等温扩增方法对7个三七病株样品进行了检测。结果表明,与实时荧光定量PCR相比,荧光定量环介导等温扩增技术速度快,还可以实现扩增与检测同步进行。3.为了快速检测三七黑斑病,本实验建立了检测三七黑斑病病原菌人参链格孢(Alternaria panax)的实时荧光定量PCR方法。通过基因筛选得到了特异扩增人参链格孢的3-磷酸甘油醛脱氢酶GPD基因。并制备了该基因的重组质粒标准品,构建了定量检测的标准曲线,最终建立了人参链格孢的实时荧光定量PCR检测方法。采样检测结果表明三七黑斑病植株中的人参链格孢含量显著高于健康植株且该方法灵敏度高,检测模板浓度可低至0.19 pg/μL。4.链格孢属真菌广泛存在于自然界,是一种重要的作物病害。本实验采集了三七主产区的一种三七叶部病害的样本,从中分离到了一株病原菌,对它进行了致病性分析、形态学鉴定、多位点序列分析和系统进化树构建。分析结果表明本研究分离到的病原菌为交链格孢(A.alternata)。另外,本研究建立了交链格孢的实时荧光定量PCR快速检测体系,采样检测的结果表明这种三七叶部病害植株中的交链格孢的含量显著高于健康植株。综上所述,本研究建立的快速分子检测体系反应快,灵敏度高,能明确三七病株及三七种植土壤中病原真菌数量的动态变化。可以为三七土壤处理、根腐病、黑斑病的早期诊断和动态监测以及后续进一步认识病原真菌的致病机制,并有针对性地研究高效、环保的防治方法提供技术支持。本研究为深入认识三七叶部病害的病原菌及其分子检测奠定了基础。更多还原

【Abstract】 Panax notoginseng(Burk.).F.H.Chen is a precious Chinese herbal medicine,which has been used for a long time to promote blood circulation and treat bruises.Researchs showed that P.notoginseng have many pharmacological effects,such as antiplatelet aggregation,thrombolysis,lowering blood pressure,anti-inflammatory and so on,and its clinical effect is significant.However,the long growth period and unique growth environment of P.notoginseng are easy to induce diseases.The root rot disease of P.notoginseng is a serious soil borne disease.It can happen throughout the year.The incidence rate is always 5%～20%,and it can reach 70% in severe cases.The continuous cropping obstacles caused by root rot increased the planting cost of P.notoginseng and decreased the utilization rate of soil,which is quite unfavorable to the industrialization development of P.notoginseng.The black spot is a kind of leaf disease that commonly occurs in the plantations of P.notoginseng.The incidence rate of black spot perennial is about 10%,which accounts for more than 60% of the total incidence at June.The pathogenic fungi live through the winter on the seeds,seedlings,roots,leaves,stumps and soil of P.notoginseng with hyphae or chlamydospores,and use them as the transmission medium for diffusion.Therefore,any rapid and accurate detection method of pathogenic fungi is of great significance to prevent the occurrence and epidemic of root rot and black spot.The main results are as follows:1.In order to establish an accurate and rapid molecular detection system for the root rot of P.notoginseng,a real-time fluorescent quantitative PCR method was developed for the rapid detection of Fusarium fungi.The specific gene loci of four species of Fusarium fungi were obtained through screened,they are aminoadipate reductase Lys2 gene of F.solani,fatty acid ω-hydroxylase gene of F.oxysporum,bleomycin hydrolase gene of F.verticillioides and abscisic acid Aba A gene of F.graminearum.Based on these specific loci,real-time fluorescent quantitative PCR primers were designed.And the quantitative detection standard curve was constructed by SYBR Green I real-time fluorescence quantitative PCR technology.Finally,a real-time fluorescent quantitative PCR method for the detection of Fusarium pathogenic fungi in P.notoginseng was established.The results showed that the content of Fusarium fungi in root rot plants was significantly higher than that in healthy plants.It is proved that Fusarium is one of the most important pathogens causing root rot of Panax notoginseng.At the same time,it also proved that the rapid molecular detection technology established in this experiment has high sensitivity and good specificity,which can provide basis and technical support for the rapid molecular detection of root rot,soil with bacteria and seeds and seedlings in the field.2.In order to make the diagnosis more convenient,we designed real-time fluorescent loop mediated isothermal amplification primers based on the specific aminoadipate reductase Lys2 gene of F.solani.Seven samples of P.notoginseng plants and soil were detected by real-time fluorescence loop-mediated isothermal amplification technology.Compared with real-time PCR,fluorescent quantitative loop mediated isothermal amplification is not only fast and highly specific,but also can achieve simultaneous amplification and detection.3.In order to detect the black spot of P.notoginseng quickly,fluorescence quantitative PCR method for the detection of A.panax was established.A specific gene,glyceraldehyde 3-phosphate dehydrogenase gene of A.panax,was obtained through screening genes.The recombinant plasmid standard of the gene was prepared,the standard curve of quantitative detection was constructed,and the real-time fluorescent quantitative PCR method to detection A.panax was finally established.The results showed that the content of A.panax in black spot plants was significantly higher than that in healthy plants.In addition,the method has high sensitivity and the concentration of detection template can be as low as 0.19 pg /μL.4.Alternaria fungi widely exist in nature,and it is an important crop disease.In this study,the samples of leaf disease of P.notoginseng were collected and the pathogenic fungi were isolated.The pathogenic inoculation experiment,morphological identification,multi site sequence analysis and phylogenetic tree study were carried out in this study.The results showed that the pathogen was Alternaria alternata.In addition,a real-time PCR system for A.alternata was established.The results showed that the content of A.alternata in the diseased plants was significantly higher than that in the healthy plants and the sensitivity of this method was high.In conclusion,the rapid molecular detection method established in this study is highly specific and sensitive,and can be used to reveal the dynamic changes of pathogenic fungi concentration in the complex P.notoginseng plants and planting soil.Furthermore,it can provide technical supports for the soil treatment,early diagnosis and dynamic monitoring of root rot and black spot,and the follow-up further understanding of the pathogenic process and mechanism of pathogenic fungi,and targeted research on efficient and environmental protection methods to provide technical support.This study laid a foundation for further understanding of the pathogen and molecular detection of the disease of P.notoginseng.更多还原