金钗石斛N6组培体系的建立及生物碱合成关键酶基因的挖掘

【作者】 刘媛媛；

【导师】 丁小余；

【作者基本信息】 南京师范大学 ， 植物学， 2020， 硕士

【摘要】 兰科石斛属植物金钗石斛(Dendrobium nobile Lindl.),主要分布于我国广西、贵州、海南等地,具有滋阴清热、益胃生津等作用。相比石斛属的其他植物,金钗石斛的生物碱含量较高,因此具有珍贵的药用价值。由于其野生资源较少,再加上人为的发掘开采,金钗石斛现已被列入濒危物种名单。为扩大金钗石斛的产业化培养,提高其药用价值,本论文对其进行了如下研究:(1)金钗石斛组织培养体系的研究。在本研究中,对金钗石斛萌发、增殖、壮苗和生根阶段的基本培养基和培养基中的添加物进行筛选,通过比较生物量、生长形态和药用成分等相关参数,筛选出最适的培养基及添加物。在种子萌发阶段,发现相较于B5和MS培养基,N6培养基更适合作为基本培养基,并且利用正交试验对培养基中天然添加物椰汁和土豆提取液的浓度进行了筛选,确定最适合金钗石斛种子萌发的培养基为:N6+15%椰汁(添加20 g/L蔗糖和7.2 g/L琼脂)。接着在原球茎增殖分化阶段筛选到最适培养基为:N6+10%椰汁+6%土豆提取液(添加20 g/L蔗糖、7.2 g/L琼脂和1 g/L蛋白胨)。在金钗石斛壮苗阶段不需要添加基本培养基,对天然添加物进行正交试验发现最适培养基为:8%香蕉提取液+8%土豆提取液(添加20 g/L蔗糖、7.2 g/L琼脂、1 g/L蛋白胨、3 g/L花宝二号和1.5 g/L活性炭)。最后,对生根阶段的基本培养基和添加物浓度进行筛选,发现最适培养基为:N6+6%香蕉提取液+4%土豆提取液(添加20 g/L蔗糖、7.2g/L琼脂、1 g/L蛋白胨、1.5 g/L活性炭和3 g/L花宝二号)。(2)外源激素诱导对金钗石斛有效药用成分积累的影响。金钗石斛中的生物碱和黄酮类等成分含量较高,具有重要的价值,目前大量研究致力于提高其药用代谢物的含量,实现产业化生产。本研究在培养基中添加不同浓度的茉莉酸甲酯和水杨酸,通过测定其药用成分的含量,并辅以生长形态和生物量等数值来筛选出诱导药用活性物质的最适外源激素。结果发现,用25μmol/L茉莉酸甲酯处理40天时,金钗石斛的组培苗中药用成分含量最高,且远高于对照组。(3)金钗石斛生物碱合成关键酶基因的挖掘。本研究中,以25μmol/L茉莉酸甲酯处理40天时的壮苗阶段的金钗石斛组培苗作为处理组,未添加茉莉酸甲酯的组培苗作为对照组,进行三代转录组测序,共获得15,089条isoform,平均长度为1,403 bp。其中98.28%的isoform都能被注释到NR、GO、KEGG、COG/KOG和Swiss-Prot数据库中,分别有14,830、10,369、7,810、9,638和12,894条。接着,共发现DEG 3,284个,其中表达上调的有1,433个,表达下调的有1,851个。在KEGG差异富集分析的结果中找出与生物碱合成相关的代谢通路有5条,并在这些通路表达上调的基因中找出6个生物碱合成关键酶基因:CYP701、QHS1、TPS21、ato B、DXR和isp G。利用Real-time PCR方法检测各基因在对照组和处理组中的表达量,发现这些基因在处理组中的表达量均高于对照组,验证了转录组分析结果。对金钗石斛进行代谢组学分析,其结果显示在对照组和处理组中与生物碱合成相关的代谢物表达量相差较大,处理组的表达量显著高于对照组,这一结果佐证了转录组分析中CYP701、QHS1、TPS21、ato B、DXR和isp G这6个基因与金钗石斛的生物碱合成相关。本论文通过对金钗石斛不同阶段基本培养基和天然添加物的筛选,建立了适合金钗石斛各阶段组培苗生长的培养基,为其他石斛属植物的组织培养提供了参考;对诱导金钗石斛中药用成分含量增加的外源激素进行了探索,为提高植物中有效药用成分含量的研究提供了借鉴;同时,对金钗石斛中生物碱合成关键酶基因的挖掘,为金钗石斛中生物碱的合成机制提供了线索,进而为高产生物碱的金钗石斛优质种苗的培育提供了理论基础,具有一定研究和应用价值。更多还原

【Abstract】 Dendrobium nobile Lindl.is one of species in Dendrobium,Orchidaceae,mainly distributed in Guangxi,Guizhou,Hainan and other places of China.It has a lot of functions such as nourishing yin,clearing heat,benefiting stomach and invigorating.D.nobile has great medicinal value because of its high content of alkaloid.Now,D.nobile is on the list of endangered species due to its few wild resources and excessive excavation.In order to expand the industrial cultivation of D.nobile and increase its medicinal value,this study conducted the following studies:(1)The research on the tissue culture system of D.nobile was carried out.In this study,the basic medium and additives for the germination,proliferation,seedling growth and rooting stages of D.nobile were screened by comparing the relevant parameters such as the biomass,growth morphology and medicinal components.During the seed germination stage,it was found that N6 medium was more suitable as the basic medium than B5 and MS medium.And the concentration of natural additives(coconut juice and potato extract)in the medium was screened and determined by orthogonal test.The best medium for seed germination of D.nobile was:N6+15%coconut juice,meanwhile supplemented with sugar 20 g/L,agar 7.2g/L.Then,the optimal medium was selected at the stage of protocorm proliferation:N6+10%coconut juice+6%potato extract,meanwhile supplemented with sugar 20g/L,agar 7.2 g/L,and peptone 1 g/L.At the stage of seedling growth of D.nobile,it was not necessary to add basic medium,and the orthogonal test of natural additives showed that the optimal medium was:N6+8%banana extract+8%potato extract,meanwhile supplemented with sugar 20 g/L,agar 7.2 g/L,peptone 1 g/L,Hyponex NO.2 3 g/L and activated carbon 1.5 g/L.Finally,the optimal basic medium and the concentration of additives in the rooting stage was found:N6+6%banana extract+4%potato extract,meanwhile supplemented with sugar 20 g/L,agar 7.2 g/L,peptone 1g/L,activated carbon 1.5 g/L,Hyponex NO.2 3 g/L.(2)The effect of exogenous hormone stimulation on the accumulation of effective medicinal components of D.nobile has been explored.At present,a lot of researches were devoted to increasing the content of medicinal metabolites and realizing industrial production.In this study,we added different concentrations of methyl jasmonate and salicylic acid to the culture medium,and the most suitable exogenous hormone for inducing the synthesis of secondary metabolites was selected by measuring the content of its medicinal ingredients,supplemented by growth morphology and biomass.It was found that when the tissue culture seedlings were treated with 25μmol/L methyl jasmonate for 40 days,their content of medicinal ingredients was the highest and it was much higher than that in the control.(3)Screening of key enzyme-coding genes related to alkaloid synthesis has been conducted in D.nobile.In this study,the tissue culture seedlings of D.nobile treated with 25μmol/L methyl jasmonate for 40 days were choosed as the treatment,while the tissue culture seedlings without methyl jasmonate were used as the control.In total,we obtained 15,089 isoforms,and the average length was 1,403 bp after removing redundancy.98.28%of the isoforms were annotated by using NR,GO,KEGG,COG/KOG and Swiss-Prot databases respectively.Next,3,284 DEGs were found by classifying statistics on differentially expressed genes,of which 1,433 DEGs were up-regulated and 1,851 DEGs were down-regulated.According to KEGG differential enrichment analysis,5 metabolic pathways related to alkaloid synthesis were found,and 6 key enzyme-coding genes(CYP701,QHS1,TPS21,ato B,DXR and isp G)for alkaloid synthesis were identified among genes whose expression was up-regulated in these pathways.Real-time PCR was used to detect the expression of each gene between the control and the treatment.It was found that the expression of these genes in the treatment were higher than those in the control,which verified the result of transcriptome.The result of metabolomics of D.nobile showed that the expression level of metabolites related to alkaloid between the control and treatment differed greatly and the expression level of treatment was significantly higher than the control.This result corroborates that these genes(CYP701,QHS1,TPS21,ato B,DXR and isp G)in transcriptome analysis were related to the alkaloid synthesis of D.nobile.In this study,by screening the basic medium and natural additives of D.nobile at different stages,the medium suitable for the growth of tissue culture seedlings of D.nobile was optimized,which provided a reference for other Dendrobium species;The effect of exogenous hormones involved in the increasing of medicinal ingredients in D.nobile has been explored,which provides a template for the research on improving the content of effective medicinal ingredients in other plants.At the same time,the discovery of the key enzyme-coding genes related to alkaloid synthesis in D.nobile has provided clues for the synthesis mechanism of active ingredients in other medicinal plants and the screening of key enzyme-coding genes.更多还原