【作者】 谢晓亮；

【导师】 刘玉军；

【作者基本信息】 北京林业大学 ， 植物学， 2008， 博士

【摘要】 丹参(Salvia miltorrhiza Bge.)为唇形科(Labiatae Juss)鼠尾草属(SalviaL)多年生草本药用植物,以根入药,是我国一种常用大宗中草药,主要用于治疗心脑血管系统疾病。河北是丹参药材主产区之一,年种植面积较大。近年来发现,丹参上有一种病毒引起丹参退化,该病田间发病率较高,有些田块发病率竞高达60-80%,表现为花叶、斑驳、卷叶、黄化、矮化等典型症状。感病植株的根系细小,产量大幅度下降,药用成分含量降低。经抽查分析发现:安国栽培丹参药用成分丹参酮ⅡA含量平均值仅为0.135%,低于国家《药典》0.2%的规定标准,不属于优质丹参。病毒感染、品种退化是造成丹参质量差、产量低、病害严重的主要原因之一,而国内外至今没有关于丹参病毒病害方面的研究报道。目前,病毒病害基本没有有效的药剂可以防治,查明丹参病毒病原,脱除病毒,进行提纯复壮,是防治丹参病毒病害,从根本上提高丹参产量、质量的有效措施,也是生产上迫切需要解决的问题。本项研究主要分丹参病毒病原鉴定、脱病毒技术建立、脱毒丹参农艺性状比较评价三个部分:Ⅰ.通过生物学鉴定、电镜观察和DAS-ELISA检测,初步确定与丹参病毒病相关的病毒。进而对该病毒病进行了分子生物学研究,克隆病毒基因并进行序列分析,确定病毒病原。根据国际病毒分类原则确定其分类地位;同时,针对特异基因组序列设计引物,建立起该病毒的RT-PCR分子检测技术。Ⅱ.脱病毒研究,切取大田丹参植株顶芽,接种获得丹参组培苗;切取根段,诱导愈伤,获得丹参愈伤组织。利用常规热处理和茎尖培养相结合的方法,以及微细胞团块诱导再生的方法进行脱除丹参病毒技术研究,检测确定脱毒株系;在此基础上,开展脱毒丹参组培快繁技术研究,建立起丹参脱毒种苗快速繁育技术体系,批量生产脱毒丹参种苗。Ⅲ.对脱病毒丹参进行农艺性状、生药学性状的比较评价。通过对脱毒丹参和未脱毒丹参在生长发育习性、苗期性状、生物学特性、产量性状、生药学性状以及药用成分等方面的比较分析,明确脱毒丹参性状表现。研究结果:1、生物摩擦接种表明,在指示植物白肋烟(Nicotiana tabacum-White Burley.)表现系统花叶、褪绿斑,三生烟(N.tabacum-Samsun)植株表现局部枯斑、卷叶系统症状,表明丹参大田病症,可以通过机械接种传播,不是生理病害,是病毒病害。大田病症和在指示植物上的病症表现与CMV(黄瓜花叶病毒)的致病症状报道较一致;电镜观测病毒粒子形态为颗粒状病毒,病毒粒子直径约29-30nm,与CMV病毒粒子相近,初步确认侵染丹参的病毒与黄瓜花叶病毒密切相关;CMV抗血清DAS-ELISA检测发现,15个丹参样品中10个呈现阳性,由此,确定侵染丹参的病毒为黄瓜花叶病毒(CMV)。2、通过对丹参CMV病毒进行外壳蛋白基因克隆和序列分析表明,证明丹参病毒确为CMV病毒一个新的株系,该基因被国际基因库收录(国际基因库收录号:AY600989),为具有自主知识产权的基因。根据病毒命名原则,将其命名的CMV—DS株系(黄瓜花叶病毒丹参株系),该研究成果为丹参脱毒和丹参抗病毒基因工程研究奠定了基础。3、创立了“微细胞团快再生法脱除丹参病毒新技术”,脱除了丹参病毒,脱毒率达75%以上,该技术2006年9月申报了国家发明专利,专利登记号为:200610048342.6。该技术解决了丹参采用常规脱毒不耐高温热处理问题,大大提高了丹参脱毒效率。同时,这种思路和方法可为其它植物脱病毒提供借鉴。4、建立了脱毒丹参组培快繁技术体系:丹参茎尖诱导不定芽的初代培养基为MS+BA1.5mg/L+IBA0.02 mg/L+蔗糖30g/L+琼脂5g/L,pH5.8;丛生芽分化最适宜培养基为MS+BA1.0 mg/L+IBA0.2mg/L+蔗糖30g/L+琼脂5g/L,pH5.8;生根最适宜培养基为1/2MS+IBA 0.5mg/L+蔗糖20g/L+琼脂5g/L,pH5.8;试管苗炼苗适宜基质为蛭石;培养条件为温度25-28℃,光照2000—3000Lx培养,芽体在分化培养基上25～30天继代一次,芽增殖系数基本保持在6.0以上,瓶苗接种到生根培养基上一般20天即可全部生根;炼苗方法是将蛭石基质经0.1%高锰酸钾彻底消毒,洗净丹参瓶苗根部培养基后移栽到蛭石基质上炼苗30天,然后控制水分1—2周,使根老化。炼苗期间每3～5天喷施多菌灵或甲基托布津等杀菌剂一次,防止病菌感染。炼苗温度20～30℃,湿度90%以上。按上述优化条件,组培生产丹参脱毒苗5万株。5、脱毒丹参产量明显提高:脱毒丹参与未脱毒丹参生物学特性、生育期基本一致。脱毒丹参苗大田栽植后,生长整齐一致,地下部根膨大速度快,根条均匀、商品根多,生药学性状好。脱病毒丹参从地下根数、根粗、单株根重、有效成分含量上都优于未脱毒丹参,试验小区产量比较表明,脱毒丹参产量比对照提高了86%。6、脱毒丹参药用成分丹参酮ⅡA含量明显提高:脱毒丹参与对照未脱毒丹参生药学性状基本一致,药用成分丹参酮ⅡA含量平均为0.376%,对照未脱毒丹参平均为0.192%,脱毒丹参药用成分丹参酮ⅡA含量比《药典》0.2%标准提高了88%,比对照提高了95.8%,是对照未脱毒丹参的2倍左右。脱毒丹参的推广应用,对从根本上解决大田丹参药材含量不符合《药典》标准的问题奠定了基础。更多还原

【Abstract】 Danshen(Salvia miltiorrhiza Bge.)is perennial herb specie of Salvia L.in Labiata,and its root as the medicinal part is a commonly used traditional Chinese medicine that has been mainly used for the prevention and treatment of vascular diseases in the clinics.Hebei province is the main producing area in China.In recent years,a disease caused by a kind of virus can cause the degeneration of Salvia miltiorrhiza Bunge and the incidence is very high and reaches up to 60-80%in the fields.The symptoms include mosaic leaves,mottling,leafrolling,yellowing,and stunting,etc.The roots of the susceptible plants are always very fine,the yield is reduced drastically,the quality becomes deterioration and the contents of medicinal ingredients decreased greatly.The continuous random sampling testing,showed that the mean value of medicianl ingredient of TanshinoneⅡA in Anguo County of Hebei is only 0.135%,less than 0.2%of the standard value set by the pharmacopoeia of China.So it is not high-quality Danshen.The virus infection and variety degeneratiaon are the main reasons what causing the bad quality,low output and serious plant disease of Salvia miltiorrhiza.And there aren’t any reports about the occurrence of Salvia miltiorrhiza virus disease.Recently,there isn’t any effective medicaments than control the virus disease.So the effective measures for radically improving the yielding and quality and controlling the disease of Salvia miltiorrhiza are found out the pathogens of virus diseases,removing the virus and purifing and rejuvenating,and this is also the urgent problem that must be settled in Danshen production.This study is mainly divided into three parts:the identification of virus pathogen,the study of removing virus technology and the agronomic traits comparative evaluation of virus-free Danshen.Ⅰ.A preliminary certainty about the virus pathogen of Salvia miltiorrhiza has been made by using the technology of biology assay,electron microscopy(EM)and double-antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA).And the molecular biology technology has also been used for the study of the virus disease,cloning the genes of the virus and analyzing the sequences so as to identifing the virus.According to specific viral gene sequence,primer was designed and then established the virus molecular detection technology based on reverse transcription polymerase chain reaction(RT-PCR)technique.Ⅱ.Virus-free methods researchs have been done,the apical buds were removed from the Salvia miltiorrhiza in the field and inoculated to the culture medium to get the tissue culture plantlets;the root segments were mechanically inoculated on the induced culture medium to get callus.By the method of the heat treatment with combing the shoot tip meristem culture,and the microcell clusters inductive regeneration technology,the virus was eliminated from Salvia miltiorrhiza plantlets.The virus was detected by DAS-ELISA and RT-PCR so as to gaining the virus-free plantlets.Based on these plantlets,we founed the systems of rapid propagation technology of virus-free Danshen plantlets and used for large-scale production of seedlings.Ⅲ.The agronomic traits and pharmacognosy characteristics comparative evaluation of virus-free plants from Salvia miltiorrhiza were conducted between virus-free and virus infected Salvia miltiorrhiza.Through the comparative analysises of growth and development characteristics, characters of seedling stage,biological characteristics,yileding characters,pharmacognosy characteristics and medical ingredients contents,the characteristics and its field performance of virus-free Salvia miltiorrhiza were made clear.The results showed that:Ⅰ.The biological inoculation asssy results showed that the inoclulated Nicotiana tabacum -White Burley showed system mosaic,chlorosis and mottle,and N.tabacum-Samsun showed partial mottling and leaf rolling,etc.This indicate that the Salvia miltiorrhiza disease can infect other species plant and it is not physiological disease but viral disease.The infection symptoms of Danshen both in field and indicator plant showed similarity to the symptoms caused by Cucumber mosaic virus(CMV)or Tobacco mosaic virus(TMV).The parlticle diameter of viruses observed by EM was 29-30nm which its size was similar to that of CMV.The DAS-ELISA detection with CMV antiserum showed that 10 samples were detected positively in all 15 samples.And the preliminary result showed that the main viral pathogen of Danshen was a isolate of CMV. Ⅱ.The CMV coat protein gene from Danshen was cloned and sequenced,the analysis showed the viral pathogen of Danshen was a new CMV strain.This coat protein gene wasdeposited by the international GenBank(the accession number is AY600989),which was the independent intelligent property right gene.According to the naming standard for the classification of International Committee on Taxonomy of Virus(ICTV),we named this new isolate as CMV-DS strain,these played a foundation on removing the virus from Danshen and the research of plant transgenetic engineering for resistance the virus.Ⅲ.In this research,we have founded "a new virus-free methods of mcirocell clusters inductive regeneration of Salvia miltiorrhiza",and it was more effective than the other methods such as heat treatment and shoot tip culture method,and its virus-free plants reached up to 75%.The method has been applied for the national invention patent in September 2006,and the patent accession mumber is 200610048342.6.This method solved the problems that Salvia miltiorrhiza cannot tolerant to heat treatment with normal virus-free methods and highly improved the virus-free efficiency.This thought and method can also be used to removing the virus from other plant species.Ⅳ.The tissue culture and quick propagation technology of Danshen was found:The best cuture medium of inducing buds from roots or stem segements was 1/3MS+BA 1.0mg/L+IBA 0.02mg/L+Cane sugar 30g/L,pH5.8;the most suitable culure medium for buds growth was MS+BA1.5 mg/L+IBA 0.2mg/L+Cane sugar 30g/L,pH5.8;the most suitable culture medium for rooting was 1/2MS+IAA 1.0mg/L+Cane sugar 20g/L,pH5.8;the optimum tempreture was 25-28℃and the optimum light intensity was 2000-3000 Lx.And the buds differentiation subculture should be one time with 25-30 days.The propagation coefficient was more than 6.0.The plantlets in vitro could root in about 20 days after inoculated on the rooting culture medium,and after 30 days,the rooting plantlets from the medium began to be cultued on the sterilization vermiculite in greenhouse where the temperature was keeping between 20-30℃,and the humidity was over 90%.After 30 days,the seedling training was controlled with water and sustained for 1-2 weeks to make the roots become older,during the seedling training,Carbendazim or thiophanate methyl are spraying once on the seedlings per 3-5 dsys in order to prevent from the infecting pathogens,and then the seedlings can be transplanted in the field.Ⅴ.The yield of virus-free Danshen was increased significantly:the biological characteristics and the growth period are similar beteen the virus-free and infecting Salvia miltiorrhiza,the virus-free seedlings that planted in the fields growing coordinately with status,the roots enlargment course are faster,the root slips are uniformed and the commodity roots pharmacognosy characteristics are better than the virus-infecint Danshen.From the roots numbers,roots diameter,roots weight per plants and the medicinal ingredients contents,the virus-free Danshen showed more superior to infecting plants,which make the production improved by 86.6%than the control.Ⅵ.The medicinal ingredient contents are improved markedly:thepharmacognosy characteristics are unifom between the the virus-free and infecting Salvia miltiorrhiza,analysis of medical compositions indicated that the average contents of tanshinoneⅡA is 0.376%in virus-free Salvia miltiorrhiza,and the control is 0.192%,which was nearly 2 times of the infecting plants and the production was improved by 88%which is regulated by Chinese Medicinal Dictionary.The extension and application of virus-free Salvia miltiorrhiza will lay a foundation for resolving radicaly the lower medicinal ingredient contents which don’t accordance with the Chinese Medicinal Dictionary.更多还原