【作者】 马平霞；

【导师】 赵庆芳；

【作者基本信息】 西北师范大学 ， 植物学， 2007， 硕士

【摘要】 百合(Lilium spp.)属百合科(Liliaceae)多年生草本,是我国重要的观赏类经济植物。近年来,从国外引进作为鲜切花的百合品种越来越多,其栽培规模也不断扩大。然而,百合由于遭受病毒侵染并出现生长势降低,枝、叶、花畸形等衰退现象的情况越来越严重,给相关产业造成了巨大的经济损失。因此,进行病毒脱除处理、推广无毒化栽培技术对提高百合的观赏价值和经济价值有着重要的现实意义。严重危害百合的病毒主要有黄瓜花叶病毒(CMV)、百合无症病毒(LSV)和百合斑驳病毒(LMoV)。国内外对百合也做了一些脱毒及病毒检测的研究,但是没有对西伯利亚百合进行过病毒脱除研究,对百合其它品种的脱毒多采用生物学或血清学方法检测其效果,现已证明分子生物学方法的检测灵敏度远远高于这两种方法,而单一的采用生物学或血清学方法检测脱毒结果有可能因植物体内病毒浓度低而出现假阴性的现象,得出错误的病毒脱除结论。本文则系统的研究了感染西伯利亚百合的CMV、LSV、LMoV的脱除及分子生物学检测方法,为西伯利亚百合生产应用提供可靠的指导。主要实验结果如下:1.建立了西伯利亚百合品种的组织培养体系。研究得出MS为百合组织培养的最适培养基,培养基各成分最适组为:鳞片和茎段的最佳诱导培养基为MS+0.1～1.2mg/L BA+0.2 mg/L NAA;丛芽增殖最佳培养基为MS+0.05～0.5mg/L BA(或KT)+0.02 mg/L TAA或0.05mg/L NAA;生根诱导最佳培养基为MS+0.05mg/L BA+0.5 mg/L NAA。2.系统研究了茎尖培养、热处理结合茎尖培养、病毒唑结合茎尖培养、病毒唑加热处理结合茎尖培养四种脱除CMV,LSV,LMoV三种病毒的方法,并对这四种脱毒方法进行了比较,综合考虑存活率和脱毒率认为:病毒唑加热处理结合茎尖培养的脱毒率最高,最高达到67.5%,热处理结合茎尖培养次之,而病毒唑结合茎尖培养又优于直接取茎尖培养。3.采用电镜观察和分子生物学RT-PCR(逆转录-聚合酶链式反应)技术检测病毒脱除效果。而RT-PCR技术首先要求高质量的RNA作为模板,由于百合组织富含多糖,难以得到高质量的RNA,故本文改良了RNA提取方法,使其具有如下优点:无需用DEPC水处理和高温烘器皿,无需液氮研磨材料,省去提取前的很多准备工作,采用该法从百合叶片中提取总RNA,整个过程及后继反应均不需使用RNasin,降低了成本,节约了资源和时间,且提取出的RNA质量高,适于后期RT-PCR的检测,实验证明这种方法更适合于百合幼嫩叶片和试管苗RNA的提取。4.优化了CMV、LSV、LMoV的RT-PCR检测体系。运用一步法RT-PCR节省了药品,并且操作过程中不易被污染。最优反应体系为:M-MLV 5×ReactionBuffer 1μ1、10×PCR buffer0.5μl、2.5mM dNTPs(each)0.8μl、10pmol/μl下游引物0.2μl、10pmol/μl上游引物0.2μl、M-MLVRT 200U/μl 0.1μl、2.5U/μl Taqpolymerase 0.1μl;0.4μl感病材料RNA提取液,剩余部分用无菌双蒸水补足,使反应体系达到10ul。更多还原

【Abstract】 Lily is a perennial herb of Liliaceae and it’s an important economic plant, widely cultivated in China. Many new varieties have been introduced into China in recent years , however viral diseases occur in some areas where lily is planted and the influence of viral diseases is becoming more and more obvious. Virus-infected trees show reduced growth, abnormal leaves and flowers. So it’s important and urgent to eliminate severe viruses.CMV、LSV and LMoV are three viruses that damage lily seriously. Some research work has been done on Chinese lily and lily in Europe, in which biological or serological methods were adopted to detect the virus, but no virus elimination research has been carried out on Siberia. It has been proved that molecular biology methods are more sensitive than biological and serological methods which may lead to wrong conclusion because of low virus concentration in plant. Elimination of virus such as CMV、LSV and LmoV of Siberia and molecular biology detection methods have been researched systematically in this paper.1. The establishment of tissue culture system of Siberia. Results showed that MS was the most suitable culture medium for the lily tissue culture. The most suitable groups of various ingredients of the culture medium are listed as follows: The best induction culture medium of squama and stem section was MS+ 0.1～1.2 mg/L BA+0.2 mg/L NAA;MS+0.05～0.5 mg/L BA(or KT)+0.02 mg/L TAA or 0.05mg/L NAA is the best for multiplication induction; The best induction culture medium for roots is MS+ 0.05mg/L BA+0.5 mg/L NAA.2. The systemica study of four methods that can eliminate the viruses of CMV、LSV and LMoV .To be specific, they are stem point culture; stem point culture combined with heat treatment; stem point culture combined with ribavirin; stem point culture combined with ribavirin and heat treatment. On the comparison of the four methods and the consideration of the survival percentage and the rate of elimination, it can be concluded that the virus elimination rate of stem point culture combined with ribavirin and heat treatment method reaches the highest degree of 67.5%, with the stem point culture combined with heat treatment comes to the next, whereas the stem point culture combined with ribavirin is superior to stem point culture. 3. The application of electronic microscope and molecular biology method of RT-PCR technology to exam the effect of virus elimination. As the lily tissue contains rich polysaccharide, it’s difficult to extract RNA of high quality , In this research paper, the author designed a new method of RNA extraction that has the following merits: it neither needs to dry the household utensils with the DEPC water treatment and high temperature, nor needs the fluid nitrogen to grind the material, which save a lot of preparatory work. The application of this method to extract RNA from the lily leaves without using RNasin in the whole process and its successive reaction will reduce the cost, save resources and time, and is more suitable for the extraction of the young tender lily leaves and test tube seedling RNA. What’s more, the RNA extracted has a high quality, is suitable for the later RT-PCR examination,4. The optimization of the RT-PCR examination system of CMV, LSV, and LMoV. By using the one-stage process of RT-PCR, this method will save drugs and the operating process will not be contaminated easily. The optimized reacting system is M-MLV 5×Reaction Buffer 1μl、10×PCR buffer0.5μl、2. 5mM dNTPs (each) 0.8μl、10pmol/μl Down primer 0.2μl、10pmol/μl Up primer 0.2μl、M-MLVRT 200U/μl 0.1μl、2.5U/μl Taq polymerase 0.1μl; 0.4μl ;Infected materials such as extracted liquid RNA. Surplus part can be supplemented by aseptic double steam water to enables the reacting system to reach 10ul.更多还原