【作者】 王鹏飞；

【导师】 陈新建；

【作者基本信息】 河南农业大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 地黄(Rehmannia glutinosa Libosch.)属于玄参科地黄属多年生草本植物,是我国传统的大宗道地中药材,在我国有着悠久的栽培历史和广阔的种植面积。但是,地黄的生产过程中存在着严重的连作障碍问题,连作地黄植株生长不良,病虫害加重,块根不能正常膨大,甚至绝收,且每茬收获后须隔8-10年方可再种。连作障碍已经严重影响了地黄的可持续生产,成为制约当地中药农业和区域经济发展的重大问题。为了更深入的研究地黄连作障碍发生的分子机制并在此基础上提出相应的消减技术,本研究在课题组前期构建的块根膨大初期头茬与连作地黄大容量转录组文库基础上,以“温85-5”地黄品种为实验材料,进行如下相关研究：1.利用solexa/Illumina高通量测序技术构建块根膨大初期头茬与重茬地黄块根基因差异表达谱,筛选响应连作胁迫的差异表达基因。其中与钙信号编码蛋白(钙通道与“钙泵”)和解码蛋白(钙靶蛋白)相关的基因表达差异显著,钙离子通道蛋白基因和细胞内钙靶蛋白基因多数被显著上调表达,而“钙泵”基因被下调,由此推测钙信号可能参与了地黄连作障碍响应。利用定量PCR技术对头茬与连作地黄体内钙信号相关基因时空表达模式进行分析,结果显示各个生长时期钙信号相关基因在头茬与连作地黄体内表达差异显著,具体表现为在连作地黄体内钙离子通道蛋白基因显著上调表达,Ca2+-ATPase基因下调表达,主要的钙靶蛋白基因也出现上调表达,这与基因差异表达谱结果一致。说明在地黄连作障碍的胁迫下,钙信号相关基因表达模式发生变化,钙信号可能参与了地黄连作障碍的响应与应答。2.为验证这一个观点,本研究利用焦锑酸盐钙离子原位沉淀法,结合透射电镜检测地黄关键生长时期拉线期和块根膨大初期,地黄块根形成层细胞内钙离子浓度变化与分布；利用钙荧光指示剂法,结合共聚焦荧光显微镜检测这两个时期头茬与连作地黄根尖组织细胞内的游离钙离子浓度。结果显示连作地黄块根分生区细胞内存在大量的钙离子沉淀,而头茬地黄细胞内则较少；连作地黄根尖组织细胞内游离钙离子浓度显著大于头茬,并且钙离子在细胞内的分布散乱。连作引起地黄细胞内钙离子的积累和散乱分布,这一结果为第一部分提出的推论提供了客观且直接的证据。说明连作胁迫引起地黄细胞内钙离子浓度变化和分布异常,导致钙信号系统的“紊乱”,对植株造成伤害。3.利用钙信号阻断剂来处理连作盆栽地黄,通过与头茬和未处理连作地黄进行对比,结果显示：钙信号阻断剂的施用,对于缓解连作障碍引起的毒害有明显效果。其中钙离子专一螯合剂EGTA的效果最为明显,其处理的连作地黄,生长情况良好,基本没有连作毒害现象发生,并且成熟期的块根生物量接近于头茬地黄；钙离子通道抑制剂异搏定和钙调素拮抗剂三氟啦嗪处理的连作地黄生长情况也明显好转,其生物量统计也有大幅度的提高。利用定量PCR技术验证了不同钙信号阻断剂对响应连作障碍的相关基因表达模式的影响,结果显示钙信号阻断剂能够调控相关基因的表达情况,使连作导致的“非正常”表达基因恢复“正常”。表明钙信号阻断剂对“非正常”钙信号的阻断作用,能有效缓解连作障碍引起的毒害。这为连作障碍消减技术的研究做了有益的探索,同时也反证了之前推论的正确性。本研究通过分析连作地黄与头茬地黄块根基因差异表达谱,首次提出钙信号参与地黄连作障碍的响应与应答这一观点,并通过焦锑酸盐钙离子原位沉淀法和钙荧光指示剂法直接验证了这一观点,利用钙信号阻断剂的使用反证了这一观点。为揭示钙信号参与响应连作障碍的作用机理研究和深入研究地黄连作障碍成因、解读连作障碍发生的分子机理奠定了基础,同时也为地黄连作毒害消减技术研究做了有益的探索。更多还原

【Abstract】 Rehmannia glutinosa L is a perennial herbaceous species belonging to the Scrophulariaceae family. It is a traditional Chinese medicine and one of the famous "Four Huai Medicines". This specie has been extensively cultivated for a long history. However, a serious consecutive monoculture problem is identified in the R. glutinosa production process. The R. glutinosa continuously cultivated grows poorly, and tuberous root can not normally expand or even without yield. This phenomenon can sustain for a period of 8-10 years.The consecutive monoculture problem has severely obstructed the sustainable production of R. glutinosa. It has become an urgent problem to restrict the development of Chinese medicine agriculture production and regional economy.So far, the researches of R. glutinosa consecutive monoculture problem are mainly focused on the causes of this phenomenon, the changes of soil microorganisms environment and the allelopathic substances in root exudates.The molecular mechanismof R. glutinosa responding to consecutive monoculture and the signal transduction pathways remain unclear.In order to study the molecular mechanism about consecutive monoculture problem of R. glutinosa,based on the early transcriptome of normal and consecutive cultivated R. glutinosa at the earlier tuberous root expansion stage, we selected the R. glutinosa cultivar"Wen 85-5" asexperimental material and conducted the relative researches as follows:Utilizing the Solexa/Illumina sequencing technology, we constructed and analyzed the Digital Gene Expression Profiling (DGE) of the normal and consecutive cultivated R.glutinosa, and the candidates were collected for consecutivecultivatedrelated genes. The expression of genes related to the calcium signal coding protein (calcium channel and "calcium pump") and decoding protein (calcium target protein) were significantly different, the genes of calcium ion channel protein and the intracellular calcium target protein were up-regulated, and "calcium pump" genes were down-regulated. The spatial and temporal expression profiles of key genes in calcium signaling system in the normal and consecutive cultivated R. glutinosa at different growth period were analyzed by qRT-PCR and the data were consistent with that in DGE.Our results indicated that the calcium signal system might be involved in R.glutinosa consecutive monoculture problem responsive processes.To confirm above results, Ca2+ localization and distribution in tuberous root cambial cells in the normal and consecutive cultivated R. glutinosa were investigated with antimonite precipitate-electromicroscopic-cytochemical method at the tuberous root elongating and the early expansion stage. The concentration and the change of free calcium ions in the root tip cells were measured by the calcium fluorescent indicator method combining with confocal fluorescence microscope technology.Results showed that a large amounts of calcium ions precipitation assembled in the consecutive cultivated R. glutinosa tuberous root meristematic cells, whereas it was on the contrary in the normal cultivated R. glutinosa root cells. The concentration of free calcium ions in the consecutive cultivated R. glutinosa root tip cells was higher than that in the normal cultivated, and the distribution of calcium ions was dispersive.The results showed that the consecutive monoculture caused intracellular Ca2+ accumulation and scattered distribution in the R. glutinosacells. It provided an objective and direct evidence for the idea mentioned above. The consecutive monoculture problem caused the concentration changes and abnormal distribution of the intracellular calcium ion, resulting in calcium signal system "disorder" and plants appeared abnormal growth.Moreover, the consecutive cultivated R. glutinosa was treated by calcium signal blocking agents. The results showed that the application of calcium signal blockers had good effect on alleviating symptom of the consecutive monoculture. In particular, the calcium chelator EGTA had most obvious effects, the treated plants grew well and consecutive monoculture poisoning phenomenon was blocked, and the root biomass at maturity was close to the normal cultivated R. glutinosa. The growth of plants treated with calcium channel inhibitor Verapamil and calmodulin antagonist trifluoperazine were significantly improved, and biomass statistics were greatly increased. Quantitative PCR technology was used to verify the influence of the different calcium signal blocking agent on the expression patterns of the related genes which are related to consecutive monoculture problem. The data showed that calcium signal blocker could regulate the abnormally expressed genes caused by consecutive cultivated return to the normal expression. The results demonstrated that the blocking effect of the calcium signal blocker on abnormal calcium signal could effectively relieve the poison caused by the consecutive monoculture problem.According to the analysis of the different genes expression profiles of the normal and consecutive cultivated R. glutinosa tuberous in root, we think that the calcium signal is involved in the response to consecutive monoculture problem of R. glutinosa. This opinion was directly demonstrated through the calcium antimonite precipitate method and the calcium fluorescent indicator technology. We also confirmed the correctness of this opinion according to the effect of calcium signal blocking agents. This research preliminarily revealed the action mechanism of calcium signal in response to consecutive monoculture problem of R, glutinosa. It laid the theoretical foundation for further research on the causes of consecutive monoculture problem. It also was beneficial to reveal the molecular mechanism of consecutive monoculture problem and to develop the effective method of alleviating the harm of consecutive monoculture problem.更多还原