钙信号系统在地黄连作障碍过程中的异常响应

发布时间：2018-01-12 07:18

本文关键词：钙信号系统在地黄连作障碍过程中的异常响应　出处：《河南农业大学》2014年硕士论文　论文类型：学位论文

【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。
[Abstract]:(Rehmannia glutinosa Libosch.) belongs to Rehmannia Scrophulariaceae Rehmannia is a perennial herb, is a traditional Chinese medicine in Zong Dao, in our country has a long history of cultivation and broad planting area. However, with the continuous cropping obstacle of serious problems exist in the production process of continuous cropping Rehmannia rehmannia, poor plant growth, plant diseases and insect pests aggravated not normal, tuber enlargement, and even crops, and each harvest after it 8-10 years before then. Continuous cropping obstacle has seriously affected the sustainable production of Rehmannia glutinosa, become the major issues of local Chinese agriculture and regional economic development. In order to further study the molecular mechanism of continuous cropping obstacles and on the basis of the reduction of related techniques, the research in the prophase construction of early head stubble and root enlargement of Rehmannia glutinosa large transcriptome library on the basis of the "temperature 85-5" yellow goods As experimental materials, the following research: 1. using solexa/Illumina high-throughput sequencing expression profile of early root enlargement and continuous cropping rehmannia root toucha differential gene construct, screening the response difference of continuous cropping stress gene expression. The protein and calcium signal encoding (calcium channel and calcium pump) and protein (calcium binding protein) decoding differential expression of related genes significantly, calcium channel was significantly increased expression of calcium binding proteins and intracellular protein gene most, and calcium pump gene is down regulated, which indicated that calcium signaling may be involved in the response of continuous cropping obstacles. Analyzed by quantitative PCR technology of head stubble closely related to calcium signal in vivo gene expression of Rehmannia glutinosa model results show that each growth period of calcium signaling related genes in the first crop and Rehmannia glutinosa in vivo expression significantly, which is manifested in the continuous cropping of luteal calcium The expression of ion channel protein gene was significantly up-regulated, down regulated expression of Ca2+-ATPase gene, calcium binding proteins also up-regulated expression of genes, and the differentially expressed genes showed consistent results. Under continuous cropping obstacles under the calcium signal related gene expression patterns change, response and.2. response to calcium signaling may be involved in the continuous cropping obstacle in order to verify this view, this study used calcium pyroantimonate precipitation in situ transmission electron microscopy to detect early growth period of Rehmannia key period and cable root enlargement, root tuber form calcium ion concentration and distribution of cells; using calcium fluorescent indicator method, combined with the concentration of free calcium ion confocal fluorescence microscope detection the two period of head stubble and Rehmannia glutinosa root tissue cells. The results showed that continuous cropping of rehmannia root meristematic cells in the presence of a large number of calciumion Precipitation, and the first crop cells less Rehmannia; free calcium concentration of Rehmannia glutinosa root tissue cells was significantly greater than that of the first stubble, and the distribution of calcium ions in cells scattered. Caused by continuous cropping Rehmannia intracellular calcium accumulation and scattered distribution, the results provide objective and direct evidence put forward in the first part conclusion. It showed that continuous cropping stress caused by the change of calcium concentration and distribution of digitalis abnormal intracellular calcium signaling system, resulting in "disorder", causing.3. damage by calcium signal blocking agent to deal with continuous cropping Rehmannia on potted plants, by displaying the results with the first crop and untreated Rehmannia glutinosa were compared: calcium signal blocker application. It is obvious to ease the continuous cropping obstacles caused by poison. The calcium chelator EGTA has the most obvious effect, the treatment of Rehmannia glutinosa, growth is good, the no Occurrence of continuous cropping poison phenomenon, and the amount of root biological maturity is close to the first crop of Rehmannia glutinosa; calcium channel inhibitor verapamil and calmodulin antagonist chlorpromazine. The growth of three fluorine treatment of Rehmannia glutinosa is significantly improved, the biomass statistics have greatly improved. To verify the different calcium signal blocking agent the gene expression patterns in response to continuous cropping obstacles using quantitative PCR technology, results showed that calcium signal blocker can regulate gene expression, so that continuous cropping caused abnormal gene expression returned to "normal". Show that calcium signal blocking blocking effect on the abnormal calcium signaling agent can effectively. Ease of continuous cropping obstacles caused by poison. This research for abatement technology of continuous cropping obstacles made beneficial exploration, but also proving the correctness before deduction. Through the analysis of Rehmannia glutinosa and toucha Rehmannia Expression of root genetic difference, first proposed the idea of response and response calcium signals involved in continuous cropping obstacles, and through the calcium pyroantimonate precipitation in situ and calcium fluorescent indicator method confirmed this view, blocker use proves this view using the calcium signal. In order to reveal the causes involved in calcium signaling the response mechanism of continuous cropping obstacles and in-depth study of continuous cropping obstacles, laid the foundation for molecular mechanism explains the continuous cropping obstacle, and also make a beneficial exploration for continuous cropping poison abatement technology research.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S567.234

【相似文献】