甘蓝型油菜LPR家族基因的特征及其BnaA07LPR2的功能初探

发布时间：2018-04-20 13:01

  本文选题：甘蓝型油菜 + 磷高效　； 参考：《华中农业大学》2017年硕士论文

【摘要】：磷是植物生长发育不可缺少的矿质营养元素,土壤中磷的有效性低已成为作物生长的重要限制因子。油菜是我国重要的油料作物,因需磷较多,对缺磷敏感。探究油菜磷营养高效的生理和分子机制,为改良油菜的磷效率和挖掘油菜磷高效的基因资源具有重要的理论和实践意义。本课题以油菜基因组数据库和课题组前期油菜响应缺磷的RNA-seq数据为基础,根据拟南芥控制低磷条件下根构型相关的基因LPR(Low Phosphate Root)序列信息,利用生物信息学技术分析甘蓝型油菜LPR家族基因的特征,结合qRT-PCR、拟南芥遗传转化等方法,研究BnaLPRs家族基因在低磷胁迫下表达模式及其BnaA07LPR2基因的功能验证,揭示BnaLPRs家族基因参与油菜耐低磷胁迫的分子机理。主要研究结果如下:1、甘蓝型油菜短期无磷饥饿和长期低磷胁迫下植株的生长和磷的吸收累积以磷高效基因型“鄂油长荚”为材料,采用营养液培养分别设置短期无磷饥饿和长期低磷胁迫处理,分析植物生长和磷的吸收累积。当外界磷源充足时,大部分磷素与光合作用产物用于地上部生长,供应根系的相对较少,根冠比较低;而在外界磷源减少时,首先满足根系的生长发育,运输到地上部的磷源减少,导致根冠比增大。在短期无磷饥饿和长期低磷胁迫下,磷利用效率较正常磷处理都有显著的提高,但根系和地上部的磷利用效率变化有所不同,这表明磷胁迫能促进体内磷的再利用率,但同一基因型材料的不同部位对磷的利用和分配存在差异。2、甘蓝型油菜LPR家族基因的特征利用拟南芥LPR1和LPR2的基因序列信息和已公布的甘蓝型油菜全基因组序列信息,在甘蓝型油菜中找到4个LPR的同源基因,分别分布在A07、A09、C05、C06四条染色体上。它们氨基酸序列相似性在77-99%之间,核酸序列一致性在36-95%,其中属BnaC06LPR2和BnaA07LPR2两个基因之间氨基酸序列和核酸序列的一致性最高。BnaC06LPR2和BnaA07LPR2基因结构相似,只有2个内含子,3个外显子,但不同于AtLPR2,说明在甘蓝型油菜进化过程中BnaLPR2s功能发生分化。同时通过分析保守结构域,发现LPR基因家族都含有3个多铜氧化酶结构域,通过系统进化树分析进一步证实了基因结构及蛋白保守结构域分析的可靠性。3、甘蓝型油菜LPR基因响应低磷胁迫的时空表达模式分析利用本实验室甘蓝型油菜苗期磷饥饿胁迫下的转录组数据,共检测到3个BnaLPR基因的表达,进一步的qRT-PCR结果表明,这种诱导具有短期持续性,而且在不同的部位对磷饥饿有不同程度的响应。在甘蓝型油菜不同组织部位的定量结果表示,异源四倍体的甘蓝型油菜中BnaLPRs家族基因在长时间的进化过程中功能发生了分化,有不同的表达模式,在磷信号调控中发挥不同的效应和生物学功能。4、甘蓝型油菜BnaA07LPR21基因的功能初步分析利用反向遗传学研究BnaLPRs在磷信号调控中的功能差异,构建35S启动子连接BnaA07LPR2 CDS的超表达载体,转化野生型拟南芥(Col-0)。通过琼脂和营养液培养实验分析野生型(Col-0)、lpr1突变体和超表达BnaA07LPR2的拟南芥转基因植株在不同磷、铁处理下的生长,发现缺磷会抑制主根生长,缺磷主根长表现为:lpr1OE-BnaA07LPR2Col-0;同时,在转基因植株的根中许多磷饥饿诱导表达基因均显著上升,说明BnaA07LPR2可能在油菜磷信号转导通路中起到一定正调控作用。所以,我们猜测甘蓝型油菜BnaA07LPR2基因与拟南芥中LPR1和LPR2有功能上的分化,超表达后能缓解部分主根缺磷抑制的现象。
[Abstract]:Phosphorus is an essential mineral nutrient element for plant growth and development. The low availability of phosphorus in the soil has become an important limiting factor for the growth of crops. Rape is an important oil crop in China. It is sensitive to phosphorus deficiency due to high phosphorus requirement. The physiological and molecular mechanism of high phosphorus nutrition in rape is explored to improve the phosphorus efficiency of rapeseed and to tap the high phosphorus of rapeseed. The effective gene resources have important theoretical and practical significance. Based on the rapeseed genome database and the RNA-seq data of the preliminary rapeseed response to the deficiency of phosphorus in the project group, according to the LPR (Low Phosphate Root) sequence information of the root configuration related to the low phosphorus condition in Arabidopsis, the bioinformatics technology is used to analyze the LP of Brassica napus. The characteristics of R family genes, combined with qRT-PCR and Arabidopsis genetic transformation, study the expression pattern of BnaLPRs family gene and the function verification of BnaA07LPR2 gene under low phosphorus stress, and reveal the molecular mechanism of BnaLPRs family genes involved in the low phosphorus stress tolerance of rape. The main results are as follows: 1, short term phosphorus free starvation and long term in Brassica napus The plant growth and phosphorus absorption and accumulation under low phosphorus stress are based on the phosphorus efficient genotype "Hubei oil long pods". The short term phosphorus free starvation and long-term low phosphorus stress treatment are set up in nutrient solution culture to analyze plant growth and phosphorus absorption accumulation. When the external phosphorus source is sufficient, the large part of phosphorus and photosynthesis products are used in the upper part of the earth. The root is relatively low and the root cap is relatively low, and when the external phosphorus source is reduced, the growth and development of the root system are first met, the phosphorus source in the upper part of the ground is reduced and the root crown ratio is increased. The phosphorus utilization efficiency is significantly higher than that of normal phosphorus treatment under the short-term phosphorus free hunger and the long-term low phosphorus stress, but the phosphorus benefit of the root and the upper part of the ground is significantly improved. The changes in efficiency vary, which indicates that phosphorus stress can promote the reutilization of phosphorus in the body, but there is a difference in the utilization and distribution of phosphorus in different parts of the same genotypic material.2. The characteristics of LPR family genes in Brassica napus are the sequence information of LPR1 and LPR2 in Arabidopsis thaliana and the whole genome sequence information of Brassica napus. 4 homologous genes of LPR were found in Brassica napus, which were distributed on the four chromosomes of A07, A09, C05 and C06 respectively. Their amino acid sequence similarity was between 77-99% and nucleic acid sequence consistency in 36-95%, which was the highest consistency of the amino acid sequence and nucleic acid sequence between the two genes of BnaC06LPR2 and BnaA07LPR2, and the highest.BnaC06LPR2 and BnaA07LPR2. The gene structure is similar, with only 2 introns and 3 exons, but different from AtLPR2, it shows that BnaLPR2s function differentiation in the process of Brassica napus evolution. At the same time, through the analysis of the conservative domain, it is found that the LPR gene family contains 3 multi copper oxidase domains, and the gene structure and protein are further confirmed by phylogenetic tree analysis. The reliability of the conservative domain analysis (.3), the spatio-temporal expression pattern of LPR gene in Brassica napus was analyzed by the time and space expression pattern of low phosphorus stress. The expression of 3 BnaLPR genes was detected by the transcriptional data of phosphorus starvation under the seedling stage of Brassica napus in the laboratory. The further qRT-PCR results showed that the induction had short duration and was not at the same time. The same parts respond to phosphorus starvation in varying degrees. Quantitative results in different tissue parts of Brassica napus indicate that the BnaLPRs family genes in the allotetraploid Brassica napus are differentiated in a long period of evolution, with different expression patterns and different effects and biological functions in the regulation of phosphorus letter numbers. .4, preliminary analysis of the function of BnaA07LPR21 gene in Brassica napus (Brassica napus) using reverse genetics to study the function difference of BnaLPRs in the regulation of phosphorus signal, construct the overexpression vector of 35S promoter to connect BnaA07LPR2 CDS, transform wild type Arabidopsis (Col-0). Analysis of wild type (Col-0), lpr1 mutant and overwatch through agar and nutrient solution culture experiment. The growth of BnaA07LPR2 transgenic plants under different phosphorus and iron treatments showed that phosphorus deficiency could inhibit the growth of the main root, and the main root length of phosphorus deficiency was lpr1OE-BnaA07LPR2Col-0. At the same time, many phosphorous starvation induced genes increased significantly in the roots of the transgenic plants, indicating that BnaA07LPR2 may play a role in the phosphorous signal transduction pathway of rapeseed. Therefore, we speculate that the BnaA07LPR2 gene of Brassica napus has functional differentiation with LPR1 and LPR2 in Arabidopsis thaliana. After overexpression, it can alleviate the inhibition of phosphorus deficiency in some main roots.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4

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