【作者】 李利华；

【导师】 张启发； 练兴明；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 磷是植物体内重要的营养元素。土壤中总磷含量丰富,但能被植物直接吸收利用的可溶性磷含量却很低,成为制约农作物产量的重要因素。低磷胁迫条件下,植物体可通过改变其形态结构及生理、生化代谢途径获取磷酸盐。目前植物适应低磷胁迫的分子机制尚不清楚。本研究利用水稻基因组寡核苷酸芯片研究了耐低磷水稻中早18和不耐低磷水稻Lagrue在正常营养条件和低磷胁迫后6小时、24小时、72小时3个时间点的全基因组表达谱。芯片杂交实验设计有3次生物学重复,每次生物学重复中又设有两次技术重复。研究结果共鉴定出两种水稻低磷胁迫根部、地上部差异表达基因共5687个。其中,水稻中早18出现差异表达基因2316个,水稻Lagrue出现差异表达基因3731个。差异表达基因依据功能注释依次分为磷酸盐转运、其它转运、磷酸酶、其它酶、主要代谢、次生代谢、蛋白质降解、蛋白质合成等类别。水稻中早18和水稻Lagrue针对低磷胁迫反应在表达谱上存在一些共同点：具体表现如下：1.根部磷酸盐转运蛋白基因、磷酸酶基因、核酸酶基因等上升表达。2.根部N吸收和利用相关基因下降表达3.根部脂代谢相关基因表达的改变4.根部蛋白质降解、细胞衰老相关基因上升表达5.根部氧化胁迫相关基因上升表达6.根部跨膜转运蛋白基因上升表达7.信号传导相关基因表达的改变8.转座因子表达的改变耐低磷水稻中早18和不耐低磷水稻Lagrue低磷胁迫时,在表达谱上还存在许多差异。在低磷胁迫早期,耐低磷水稻中早18根部通过加强糖酵解途径为其以后侧根和根毛生长提供尽可能多的物质和能量。随着磷素的进一步缺乏,水稻根部和地上部糖酵解都减弱。而不耐低磷水稻Lagrue低磷胁迫时,糖酵解就开始减弱,没有糖酵解加强这一过程。耐低磷水稻中早18在低磷胁迫开始时,三羧酸循环(TCA)加强,加强的三羧酸循环(TCA)产生更多的有机酸,分泌到土壤中,活化土壤中的难溶性磷,这样有利于更好的吸收磷。同时低磷胁迫72h磷酸烯醇式丙酮酸羧化酶(PEPC)上升表达,为三羧酸循环(TCA)产生有机酸提供足够的C源。而不耐低磷水稻Lagrue在低磷胁迫时三羧酸循环(TCA)始终减弱,不能产生有机酸去活化土壤中的难溶性磷。从这些差异表达基因中选择部分基因,可以作为遗传改良侯选基因,希望能对我国水稻磷营养高效的利用提供有用的资源。更多还原

【Abstract】 Phosphate (Pi) is one of the essential nutrients required by plants. Phosphorus that can be absorbed by plants directly is very low although it is rich in soil. Sub-optimal P nutrition can lead to yield losses.Plants have developed numerous morphological, physiological, biochemical adaptations to acquire phosphate. However, the molecular bases of these responses to Pi deficiency are not thoroughly elucidated. Expression profile in response to low phosphate was investigated for rice Zhongzao 18 (low-phosphate tolerant) and rice Lagrue(not low-phosphate tolerant) at 6hr,24hr and 72 hr after low phosphate stress with the normal phosphate as the control with DNA chip of 60,000 oligos (70 mer) representing all putative genes of rice genome purchased from the Beijing Genomic institute. The planting and harvesting were conducted trebly with an interval of 5 days for three biological repeats. Each of the hybridizations was performed with 2 technical repeats using independent samples from three different plantings. A total of 5687 genes exhibited alteration in expression in response to low P stress in at least one of the three time points in rice Zhongzao 18 and in rice Lagrue. The number of differentially expressed genes was 2316,3731 in rice Zhongzao 18, rice Lagrue, respectively. The differentially expressed genes were classified into Pi transport、transport except Pi transport、phosphatase、enzyme except phosphatase、primary metabolism、secondary metabolism、protein degradation、protein synthesis et al. according to the genes function annotation.There were some common areas in the expression profile between in rice Zhongzao 18 and in rice Lagrue in response to low-phosphorus stress. Specific performance was as follows:1. The genes of phosphate transporter, acid phosphatases, nuclease were up-regulated in rice roots.2. Genes involved in N metabolism were down-regulated in rice roots.3. Several genes involved in lipid metabolism changed their expression in rice roots4. Some genes involved in cell senescence and protein degradation were up-regulated;5. Oxidative stress-related genes were up-regulated in rice roots6. Some transmembrane transporter genes were up-regulated.7. Some signal transduction-related genes changed their expression in rice8. Some transposable element genes changed their expression in riceThere was significant difference in the expression profile between in rice Zhongzao 18 and in rice Lagrue in response to low-phosphorus stress. Increased glycolysis helped to provide as much material and energy to lateral root and root hair for growth in rice Zhongzao 18 roots during early stages of Pi deficiency treatments. As a further lack of phosphorus, the glycolysis decreased in rice Zhongzao 18 shoot and roots. The process of strengthen glycolysis did not exist in rice Lagrue roots during early or late stages of Pi deficiency treatments. The TCA cycle was strengthen after low-phosphorus stress treatments started in rice Zhongzao 18 roots. Thus the more organic acids was produced and released into the soil to activate the insoluble phosphorus. And The increased expression of phosphoenolpyruvate carboxylase genes helps to supply sufficient carbon source to promote the TCA cycle in rice Zhongzao 18 roots.the TCA cycle decreased during early or late stages of Pi deficiency treatments in rice Lagrue roots. So Organic acids can not be produced and released into the soil to activate the insoluble phosphorus in rice Lagrue roots.The identification of differentially expressed genes can provide useful information to further study the molecular mechanism of plant adaptation to low phosphorus. And these differentially expressed genes can be used as candidate genes to improve Pi use by crop species.更多还原