【作者】 林海建；

【导师】 潘光堂；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 土壤中有效磷的缺乏是一个全球性的问题,是制约农业生产和作物产量提高的重要限制性因子。培育作物磷高效吸收利用型品种是解决由土壤有效磷缺乏而带来的一系列问题的重要途径之一。在我国和世界其他国家的主要玉米产区,土壤有效磷缺乏已成为一个不争的事实,严重制约了玉米产量的增加。培育和推广玉米磷高效吸收利用型品种是今后这些地区玉米生产实现低投入稳产出、农业生态和谐发展的有效保证。因此,鉴定和筛选玉米耐低磷种质资源、寻找低磷胁迫响应的重要性状及关键基因、探明玉米耐低磷的分子调控机制,并借助传统育种方法和现代分子生物学技术是寻找和培育高效、稳定和安全的磷高效玉米品种的有效途径。本研究在前期耐低磷材料筛选鉴定的基础上,对不同磷营养条件下玉米自交系在低磷胁迫下根系性状的变化情况进行了考察,同时利用基因芯片技术和miRNA克隆的方法,对磷高效吸收利用型玉米自交系178的根系在低磷胁迫下差异基因和相关miRNA进行了鉴定与分析,并结合酶活性、半定量RT-PCR和实时荧光定量PCR (Real-time PCR)等方法对部分候选基因和miRNA进行了分析,得到的主要结果如下：1.采用大田和沙土培养的方式,对前期大田低磷胁迫筛选得到的20份表现差异的玉米自交系进行了重复性的鉴定,结果与课题组前期鉴定结果基本一致。鉴定筛选得到了磷高效吸收利用玉米自交系3份,分别是178、RP125和99S2052-2；磷低效自交系10份。2.采用沙土培养和营养液培养的方式,对鉴定出的3份磷高效吸收利用玉米自交系和3份磷低效自交系在低磷胁迫下的根系性状进行了分析,研究发现3份磷高效吸收利用型自交系在根系总长度、根总条数和根表面积的绝对值以及增长率上均优于3份磷低效自交系。3.利用Affymetrix玉米基因组表达谱芯片杂交技术,筛选得到487个在178低磷胁迫处理3d后根系中差异表达的基因,其中上调表达的基因有349个,下调表达的基因有138个。对这些差异表达基因的功能进行分类注释后发现,这些基因涉及物质与能量代谢、转录调控、物质的吸收与转运、胁迫响应、激素信号转导和电子传递等过程。4.采用Real-time PCR方法对玉米无机磷转运因子、酸性磷酸酶、植酸酶、2-脱氧麦根酸合成酶、过氧化物酶、MYB转录因子等7个重要候选基因在低磷胁迫下178根系中的差异表达情况进行了分析,结果表明7个候选基因的总体表达趋势与基因芯片所揭示的表达趋势基本吻合,不同基因间在低磷胁迫后不同时间段的表达趋势存在差异。5.对同期处理的玉米自交系178根系中磷含量,酸性磷酸酶(胞内／胞外)、植酸酶、过氧化物酶及根系活力等特征进行了检测,结果表明低磷胁迫下根系中含磷量呈现出了明显的下降趋势,胞内酸性磷酸酶活性受低磷胁迫诱导,其增长趋势较为明显,而胞外酸性磷酸酶变化并不十分明显,POD活性明显增加,植酸酶活性上升平稳,根系活力呈下降趋势。6.成功构建了低磷胁迫下磷高效吸收利用型玉米自交系178根系的小分子RNA文库,菌液PCR检测后共获得263个克隆,经测序及序列生物信息学分析后共得到12条符合标准的候选miRNA序列,进一步比对分析后发现12条候选miRNA中有2个已知miRNA(miRNA156和miRNA399)和10个新的miRNA。7.利用生物信息学分析方法对候选miRNA进行物种间保守性分析,结果发现除2个已知miRNA在植物物种中具有较高的保守性外,新发现了1个miRNA (Zm-miRNA2)在禾本科植物上具有一定的保守性。同时,序列比对还发现Zm-miRNA9为玉米miRNA169家族新的成员。8.采用RT-PCR方法对12个候选miRNA的真实性和表达特征进行了分析,结果表明除Zm-miRNA6只能在低磷胁迫处理后12 h、24 h和48 h被检测到外,其余miRNA均能在大部分处理时间段(0h、12 h、24 h、48 h、72 h、96 h)中被检测到。Zm-miRNA2、Zm-miRNA3、Zm-miRNA5、Zm-miRNA6、Zm-miRNA9和miRNA399b可能存在诱导表达;Zm-miRNAl和Zm-miRNA7可能存在着下调表达；另外Zm-miRNA8和Zm-miRNA10可能存在先上调后下调的表达趋势,而]miRNA156的表达则无明显差异。9.利用生物信息学在线miRNA靶基因预测软件,对12个候选miRNA进行了靶基因预测,结果表明,除Zm-miRNA9未找到靶基因外,其余11个miRNA共预测得到57个靶基因,涉及物质与能量代谢、转录调控、物质转运、胁迫响应和信号转导等过程。进一步分析发现预测获得的靶基因与基因芯片筛选得到的差异表达基因部分相同,如miRNA399b和Zm-miRNA3靶向的无机磷转运子(TC420181), Zm-miRNA2靶向的bZIP转录因子(EE023309), Zm-miRNA5靶向的Ubiquitin carrier protein (TC432098)、Zm-miRNA6靶向的GST (TC385801)等。10.采用Real-time PCR对miRNA399b和Zm-miRNA3及其可能的靶基因ZmPT1/2的表达特征进行了分析,结果表明miRNA399b在玉米根系响应低磷胁迫的过程具有明显的被诱导表达趋势,Zm-miRNA3的表达表现出先诱导后抑制的趋势,整体表现出诱导表达的趋势。11.结合基因芯片和miRNA靶基因预测的结果,参考水稻和拟南芥在低磷胁迫下可能存在的调控机制和信号通路,阐述了玉米在低磷胁迫下,其根系产生的可能适应性机制,并模拟了几种代谢途径和信号通路及miRNA介导的表达调控途径在玉米耐低磷胁迫中的调控模式,为今后阐明玉米根系响应低磷胁迫的分子机理和miRNA介导的调控机制奠定基础。更多还原

【Abstract】 The deficiency of available phosphorus in soils is a globle problem and becoming an important limeted facor for agricultural production and crop yiled increase. Cultivation of crop varieties with high efficiency for phosphorus absorbing and utilization is one of the important approaches to solve the aboving problem. In the main maize production areas of China and other countries, the lack of available phosphorus is becoming an indisputable fact and restricts the maize production badly. The cultivation and extension of maize varieties with high efficiency for phosphorus absorbing and utilization is the vital guarantee of concept achievement on’low imput, environmentally-friendly with steady maize production’. So, screening and identification of maize phosohorus high efficieny germplasm resource, validation of important traits and key genes related to low phosphorus stress resopnse, verification of the molecular regulation mechanism of tolerance to low phosphorus and facilitated by traditional breeding methods and molecular biology technologies are important approaches for cultivation of maize varieties with effective, stability and safety. Based on the sreening and identification of maize inbred lines, this paper analyzed the changes of maize roots under low phosphorus stress, and then microarray and miRNA cloning methods were used for indentication of the different expression genes and related miRNAs in elite line 178 roots responsed to low phosphorus stress. Enzyme activity test, semiquantitativeRT-PCR and Real-time PCR analysis were applicated for further validation of some candidate genes. In summary, we have obtained the following results:1. The reduplicative identification of 20 maize inbred lines screened before in filed tolerance to low phosphorus stress by sand pot and field approaches and obtained in rough agreement with the before results. We have obtained 3 high phosphorus efficiency inbred lines as followings,178, RP125 and 99S2052-2 and other 10 susceptive lines to low phosphorus stress.2. The root characters were analyzed using 3 tolerant and 3 susceptive lines which random selected from 10 susceptive lines under low phosphorus stress. The results indicated that the absolute values of root total length, total number and total superficial area of high phosphorus efficiency inbred lines exceeded that of susceptive lines in sand pot. And in hydroponics, both the absolute values and growth rates of root total length, total number and total superficial area of high phosphorus efficiency inbred lines exceeded that of susceptive lines. 3. Affymetrix maize genome expression microarray was used for screening the different genes in maize roots of 178 under low phosphorus stress for 3 days.487 different expression genes were obtained and 349 genes were induced and 138 genes were repressed in response to low phosphorus stress respectively. These different genes were conducted for function annotation, which indicated that the functions of these genes were involvement in substance and energy metabolism, substances absorption and transport, transcriptional regulation, stress response, phytohormone signal transduction, electron transfer and other processes related to growth and development.4. The expression patterns of maize inorganic phosphorus transporters, acid phosphatase, phytase,2-deoxymugineic acid synthasel, POD and MYB transcription factor were validated in 178 roots responsed to low phosphorus stress by Real-time PCR. The results indicated that the expression tendencies of these candidate genes were in rough coincident with the result of microarray analysis and with different expression patterns among these genes after the on set of low phosphorus stress.5. Phosphorus content and related enzyme activities including Apase(inner and outside), phytase, POD and the activity of root in maize roots were analyzed using inbred line 178 with the identical treated time like aboving material. The results indicated the content of phosphorus was decreased and the inner activity of Apase was induced, but the outside activity of Apase was changed unnoticeably under low phosphorus. Also, the activity of POD was increased and the activity of phytase was ascended placidly, and the activity of root was decreased under low phosphorus.6. We have successfully constructed a small RNA library of 178 roots from 6 time points under low phosphorus stress.263 clones were obtained by RT-PCR detection and selected for sequencing analysis. Sequence blast and miRNA structure prediction indicated that a total of 12 sequences were conforming to the evaluation criterion of miRNAs. Further analysis found that 2 miRNA was identical with the sequence of maize known miRNAs (miRNA156 and miRNA399), and other miRNAs were deemed to new miRNAs in maize.7. The conservation of these candidate miRNAs among plant species were analyzed by bioinformatic tools. The results indicted that exception of the two known miRNAs were conserved among plant species, another new cloned miRNA, Zm-miRNA2 was found to be conserved among gramineous species. Simultaneity, Zm-miRNA9 was also found to be with the identical sequence with known maize miRNA169 family members’exception of specific base and was falled under new member of miRNA169.8. The authenticities and expression patterns of these 12 miRNAs were analyzed by polyA-tailed RT-PCR. It indicated that exception of Zm-miRNA6, which was detected only in 12 h,24 h, and 48 h treated time point, other miRNAs could be detected at the majority of treated time point (0h,12 h,24 h,48 h,72 h, and 96 h). Expression pattern indicated that Zm-miRNA2, Zm-miRNA3, Zm-miRNA5, Zm-miRNA6, Zm-miRNA9, and miRNA399b could be induced and Zm-miRNA8 and Zm-miRNA10 could be repressed under low phosphorus stress. Other two miRNAs, Zm-miRNA8 and Zm-miRNA10 may be induced first then repressed. However, the expression of miRNA156 was no significant change.9. The targets of 12 miRNAs were predicted by web based miRNA prediction software. The results were indicated that a total of 57 putative target genes were predicted for 11 miRNAs, except of Zm-miRNA9. These genes were referring to diverse gene families and different biological functions including substances and energy metabolism, transcription regulation, transport, stress response, signal transduction and so on. Further analysis found that some of these genes were also found with different expression in microarray analysis. For example, maize inorganic transporters (TC420181) were targeted by miRNA399b and Zm-miRNA3. Zm-miRNA2, Zm-miRNA5 and Zm-miRNA6 target bZIP (EE023309), ubiquitin carrier protein (TC432098) and GST (TC385801), respectively.10. The expression patterns of miRNA399, Zm-miRNA3 and theirs targets, ZmPT1/2 were analyzed by Real-time PCR, which indicated that expression pattern of miRNA399b was induced obviously in response to low phosphorus stress. The expression pattern of Zm-miRNA3 could be induced first, and then went mild and the overall trendency was induced.11. Based on the known regulatory mechanism and signal pathway in arabidopsis and rice in response to low phosphorus, the putative adaptation mechanism of maize root was investigated and the regulatory models of metabolism pathway and signal transduction was simulated according to the result of microarray and miRNA target prediction. These findings could be used for reference for further understanding the whole molecular mechanism and regulation processes mediated by miRNAs in maize roots responsed to low phosphorus stress.更多还原