【作者】 马建华；

【导师】 王玉国； 孙毅；

【作者基本信息】 山西农业大学 ， 作物栽培学与耕作学， 2014， 博士

【摘要】 氮、磷是作物生长发育所必需的营养元素,是蛋白质、核酸、氨基酸和叶绿素等的组成成分。氮、磷的缺乏是影响作物产量的重要限制因子。农业生产中,常常通过大量施用氮肥和磷肥来保证作物高产、稳产,氮、磷肥的大量施用导致我国氮、磷肥利用效率低且环境污染严重等问题。因此,鉴定和筛选作物耐低磷、低氮种质资源、研究作物耐低磷、低氮的分子调控机制,培育氮、磷高效作物品种是农业可持续发展的必由之路。高粱作为许多国家的主要农作物之一,具有高度耐旱、耐涝及耐瘠薄等特性,因此本研究以高粱为材料,研究了低氮、低磷对高粱幼苗形态及生理指标的影响,同时利用高通量测序技术,鉴定了高粱低氮胁迫响应的miRNAs,并用荧光定量PCR对候选的miRNAs及其靶基因的表达进行了验证,主要结果如下：1、通过蛭石培养的方式,研究不同磷浓度对高粱幼苗的影响,结果表明,不同水平磷处理对高粱苗期各形态指标均有极显著影响,低磷对高粱生理指标MDA和POD影响较大,对叶绿素、可溶性蛋白和SOD的影响不明显；其中0.25 mmol/L和1 mmol/L可作为高粱蛭石培养的低磷胁迫水平和正常磷水平；干重和植株地上部含磷量可作为高粱苗期耐低磷基因型筛选指标。从14个高粱品种中筛选出耐低磷高粱为八月齐,低磷敏感型高粱为农858。2、通过水培方式,研究不同氮浓度对高粱幼苗形态及生理指标的影响,发现株高、茎粗、总干重、主根长、叶绿素和可溶性蛋白含量等指标在4 mmol/L和0.04 mmol/L氮浓度下差异均达显著或极显著水平。由此确定0.04 mmol/L和4 mmol/L的硝酸盐浓度适合作为高粱低氮胁迫的低氮处理和正常氮处理水平。3、通过高通量测序获得高粱低氮响应的已知miRNA总数为122个,具有显著或极显著差异的miRNA,总数为107个,分属于27个miRNA家族。其中根系短时胁迫下,60个差异表达的miRNA中38个为上调表达,22为下调表达；长时胁迫的41个差异表达miRNA,上调表达数22个,下调表达数19个；叶片短时胁迫下,82个差异表达miRNA中20个为上调表达,62为下调表达,长时胁迫的76个差异表达miRNA,上调表达数26个,下调表达数50个。4、用生物信息学方法对107个有差异的已知miRNA进行靶基因预测,发现78个已知的显著差异miRNA有靶基因,其靶基因共101个。这些靶基因行使的生物学功能达30多种,其中55%的靶基因为结合功能,40%的为未知功能,8%为转录因子活性,7%的水解酶活性,此外还有蛋白质二聚化、氧化还原酶、信号转导和ATP酶活性等。5、高通量测序共获得新miRNA总数123个,其中根系中短时胁迫差异表达数为16个,长时胁迫差异表达数为4个,叶片中短时胁迫差异表达数为27个,长时胁迫差异表达数为20个。根系中短时胁迫的16个差异表达miRNA中5个为上调表达,11为下调表达,长时胁迫的4个差异表达miRNA,上调表达数2个,下调表达数2个；叶片中短时胁迫的27个差异表达miRNA中4个为上调表达,23个为下调表达,长时胁迫的20个差异表达miRNA中,上调表达数7个,下调表达数13个。6、对4个新的差异miRNA进行靶基因预测,发现8个靶基因。对这些靶基因进行功能注释,发现一个靶基因功能未知,其余7个靶基因都有结合功能,即DNA结合、铁离子结合、蛋白质结合、金属离子结合和磷酸吡哆醛结合等。7、通过实时荧光定量PCR结合geNorm软件分析,确定基因GAPDH和EIF4a适合作为高粱叶片中靶基因定量PCR的内参,而sbGAPDH和EIF4a适合作为高粱根系中基因定量PCR的内参。8、我们采用实时荧光定量PCR对候选miRNA及其靶基因的表达进行验证,结果表明miRNAs的表达趋势与高通量测序结果吻合,大部分miRNA与其靶基因的表达为负调控关系。更多还原

【Abstract】 Phosphorus and nitrogen are essential elements required for plant growth and development. They are constituents of numerous important compounds including proteins, nucleic acids, amino acids and Chlorophyll. The deficiency of available phosphorus and nitrogen in soils is an important limiting factor for crop yield increase. In agricultural production, abundant nitrogenous and phosphorus fertilizer are used in order to give high and stable crop yields, which often result in low nitrogen and phosphorus use efficiency and leads to serious environmental pollution problems. Therefore, identifying crop germplasm resources for high efficiency utilization of phosphorus and nitrogen, revealing the molecular regulation mechanism of crop’s tolerance to low phosphorus and nitrogenous, and breeding for phosphorus and nitrogen high-efficient crop varieties are essential for the sustainable agriculture. Sorghum is one of a main cereal crop of many countries, which is tolerant to drought, flood and poor soil. In the present study, we investigated the effects of low P and N stress on the morphological and physiological indices of sorghum seedlings, and identified the miRNAs of sorghum under low nitrogen treatment by high-throughput sequencing, verified the expression of candidate miRNAs and their target genes by northern blot in sorghum, and obtained following results:1. Effects of various P levels on sorghum seedlings were investigated by vermiculite culture in pots, the result shows different P supply levels significantly affect the morphologies of sorghum seedlings, but the physiologies of sorghum seedlings were not significantly affected, and P levels of 0.25 mmol/L and 1 mmol/L were found to be the proper levels for low P and normal P treatments; seedling dry weight and P content in shoots were recommended as screening indices for low P-tolerance cultivars at sorghum seedling stage; Bayueqi was identified as a low-P tolerant cultivar, while Nong 858 was a low-P sensitive cultivar.2. Effects of low N stress on the morphological and physiological indices of sorghum seedlings were investigated by hydroponics, the differences of many indices were significant or highly significant between sorghum seedlings under 4 mmol/L and 0.04 mmol/L N levels, the indices included seedling height, stem diameter, plant dry weight, chlorophyll and soluble protein contents. Therefore, the low and normal nitrogen treatment levels were determined as 0.04 mmol/L and 4 mmol/L in sorghum seedling N-stress test, respectively.3. One hundred and twenty-two known miRNAs were obtained in low N-stressed sorghum with high-throughput sequencing, among them the expression differences of 107 miRNAs were observed significant or highly significant between the normal and low N treatments, which belonged to 27 miRNA families., Sixty different expression miRNAs were found in root under the stress of 8 h low N,38 of them were up-regulated, and 22 were down-regulated; when sorghum roots were stressed under 15d low N,22 miRNAs of 41 different expression miRNAs were up-regulated, and 19 were down-regulated; in sorghum leaves,82 differently-expressed miRNAs were found under the stress of 8 h low N,20 of them were up-regulated, and 62 were down-regulated; while 76 differently-expressed miRNAs were found after 15d of low N stress,26 of them were up-regulated, and 50 were down-regulated.4. Target genes of low nitrogen response miRNAs were predicted by bioinformatics approaches,101 target genes were related to 78 differently-expressed miRNAs, which play more than 30 molecular functions, including 55% binding,40% unknown,8% transcription factor activity,7% hydrolase activity, and protein dimerization activity, oxidoreductase activity; signal transducer activity; ATPase activity,etc.5. One hundred and twenty-three novel miRNAs were obtained in sorghum stressed by low N with high-throughput sequencing. In roots,16 differently-expressed miRNAs were found under the stress of 8 h low N,4 were found under the stress of 15d of low N; while in leaves,27 differently-expressed miRNAs were obtained under the stress of 8 h low N,20 were got under the stress of 15d low N. Under the stress of 8 h low N,5 miRNAs were up-regulated in roots and 11 were down-regulated; 4 miRNAs were up-regulated in leaves and 23 were down-regulated; while under the stress of 15d low N,2 miRNAs were up-regulated in roots and 2 were down-regulated,7 miRNAs were up-regulated in leaves and 13 were down-regulated.6. Eight target genes of 4 novel differently-expressed miRNAs were predicted. The molecular functions of the target genes are mainly binding that is DNA binding, protein binding, nucleotide binding; iron ion binding, metal ion binding and pyridoxal phosphate binding, etc.7. Reference genes were screened by Real-time PCR and geNorm. We found that GAPDH and EIF4a were suitable as reference genes of sorghum leaves in Real-time PCR, while sbGAPDH and EIF4a for roots.8. The expression of candidate miRNAs and their target genes in sorghum were verified by Real-time PCR. The similar expression tendencies of these miRNAs were observed with the sequencing result, and the expression levels between most miRNAs and their target genes were negatively correlated.更多还原