【作者】 郝青南；

【导师】 周新安；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2013， 博士

【摘要】 大豆（Glycine max (L.) Merr.）是世界上最广泛种植的豆科植物，是重要的蛋白质和油料来源。随着人们对蛋白质、脂肪需求量的增加，大豆在国民经济中的地位越来越重要。氮素是植物生长必需的大量元素之一，也是限制植物生长和产量形成的首要因素。大豆一生需要大量的氮素营养。虽然大豆是固氮植物，但其自身所固定的氮不能满足大豆丰产对氮素营养的需求。在苗期，大豆根瘤的数量少，植株尚不能或很少利用根瘤菌共生固氮供给的氮素，因此在这个时期吸收土壤氮以及施用氮肥显得十分重要。传统上人们通过改良土壤和施用氮肥来改善作物的氮营养状况，但氮肥利用效率较低，可被植物利用的氮在30%~40%左右，土壤中超过40%的氮素通过挥发、淋失、脱氮以及微生物分解等途径流失掉，这对环境造成了极大的污染，同时也造成了巨大的经济损失。因此，提高氮素利用效率十分重要。培育耐低氮胁迫的大豆品种是提高氮素利用效率的有效途径，利用基因工程手段来培育大豆耐低氮胁迫品种可为此开辟一条新途径。本研究以现代分子生物学和转基因等技术为主导，结合新一代高通量测序技术，系统分析与克隆了大豆中控制氮素吸收、转运、同化和再动员等过程相关的功能基因和转录因子，并进行功能验证，最终以提高大豆氮素利用效率，减少化肥投入、提高大豆产量和改善农业生态环境为目的。主要研究结果如下：1．以147个大豆品种为材料，采用温室苗期水培方法，观察各品种在低氮培养和正常供氮环境下培养时的叶片黄化程度，初步评价大豆品种苗期对低氮胁迫的反应。通过对相关指标的变异系数进行比较并结合各指标相关性分析结果，将干重、地上部分氮积累量、生物量增加值和氮吸收量作为判断耐低氮的指标，最终筛选出耐低氮型品种“坡黄”和氮敏感型品种“84-70”。2．对耐低氮型品种“坡黄”和氮敏感型品种“84-70”进行数字表达谱分析：将这两个品种进行低氮处理，对地上部分和地下部分分别取样测序。一共建立了8个库进行测序：2种基因型×2种大豆组织（地上部分，地下部分）×2个处理时期[短时期（0.5-12h）和长时期（3-12d）]。通过高通量测序分析，八个库中分别获得了5,739,999,5,846,807,5,731,901,5,970,775,5,476,878,5,900,343,5,930,716,和5,862,642个clean tags;对应224,154,162,415,191,994,181,792,204,639,206,998,233,839和257,077个distinct tags。3,231个基因在两个品种中表达差异显著，参与21个代谢和信号传导途径。这些差异表达基因可能是与氮高效利用相关的候选基因。3．通过分子克隆技术克隆得到大豆Gmduf-cbs和Gmdof1.4基因的cDNA全长序列，并利用生物信息学方法对所得序列进行氨基酸理化性质分析、蛋白质结构域及功能域预测、同源性比对及系统进化树分析等。为了验证预测结果，将GmDuf-cbs和GmDof1.4蛋白与绿色荧光蛋白（GFP）进行融合，通过基因枪轰击转化洋葱表皮细胞，融合了目的蛋白质的GmDof1.4在细胞核上有绿色荧光信号分布，从而表明GmDof1.4蛋白定位在细胞核上。融合了目的蛋白质的GmDuf-cbs在细胞核核细胞膜上均有绿色荧光信号分布，从而表明该蛋白定位在细胞膜和细胞核上。同时应用荧光定量PCR技术对Gmduf-cbs和Gmdof1.4基因在大豆不同氮浓度培养条件下不同组织不同时期表达情况进行了分析，发现这两个基因表达水平存在显著差异。4．以模式植物拟南芥为材料，利用反向遗传学的方法，即根据已知基因的表达变化情况来研究表型的变化，从而推测该已知基因的功能。采用花序浸泡法将Gmduf-cbs和Gmdof1.4转化拟南芥，经PCR及Southern杂交检测获得了转基因植株。选取3个转录水平较高的拟南芥株系进行耐低氮性分析。对Gmduf-cbs和Gmdof1.4基因所编码的蛋白在拟南芥氮吸收过程中的生物学功能进行了分析。通过氮高效相关指标的测定，结果显示野生型植株转Gmduf-cbs或Gmdof1.4基因的拟南芥株系在低氮胁迫处理下，生长14天后，转基因的拟南芥植株叶片黄化程度较低，且植株较健壮。转植株的鲜重，全氮含量，硝态氮，铵态氮以及非蛋白氮含量均高与野生型植株。从而推测过表达Gmduf-cbs或Gmdof1.4基因的转基因植株具有一定的低氮耐受性。更多还原

【Abstract】 Soybean (Glycine max (L.) Merr.) is the world’s most widely planted legumes and soybean is avaluable crop that provides protein and oil. With the increase in the demand of protein, fat, soybean isbecoming more and more important in the national economy.Plants require large amounts of nitrogen (N) for their growth and survival. This N accounts forapproximately2%of total plant dry matter. Soybean requires more N than other major crops to sustainseed growth. As a legume, soybean can acquire N for its growth via its N-fixing symbiosis withrhizobacteria, which form nodules on the roots and can fix atmospheric N. In addition, soybean candraw mineral nitrogen from the soil. These processes may not supply enough N for soybeans tomaximize yield, especially in high-yield environments. Plants can only use approximately30-40%ofthe applied N, and more than40%of the N fertilizer is lost via leakage into the atmosphere,groundwater, lakes and rivers. Such leakage results in serious environmental pollution. Improving N-useefficiency (NUE) by genetic improvement is necessary for the development of agriculture.Next-generation sequencing techniques are opening fascinating opportunities for life sciences, and havedramatically improved the efficiency and speed of gene discovery. Driven by Solexa/Illuminatechnology, DGE creates genome-wide expression profiles by sequencing.The study based on modern molecular biology and transgenic technology, and combined with anew generation of high-throughput sequencing technology. By investigating the expressions of genesrelated to N utilization, a number of candidate genes for NUE were identified and analysis. The mainresults are as follows:1. In order to obtain the different NUE soybean varieties, a total of147varieties were screened inthis study. After analyzed the coefficient of variation and correlation among the indexes, shoot nitrogenaccumulation, increase in biomass,dry weight and the amount of nitrogen absorption were chosen as theindexes for evaluating the NUE of soybean varieties. The results indicated that“Pohuang” and“84-70”had the highest and lowest relative value, respectively. So “Pohuang” was tolerantand“84-70”was sensitive to low N-stress, respectively.2. Two soybean genotypes were grown under N-limited conditions.The shoots and roots ofsoybeans were used for sequencing. Eight libraries were generated for analysis:2genotypes×2tissues(roots and shoots)×2time periods [short-term (0.5to12h) and long-term (3to12d) responses] andcompared the transcriptomes by high-throughput tag-sequencing analysis.5,739,999,5,846,807,5,731,901,5,970,775,5,476,878,5,900,343,5,930,716, and5,862,642clean tags were obtained for theeight libraries,these corresponded to224,154,162,415,191,994,181,792,204,639,206,998,233,839and257,077distinct tags,respectively. A number of soybean genes were differentially expressedbetween the low-N-tolerant and low-Nsensitive varieties under N-limited conditions. Some of thesegenes may be candidates for improving NUE.3. Two full-length cDNAs Gmduf-cbs and Gmdof1.4were cloned from soybean. Analysis of these two genes by bioinformatic, including physical and chemical properties of amino acids, protein domainsand functional domains predicted homology comparison and phylogenetic analysis. To verify thesubcellular locations of Gmduf-cbs and Gmdof1.4, a green fluorescent protein (GFP)-tagged gene wasfused to Gmduf-cbs and Gmdof1.4, respectively.They were transformed into onion epidermal cells bygene gun bombardment. A clear GFP signal was observed at the nucleus of onion epidermal cellsbombarded with the Gmdof1.4construction, and at the nucleus and cytomembrane of onion epidermalcells bombarded with the Gmduf-cbs construction whereas the signal was seen throughout cellsexpressing free GFP. Gene expression of Gmduf-cbs and Gmdof1.4in soybean were analyzed byreal-time PCR.The result showed that the expression were significantly different between two varieties.4. The Gmduf-cbs and Gmdof1.4gene were overexpressed in Arabidopsis to study their functionunder low nitrogen stress. Some transgenic positive plants were obtained and the gene successfullytranscribed by PCR and RT-PCR detection. The results showed that constitutive expression ofGmduf-cbs or Gmdof1.4in Arabidopsis not only enhanced nitrogen starvation tolerance of the cells butaccelerated the growth of the plant. We conclude that Gmduf-cbs or Gmdof1.4may serve as an excellentcandidate for breeding crops with enhanced NUE and better yield.更多还原