| 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. |
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