NMT是基因功能的活體檢測技術，已被103位諾貝爾獎得主所在單位，及北大、清華、中科院使用。
期刊：International Journal of Molecular Sciences
主題：OsCIPK2過表達促進低氮條件下水稻NO3-吸收
標題：Exploring the Potential of Overexpressed OsCIPK2 Rice as a Nitrogen Utilization E cient Crop and Analysis of Its Associated Rhizo-Compartmental Microbial Communities
影響因子：4.183
檢測指標：Ca2+、NH4+、NO3-流速
檢測樣品：水稻根尖
Ca2+、NH4+、NO3-流實驗處理方法：
幼苗長至三葉期，分別在正常氮（1.44 mM NH4NO3）和低氮（0.24 mM NH4NO3）處理
Ca2+、NH4+、NO3-流實驗測試液成份：
Ca2+: 0.1 mmol/L KCl, 0.1 mmol/L CaCl2, 0.1 mmol/L MgCl2, 0.5 mol/L NaCl, 0.3 mmol/L MES, 0.2 mmol/L Na2SO4, pH 6.0;
NO3-: 0.1 mmol/L NH4NO3, 0.1 mmol/L NH4NO3, 0.1 mmol/L KCl, 0.1 mmol/L KCl, 0.1 mmol/L KCl2, 0.3 mmol/CaCl2, 0.3 mmol/L MgCl2, 0.3 mmol；
NH4+: 0.1 mmol/L NH4NO3, 0.1 mmol/L CaCl2, 0.3 mmol/L MES, pH 6.0
作者：福建農林大學林文雄
英文摘要
Nitrogen (N) is one of the indispensable factors in rice growth and development. China holds a premier position in the production of rice and at the same time also faces higher N fertilizer costs along with serious damage to the environment. A better solution is much needed to address these issues, without disrupting the production of rice as an important cereal, while minimizing all the deleterious effects on the environment.
Two isogenic lines Kitaake (WT) and its genetically modified line CIPK2 (RC), overexpressing the gene for Calcineurin B-like interacting protein kinase 2 (OsCIPK2) with better nitrogen use efficiency (NUE), were compared for their growth and development under low versus normal levels of N. NUE is a complex trait mainly related to a plant’s efficiency in extraction, assimilation, and recycling of N from soil. The microbial population was analyzed using high-throughput Illumina Miseq 16S rRNA sequencing and found that RC with CIPK2, specifically expressed in rice root, not only performed better without nitrogen fertilizer (LN) but also increased the diversity of bacterial communities in rice rhizosphere compartments (rhizosphere, rhizoplane, and endosphere).
The relative abundance of beneficial bacteria phyla increased, which are known to promote the circulation and transformation of N in rhizosphere soil. To further explore the potential of RC regarding better performance under LN, the ion fluxes in root apical were detected by non-invasive micro-test technique (NMT). We found that RC can absorb more Ca2+ and NO3 under LN as compared to WT. Finally, compared to WT, RC plants exhibited better growth of root and shoot, and increased yield and N uptake under LN, whereas there was no significant difference in the growth of two rice lines under normal nitrogen (NN) treatment.
We are able to get preliminary results, dealing with the OsCIPK2 overexpressed rice line, by studying the rice molecular, physiological, and chemical parameters related to NUE. The results laid the foundation for further research on N absorption and utilization in rice from the soil and the interaction with microbial communities.
中文摘要（谷歌機翻）
氮（N）是水稻生長發育不可或缺的因素之一。中國在大米生產中處於領先地位，同時也面臨著更高的氮肥成本以及對環境的嚴重破壞。迫切需要一種更好的解決方案來解決這些問題，同時又不中斷作為重要穀物的大米的生產，同時將對環境的所有有害影響降至最低。
比較了兩個同基因系Kitaake（WT）及其基因改造品系CIPK2（RC），它們以較低的氮素利用效率（NUE）過量表達了鈣調磷酸酶B樣相互作用蛋白激酶2（OsCIPK2）的基因，並在低溫下對其生長發育進行了比較。與正常水平的氮相比。NUE是一個複雜的性狀，主要與植物從土壤中提取，吸收和循環利用N的效率有關。使用高通量Illumina Miseq 16S rRNA測序分析了微生物種群，發現帶有CIPK2的RC在水稻根部特異表達，不僅在不使用氮肥（LN）的情況下表現更好，而且增加了水稻根際區室細菌群落的多樣性（根際，根際和內球）。
有益菌門的相對豐度增加，已知它可以促進根際土壤中氮的循環和轉化。為了進一步探索RC在LN下獲得更好性能的潛力，通過無創微測試技術（NMT）檢測了根尖中的離子通量。我們發現，與野生型相比，RC在LN下可以吸收更多的Ca2+和NO3-。最後，與WT相比，RC植物在LN下表現出更好的根和莖生長，並增加了產量和氮素吸收，而在正常氮（NN）處理下，兩個水稻品系的生長沒有顯著差異。
通過研究與NUE相關的水稻分子，生理和化學參數，我們能夠獲得初步結果，處理過表達OsCIPK2的水稻品系。研究結果為進一步研究水稻從土壤中吸收和利用氮以及與微生物群落的相互作用奠定了基礎。
結果表明：LN（low nitrogen）處理時，RC（過表達型）根系的Ca2+吸收顯著增加，而NN（normal nitrogen）處理時無顯著差異；LN處理時，RC根系的NO3-吸收增加，而NN條件下RC和WT（野生型）根系的NO3-表現為外排，且二者並無顯著差異；至於NH4+，RC和WT均表現為外排，且無顯著差異。這說明，LN條件下，根系CIPK2基因過表達促進了根系吸收NO3-，與RC根際的硝化作用增加是符合的。