水稻抽穗期基因OsDof6功能的初步研究

【目的】以前期通过穗发育芯片筛选到一个水稻Dof家族转录因子OsDof6为研究对象,进一步探究OsDof6的表达模式与生物学功能。【方法】对OsDof6的基因、蛋白及启动子序列进行生物信息学分析,利用CRISPR/Cas9技术对该基因进行定点编辑,通过实时定量PCR和亚细胞定位技术分析该基因的表达模式。【结果】对该基因的启动子序列分析表明,该基因启动子中存在大量与光响应、激素响应及胁迫响应相关的顺式调控元件。利用CRISPR/Cas9技术获得了2种不同突变类型的功能缺失突变体9522^Dof6-3和9522^Dof6-4。对突变体T₁株系进行表型观察发现,相较于对照,两种突变体在营养生长阶段分蘖数明显降低,转入生殖生长阶段后,两种突变体的抽穗时间推迟约3 d。实时定量PCR结果显示OsDof6在水稻根、茎、叶和穗中均有不同程度的表达,在穗发育后期相对表达量明显提高;通过亚细胞定位分析发现OsDof6定位于细胞核。【结论】初步判断OsDof6基因会影响水稻分蘖数与抽穗期。     

麦/油-稻轮作下秸秆还田与氮肥管理对直播杂交稻氮素利用特征的影响

【目的】探明秸秆还田和氮肥管理对麦/油后直播杂交稻氮素积累、转运、氮肥利用效率及籽粒产量的影响。【方法】选用优质三系杂交稻宜香优2115,采用二因素裂区设计,麦、油茬田同步开展试验,处理完全一致。主区为麦/油秸秆全量翻埋还田(M₁)和秸秆不还田(对照,M₀),副区设4个氮肥管理,即不施氮对照(N₀)、m_基肥∶m_分蘖肥∶m_促花肥∶m_保花肥比例分别为1∶0∶0∶0(N₁)、3∶3∶2∶2(N₂)、2∶2∶3∶3(N₃),测定了直播杂交稻主要生育时期各器官的氮素积累量及籽粒产量。【结果】结果表明,两种轮作方式下,氮肥管理对直播杂交稻主要生育时期的氮素积累、齐穗后茎鞘、叶片的氮素转运及稻株氮素利用效率均存在显著或极显著的调控效应。秸秆还田显著提高麦/油茬杂交稻中后期的氮素积累量、茎鞘和叶片的氮素转运量以及氮肥利用效率,其中,氮肥农学利用率、氮肥偏生产力和氮肥表观利用率较秸秆不还田分别提高了34.96%/28.76%、2.52%/2.61%和31.91%/22.30%。同时,油菜秸秆还田下直播杂交稻各生育时期氮素积累和产量较麦秆还田表现更好,籽粒产量提高481 kg/hm²(5.22%)。M₁N₂处理、M₀N₃处理下,直播杂交稻各阶段的氮素积累速率明显加大,促进结实期茎鞘和叶片的氮素向穗部转运,成熟期稻株氮素积累量优势明显且有较高的氮素利用效率(麦/油茬稻氮肥农学利用率、偏生产力和表观利用率分别达17.87 kg∙kg^-1/17.85 kg∙kg^-1、67.27 kg∙kg^-1/71.28 kg∙kg^-1、74.93%/75.05%),最终实现高产。【结论】在麦/油-稻轮作下秸秆全量还田,配合N₂氮肥管理,可有效提高直播杂交稻氮素吸收、利用效率,增加籽粒产量,尤以油菜秸杆还田的效果更好。     

秸秆还田对东北半干旱区玉米‖花生轮作系统土壤水热特性及作物产量的影响

以东北半干旱区玉米‖花生轮作系统为研究对象,试验设传统玉米单作、花生单作、不同秸秆还田量下(30%、60%和100%)玉米‖花生轮作5个处理。通过连续3年田间定位试验,研究玉米秸秆不同还田量对农田土壤水热特性、产量及土地当量比(LER)的影响。结果表明,与传统单作相比,秸秆还田处理玉米区播前0～40 cm土壤平均含水量提高了1.9%～3.9%,花生区提高了11.0%～13.9%;成熟期,秸秆还田花生区0:00～8:00平均地温比传统单作提高了0.2℃～1.1℃,10:00～22:00平均地温较传统单作降低了0.5℃～1.0℃。2016年和2017年玉米‖花生轮作系统LER为1.05～1.16,2018年各处理LER均小于1。吉林半干旱区玉米‖花生轮作系统进行秸秆还田改善了作物播前的土壤水分状况,但对于作物产量的影响在不同年份有所差异。     

Dynamics of nitrogen translocation from mature leaves to new shoots and related gene expression during spring shoots development in tea plants (Camellia sinensis L.)

The contribution of N remobilization is crucial for new shoots growth and quality formation during spring tea shoots development. However, the translocation mechanism of N from source leaves to sink young shoots is not well understood. In the present study, ¹⁵N urea was applied to mature tea leaves one week before bud break to track N remobilization in a field experiment. The dynamic changes in plant ¹⁵N abundance, contents of amino acids, and the expression levels of genes related to N metabolism and translocation were followed during the 18‐d development of new spring shoots until three expanding young leaves. The results showed that during the growth of new shoots the amount of ¹⁵N in the shoots increased, whereas the N_dff (N derived from ¹⁵N‐urea) in mature leaves decreased, showing that the foliar‐applied N in mature leaves was readily exported to new shoots. This process was found to be accompanied by decline of chlorophylls. In the mature leaves, expression CsATG18a and CsSAG12 involved in autophagy was dramatically induced (> 4‐fold) at approximately nine days after the bud breaking. The genes involved in the transformation of amino acids, including primarily CsGDH2, CsGDH4, CsGLT3, CsGS1;3, and CsASN2 were upregulated by > 3‐fold after bud breaking. The expression levels of CsATG8A, CsATG9, CsSAG12, CsGS1;1, CsGDH1, and CsAAP6 correlated negatively with the N_dff in mature leaves, but positively with ¹⁵N amount and total N amount in new shoots, suggesting these genes played important roles in N export from mature leaves. In the new shoots, the expression of most genes showed two defined peaks, one on six days and one on 12 days after bud breaking. The expression of CsGS2, CsASN3, CsGLT1, and CsAAP4 positively correlated with the ¹⁵N amount and total N amount in new shoots. These genes might be involved in the transport and re‐assimilation of N from mature leaves. The overall results demonstrated that the translocation of ¹⁵N from mature leaves to new spring shoots was regulated by the genes involved in autophagy, protein degradation, amino acid transformation and transport.     

Intensive organic vegetable production increases soil organic carbon but with a lower carbon conversion efficiency than integrated management

Intensive vegetable production in greenhouses has rapidly expanded in China since the 1990s and increased to 1.3 million ha of farmland by 2016, which is the highest in the world. We conducted an 11‐year greenhouse vegetable production experiment from 2002 to 2013 to observe soil organic carbon (SOC) dynamics under three management systems, i.e., conventional (CON), integrated (ING), and intensive organic (ORG) farming. Soil samples (0–20 and 20–40 cm depth) were collected in 2002 and 2013 and separated into four particle‐size fractions, i.e., coarse sand (> 250 µm), fine sand (250–53 µm), silt (53–2 µm), and clay (< 2 µm). The SOC contents and δ¹³C values of the whole soil and the four particle‐size fractions were analyzed. After 11 years of vegetable farming, ORG and ING significantly increased SOC stocks (0–20 cm) by 4008 ± 36.6 and 2880 ± 365 kg C ha^?1 y^?1, respectively, 8.1‐ and 5.8‐times that of CON (494 ± 42.6 kg C ha^?1 y^?1). The SOC stock increase in ORG at 20–40 cm depth was 245 ± 66.4 kg C ha^?1 y^?1, significantly higher than in ING (66 ± 13.4 kg C ha^?1 y^?1) and CON (109 ± 44.8 kg C ha^?1 y^?1). Analyses of ¹³C revealed a significant increase in newly produced SOC in both soil layers in ORG. However, the carbon conversion efficiency (CE: increased organic carbon in soil divided by organic carbon input) was lower in ORG (14.4%–21.7%) than in ING (18.2%–27.4%). Among the four particle‐sizes in the 0–20 cm layer, the silt fraction exhibited the largest proportion of increase in SOC content (57.8% and 55.4% of the SOC increase in ORG and ING, respectively). A similar trend was detected in the 20–40 cm soil layer. Over all, intensive organic (ORG) vegetable production increases soil organic carbon but with a lower carbon conversion efficiency than integrated (ING) management.     

Is N‐feedback involved in the regulation of nitrogenase activity in Medicago truncatula?

To determine whether senescing leaves provoke an active nitrogen (N) remobilization that results in the reduction of nitrogenase activity, 60% of Medicago truncatula lower leaves were either darkened or individually excised for two weeks. Although a considerable amount of N was remobilized, N₂ fixation activity was found to be increased to maintain the N source/sink balance, indicating an absence of the negative N‐feedback regulation of nitrogenase activity in the senescing M. truncatula.     

不同温度下TMV侵染枯斑三生烟的LncRNA差异表达

【目的】 筛选不同温度下烟草花叶病毒（Tobacco mosaic virus,TMV）侵染后枯斑三生烟（Nicotiana tabacum var. Samsun NN）差异表达的长链非编码RNA（long non-coding RNA,lncRNA）,研究lncRNA在枯斑三生烟抗性反应中的作用。【方法】 N基因的温度敏感性使枯斑三生烟在25℃时具备对TMV的抗性、在31℃抗性丧失,在这两个温度条件下对枯斑三生烟接种TMV和磷酸盐缓冲盐水（phosphate buffered saline,PBS）,48 h后提取系统叶总RNA,构建链特异性文库后进行深度测序。对测序结果进行过滤后利用HTSeq将有效数据与近缘品种TN90（N. tabacum var. TN90）基因组比对,筛选得到lncRNA后利用FPKM法估计lncRNA的表达水平。通过edgeR筛选差异表达lncRNA（differentially expressed lncRNA,DElncRNA）,并利用qRT-PCR技术对这一结果进行验证。通过共定位及共表达分析预测DElncRNA的靶基因,通过参考基因组注释、GO和KEGG富集分析研究靶基因的功能。【结果】 4个处理共12个样本经lncRNA-seq各测得约8 000万条clean reads,共获得4 737条已知lncRNA、40 169条新lncRNA。其中64个lncRNA在不同温度条件下TMV侵染后存在差异表达,qRT-PCR测定结果显示这些lncRNA的测序正确率在80%左右,表明本研究所得测序数据具备较高的可信度。对DElncRNA进行靶基因预测,发现一些基因同时被25℃下调和31℃上调的DElncRNA靶向。靶基因注释功能丰富,主要参与植物抗病、激素和代谢等生理过程。部分可能与激素通路相关的lncRNA,在25℃下TMV侵染时呈现下调趋势,而在31℃下TMV侵染则呈现上调趋势。GO富集分析显示靶基因主要参与构成膜、囊泡等组分,具备钙、钾离子通道抑制剂活性等分子功能,使相应离子得以转运引发随后的反应,同时也参与发病、抗原加工和呈现、细胞分裂素代谢等生理过程。KEGG分析发现靶基因显著富集在植物激素信号转导通路,25℃下调和31℃上调的DElncRNA靶基因同时富集在激素信号传导、ABC运输蛋白、苯丙烷类生物合成等通路。【结论】 不同温度（25℃和31℃）条件下TMV侵染枯斑三生烟后,长链非编码RNA差异表达,DElncRNA通过作用于激素信号传导、物质转运等过程参与寄主系统获得性抗性反应。研究结果可为揭示植物系统获得性抗性中lncRNA的调控功能以及新型抗病毒技术开发提供依据。     

不同根系分泌物对土壤N2O排放及同位素特征值的影响

【目的】探究植物根系分泌的主要组分（有机酸、氨基酸、糖类）对土壤N₂O排放及其微生物过程的影响,为选择适宜的植物进而控制土壤N₂O排放提供支撑。【方法】通过室内试验分别添加草酸、丝氨酸、葡萄糖于土壤中模拟根系的3种主要分泌物,每种分泌物设置两个浓度水平：低浓度（150 μg C·d ^-1）和高浓度（300 μg C·d ^-1）,另设置添加蒸馏水的对照组,共7个处理。将土壤置于120 mL玻璃瓶中进行培养,24 h内采集气体样品7次,每次培养2 h,获取N₂O排放速率、日累积排放量和同位素特征值（δ ¹⁵N ^bulk、δ ¹⁸O和SP（site preference,SP=δ ¹⁵N ^α-δ ¹⁵N ^β））。【结果】添加3种根系分泌物组分后,土壤N₂O排放速率均逐渐升高,且均高于对照。高浓度处理组N₂O累积排放量为：葡萄糖（（3.2±1.3）mg·kg ^-1·d ^-1）处理>丝氨酸（（2.6±0.5）mg·kg ^-1·d ^-1）处理>草酸（（1.4±0.2）mg·kg ^-1·d ^-1）处理,低浓度处理组为：草酸（（2.7±1.3）mg·kg ^-1·d ^-1）处理>丝氨酸（（1.8±0.4）mg·kg ^-1·d ^-1）处理>葡萄糖（（1.6±0.8）mg·kg ^-1·d ^-1）处理;添加根系分泌物的不同处理间土壤N₂O的δ ¹⁸O值无明显差异,并稳定在24.1‰—25.6‰,且均显著高于对照（（20.1±1.5）‰）;土壤N₂O的δ ¹⁵N ^bulk值与添加根系分泌物的种类有关,其中草酸处理组为（-20.06±2.22）‰、丝氨酸处理组为（-22.33±1.10）‰、葡萄糖处理组为（-13.86±1.11）‰、对照组为（-23.14±3.72）‰。各处理土壤N₂O的SP值的变化范围为13.13‰—15.03‰,根系分泌物浓度越高,SP值越低。综合分析不同处理4个指标（N₂O排放速率、N₂O的δ ¹⁵N ^bulk、δ ¹⁸O和SP值）的不同时刻的检测值与日均值的校正系数,添加根系分泌物后第16小时各处理4个指标的校正系数最接近于1。【结论】在NH+ 4-300 mg N·kg ^-1的土壤环境下根系分泌物促进N₂O的排放,且在培养期间（24 h）土壤N₂O排放速率逐渐升高。高浓度处理组葡萄糖对土壤N₂O排放速率促进效果最强,低浓度处理组草酸对土壤N₂O排放速率促进效果最强。与对照组相比,根系分泌物的添加使N₂O的δ ¹⁸O值显著升高;与对照组相比,葡萄糖的添加使δ ¹⁵N ^bulk值显著升高。根系分泌物浓度越高,反硝化作用对N₂O的贡献越大。     

基于GIS与RS的北方防沙屏障带生态系统格局演变

研究北方防沙屏障带生态系统格局演变特征,对加强屏障带建设、改善生态环境具有重要意义。以北方防沙屏障带为研究区,根据2005、2010、2015年MODIS遥感影像,将北方防沙屏障带生态系统类型划分为森林、草地、湿地、农田、城镇、荒漠和裸地7类。采用空间统计、转移矩阵、景观指数分析法、PNTIL模型等方法,对北方防沙屏障带2005—2015年生态系统类型时空演变特征进行分析。结果表明,2005—2015年北方防沙屏障带生态系统整体呈稳定状态;从分屏障带看,内蒙古防沙屏障带荒漠面积明显减少,塔里木防沙屏障带森林面积迅速增加,而河西走廊防沙屏障带草地面积明显增加;从正/逆转换方向来看,屏障带内农田、城镇、荒漠及裸地生态系统正向转换率高于逆向转换率。10年间,在景观水平上,北方防沙屏障带斑块数量、斑块密度呈减少趋势;在类型水平上荒漠景观整体呈现破碎化萎缩的趋势特征,森林、草地向形状简单化的方向发展。研究表明,2005—2015年,北方防沙屏障带生态总体向好,治沙效果明显,但局部仍面临较大压力。