基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法

马铃薯晚疫病菌（Phytophthora infestans）能侵染多种茄科植物,它引起的马铃薯晚疫病,是马铃薯生产中的第一大病害。为了开发能在田间快速检测马铃薯晚疫病病原的方法,利用P. infestans T30-4基因组测序数据的contig 1.18131,设计qPCR和LAMP引物,优化扩增条件后得到引物的特异性和灵敏度,最后通过检测田间收获薯块,比较形态学传统方法、qPCR及LAMP的差异。特异性检测结果发现,qPCR和LAMP仅在含有P. infestans DNA模板的体系有阳性扩增,在寄主和其他微生物DNA中均无扩增;在优化的条件下,qPCR和LAMP的检测下限可达1×10 ^-6ng/μL,在有寄主和其他微生物DNA存在的条件下,引物的灵敏度没有显著差异。利用两种快速方法对在大理、丽江及昆明3个地区田间收获薯块上检测发现,qPCR和LAMP方法得到的检出率差异极为不显著（P=0.420）,两种快速检测方法和形态学鉴定方法检出率差异极显著（P=0.009）。在大理、丽江及昆明3个地区的薯块中,两种分子检测方法检出率均比形态学方法高。其中,qPCR检测方法比形态学方法分别提高了12.00%、2.00%、8.70%;LAMP检测方法比形态学方法分别提高了11.30%、2.00%、8.70%。     

基于土地利用变化的河北省坝上地区景观生态风险评价

[目的]对河北省坝上地区近40 a来的土地利用动态变化和生态风险进行分析评价并对未来趋势作出预测,为该地区生态建设和治理、可持续发展提供科学依据。[方法]基于坝上地区1980—2018年5期土地利用数据以及通过土地转移矩阵、空间相关性分析等方法揭示和预测该区1980—2026年的土地利用变化特征并评估该区生态风险水平。[结果](1)整个研究期间,坝上地区土地利用类型以耕地为主,所占比例近50%,其中,1980—2018年,耕地、林地扩张面积均超过300 km~2,草地减少近616.60 km~2,水域面积缩减36.04%,其中耕地、林地、草地之间的互相流转程度较为剧烈,空间变化上表现为各地类的重心在2000—2010年明显迁移。(2)1980—2026年,坝上地区6个时期内生态风险值全局空间自相关Moran’s I指数均在0.500左右,其空间分布表现出较高的趋同集聚性。(3)近40 a来,坝上生态风险水平升至为高风险级,其区域增加了123.22 km~2,较高风险区域分布在城镇地区,据CA-Markov模型预测,未来坝上地区中等及中等以上风险区域持续扩张,丰宁县和围场县将分别出现小规模高风险区和较高风险区。[结论](1)近40 a来坝上地区草地退化严重,水域面积显著减少,原因系安固里淖干涸所致。(2)该区生态风险水平与土地格局分布具有较强相关性,且在未来会继续升高。     

转基因甘蔗BtG-2的T-DNA侧翼序列分析及其转化事件特异性检测

转基因甘蔗BtG-2是利用农杆菌介导法把Cry1Ac-2A-gna融合抗虫基因导入‘新台糖22号’的转基因甘蔗株系,具有良好的抗虫特性和农艺性状。为了明确转基因甘蔗BtG-2的分子特征及其检测方法,推进其生物安全性评价工作,以BtG-2的T2代为研究材料,利用Southern杂交检测外源基因在转基因甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立了该转化体高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BtG-2株系;经过3次的热不对称巢式PCR扩增,获得外源基因T-DNA左边侧翼序列984 bp和右边侧翼序列705 bp;以这2个序列和相应的T-DNA的左右端序列分别设计3对检测引物对,建立了BtG-2株系的转化事件特异性PCR检测方法,扩增效率最高的引物对LS011/LA451和RS160/RA588分别扩增到440 bp和428 bp的特异片段。其中T-DNA左侧设计的LS011/LA451检测引物对扩增的灵敏度高、特异性强,能够在甘蔗BtG-2基因组DNA相对含量为0.1%的模板中检测出转基因目的成分,相当于9个单倍体基因组拷贝数。本研究完成了转基因株系BtG-2的分子特征及其转化事件特异性检测,为该转基因甘蔗及其衍生产品的检测和身份识别提供技术依据。     

卵磷脂桶混助剂的特性及其在防治稻飞虱中对氟啶虫胺腈的协同增效作用

为明确新型卵磷脂类桶混助剂“融透”的特性，采用透射电镜和激光粒度分析仪表征了其物理性质，并通过室内生物测定和田间药效试验测定了其在防治稻飞虱中对氟啶虫胺腈的协同增效作用。结果表明，“融透”中含有圆形脂质体，Z平均粒径为129.5 nm。室内生物测定结果表明，“融透”可使氟啶虫胺腈对褐飞虱的毒力增加1.67倍。田间药效试验表明，“融透”可使氟啶虫胺腈对稻飞虱的防效提高9.04%~41.77%；当氟啶虫胺腈减量40%时，添加“融透”的处理对稻飞虱的防效与未添加“融透”的常量组相当。研究结果表明，卵磷脂桶混助剂“融透”可以通过形成脂质体提高药剂的利用率，从而达到增效的作用。     

控释肥对花生氮代谢相关酶活性的影响

大田条件下,以花育22号和花育20号为试验材料,设控释肥(T1)、控释肥减施(T2)、普通肥料(T3)和不施肥(T4)处理,研究了控释肥对花生氮代谢相关酶活性的影响。结果表明,与不施肥处理相比,施肥处理花生结荚期和成熟期根系和叶片的可溶性蛋白含量、NRase、GDH、GS、GPT等活性显著增加。各施肥处理间比较,与普通肥料相比,控释肥可有效提高花生结荚期和成熟期根、叶可溶性蛋白质含量,增加氮代谢相关酶NRase、GDH、GS、GPT等活性;控释肥减施处理花生根叶可溶性蛋白含量和相关酶活性花生生育前期低于控释肥处理和普通肥料处理,但成熟期和普通肥料处理差异不显著。总之,控释肥比普通无机肥更有利于花生生育后期氮素同化和蛋白质的合成,延缓根系和叶片衰老。     

Comparison of the microbial communities of alpacas and sheep fed diets with three different ratios of corn stalk to concentrate

The objective of this study was to investigate the characteristics of ruminal microbial communities of alpacas (Lama pacos) and sheep (Ovis aries) fed three diets with varying ratios of roughage (corn stalk) to concentrate, 3:7 (LS), 5:5 (MS) and 7:3 (HS). Six alpacas (one-year-old and weighing 29.5 ± 7.1 kg) and six sheep (one-year-old and weighing 27.9 ± 2.7 kg) were used in this study, in a replicated 3 × 3 Latin square experiment. Total protozoa concentration was determined under the microscope; total fungi and methanogens were assessed using quantitative polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies; bacterial communities were investigated by targeted 16S rRNA gene (V3–V4 region) sequencing. The percentage of fungi was significantly higher in alpacas than in sheep under the LS diet, while the concentration of protozoa was significantly lower in alpacas under HS, MS and LS diets. The alpha diversity including Shannon, Chao l and ACE indices of bacterial communities was higher in alpacas than in sheep, under the LS diet. A total of 299 genera belonging to 22 phyla were observed in the forestomach of alpaca and sheep, with Bacteroidetes and Firmicutes dominating both animal species. Phyla Armatimonadetes and Fusobacteria, as well as 64 genera, were detected only in alpacas, whereas phyla Acidobacteria and Nitrospira, as well as 44 genera, were found only in sheep. The abundance of cellulolytic bacteria, including Butyrivibrio and Pseudobutyrivibrio, was higher in alpacas than in sheep under all three diets. These differences in the forestomach microbial communities partly explained why alpacas displayed a higher poor-quality roughage digestibility, and a lower methane production. Results also revealed that the adverse effects of high-concentrate diets (70%) were lesser in alpacas than in sheep.     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.     

苗后除草剂喷施时期对杂草防治及冬油菜产量和品质的影响

为了探究苗后除草剂不同施用时期对北方冬油菜田间杂草的防控效果及其对冬油菜生长、产量和品质的影响,以当地主栽甘蓝型冬油菜天油2266和白菜型冬油菜天油H614为试验材料,选用对冬油菜安全的苗后除草剂高效氟吡甲禾灵,设置4个喷施时期处理,于2018-2019年开展大田试验。结果表明,越冬后不同时期喷施苗后除草剂,甘蓝型冬油菜田间杂草总数较对照减少57.54%~92.56%,杂草总鲜重较对照降低75.40%~97.48%,第2时期（3月25日）处理产量较对照增加354.50kg/hm²;白菜型冬油菜田间杂草株防效和鲜重防效分别为55.91%~92.02%和37.17%~97.46%,产量较对照增加2.29%~16.77%。因此,在冬油菜大量返青但未封行前施用苗后除草剂,可显著防控田间杂草并增加冬油菜籽粒产量。     

基于UHPLC-Q-TOF/MS的不同产地普洱生茶化学成分差异研究

为了更加全面地了解产地对普洱生茶品质与化学成分的影响,本研究选取来自临沧市、普洱市、西双版纳州三大产区12个茶山(自然村)的普洱生茶样品,采用超高效液相色谱-四级杆-飞行时间质谱(UHPLC-Q-TOF/MS)对不同产地普洱生茶的非挥发性代谢物表型进行了研究。结果表明:不同产地普洱生茶的化学成分在含量上具有较大差异,且具有明显的产地特征。采用主成分分析,可以对来自西双版纳自治州(包括勐腊县、勐海县、景洪市)、普洱市、临沧市3个地级行政区的普洱生茶进行有效区分,也可以对来自普洱茶产区的东南、西南、西北3个区域的普洱生茶进行有效区分。进一步鉴定了普洱生茶中79种主要成分,并对其在12个茶山(自然村)的普洱生茶中的含量分布,以及与不同产地普洱生茶滋味品质的关系进行了分析。本研究表明,基于UHPLC-Q-TOF/MS的茶叶非挥发性化学成分轮廓可以作为普洱生茶产地判别的依据。     

同源四倍体及其原二倍体黑皮冬瓜光合光响应模型筛选

为了筛选出最适合黑皮冬瓜Benincasa hispida (Thunb.) Cogn.的光合光响应模型,为其育种提供理论依据,以同源四倍体及其原二倍体黑皮冬瓜为试验材料,对8种经典的光合光响应模型适用性进行了比较分析。结果表明：二次多项式能够表现出光抑制情况,但在拟合过程中出现暗呼吸速率为正值、光补偿点为负值及无法解释当光强达到饱和后光合速率快速下降的问题;直角双曲线、非直角双曲线及指数函数Ⅰ、指数函数Ⅱ无法直接求取光饱和点、光补偿点,结合常用的光饱和点的计算方法得到的光饱和点与实测值均存在较大的偏差,且指数函数Ⅱ在计算光饱和点时表现出明显的人为性,也无法拟合光抑制情况,但4种模型拟合得到的光补偿点均与实测值相差不大;指数修正模型因系数β为负值,无法求取四倍体黑皮冬瓜材料的光饱和点和最大净光合速率,且拟合得到的四倍体黑皮冬瓜的光补偿点明显低于实测值;直角双曲线修正模型计算得到的暗呼吸速率及二倍体黑皮冬瓜的光饱和点明显低于实测值,但获得的四倍体及其二倍体的最大净光合速率与实测值最接近,说明其在拟合最大净光合速率上有优势;整体上分段函数计算得到的黑皮冬瓜的各光合参数与实测值最为接近,与实测值的平均相对误差最小,也能很好的拟合发生光抑制部分的光响应曲线。分段函数拟合同源四倍体及其原二倍体黑皮冬瓜光合光响应曲线效果较其他模型效果好,分段函数模型为黑皮冬瓜最适合的光合光响应模型。