番茄乙烯信号转导相关基因EIN3（Le-EIL）的克隆和表达

摘要：根据拟南芥?穴Arabidopsis thaliana ?雪、烟草?穴Nicotiana tabacum ?雪EIN3基因保守区序列设计简并引物，采用RT-PCR扩增从番茄(Lycopersicon esculentum )果实中得到一个486 bp的同源片段。以此片段作为探针，从番茄果实cDNA文库中分离到一个拟南芥EIN3的同源基因的cDNA克隆,命名为Le-EIL。该克隆含有一个1 845 bp的开放阅读框，编码615个氨基酸。经同源分析，它与烟草TEIL基因的同源性为79%，与拟南芥EIN3、EIL1、EIL2和EIL3的同源性分别为59%、56%、52%、46%。Le-EIL基因表达与番茄果实的成熟及乙烯生成情况具有较强的相关性。在普通番茄果实的不同成熟时期Le-EIL基因都有表达，并且随着成熟度的增加基因表达有明显的增强，在转色期和粉红期Le-EIL基因的表达最强, 在全红期后减弱。在转反义ACS基因的转基因番茄果实中，只有开花后60 d的果实中Le-EIL基因有微弱的表达，在其它成熟时期均没有表达。     

黄瓜、西瓜、南瓜EIN3基因片段的克隆与序列分析

EIN3（Ethylene insensitive 3）是位于细胞核的核蛋白为乙烯信号转导的下游调控基因,根据GenBank所报道的植物EIN3基因mRNA的保守序列设计两个引物,以黄瓜、西瓜、南瓜总DNA为模板PCR扩增到3个725bp的基因片段，将片段序列在NCBI数据库中Blastn同源搜寻，显示151条有同源性的序列全部是EIN3基因，因此推断克隆的3个片段均为EIN3基因家族成员。NCBI网站的ORF Finder找到正确的开放式阅读框，翻译成为氨基酸序列，对CsEIN3、ClEIN3及CmEIN3氨基酸序列在NCBI网站进行Blastp比对, 在大分子结构数据库MMDB (Molecular Modelling Database) 搜索,3个推测蛋白保守结构域三级结构与拟南芥EIN3的DNA结合域(MMDB: 30598 PDB: 1WIJ )完全相同，系统进化分析表明，黄瓜、西瓜、南瓜与双子叶植物甜瓜、绿豆、蒺藜状苜蓿、烟草、番茄、拟南芥及单子叶水稻、桃红蝴蝶兰等的EIN3家族成员都有较高的相似性。     

番茄ACO基因的克隆及其RNAi对乙烯产量的抑制

筛选番茄幼苗cDNA文库获得与番茄抗性相关的克隆E11，设计引物，利用RT-PCR方法从受到病原浸染的番茄叶片中获得长1018 bp的候选片段，同源性分析发现该片段与其他作物上已发表的ACO基因序列高度同源：同源率从83%~99%，推断该基因为番茄ACO基因家族的新成员。在此基础上，用BP克隆的方法构建该基因的RNA干涉（RNAi）载体pD311，对番茄进行遗传转化，获得卡那霉素抗性植株27棵，分子检测证实外源片段成功导入其中番茄基因组中。对获得的转基因植株的乙烯生成量测定结果表明，RNAi结构的导入大大抑制了内源ACO基因的表达，从而导致乙烯的生成大大降低。     

黄瓜、西瓜和南瓜EIN3基因片段的克隆与序列分析

EIN3(ethylene insensitive 3)位于细胞核的核蛋白,为乙烯信号转导的下游调控基因,根据GenBank植物EIN3基因家族的保守序列设计了1对引物,以黄瓜(Cucumis sativus)、西瓜(Citrullus lanatus)和南瓜(Cucurbita maxima)总DNA为模板PCR扩增到3个725bp的基因片段CsEIN3、ClEIN3和CmEIN3,并提交GenBank,登录号分别为AY973275、DQ023225和DQ023224。将片段序列在NCBI数据库中Blastn同源搜寻,显示151条有同源性的序列全部是EIN3基因。NCBI网站的ORF(open reading frame)Finder找到正确的开放式阅读框,翻译成为氨基酸序列,对CsEIN3、ClEIN3及CmEIN3氨基酸序列在NCBI网站进行Blastp比对,在大分子结构数据库(molecular modelling database,MMDB)搜索,3个推测蛋白保守结构域三级结构与拟南芥EIN3的DNA结合域(MMDB:30598;PDB:1WIJ)完全相同,系统进化分析表明,黄瓜、西瓜和南瓜与双子叶植物甜瓜、绿豆、蒺藜状苜蓿、烟草、番茄、拟南芥及单子叶水稻、桃红蝴蝶兰等的EIN3家族成员都有较高的相似性。     

猪肥胖基因片断的克隆及不同日龄猪肥胖基因表达的差异

从杜长嘉(Duroex Landracex Taihu)猪的皮下脂肪中提取基因组RNA，用RT-PCR扩增肥胖基因(obesegene；ob)，获得1条504bp的片断，以pGEM-T Easy Vector为载体，将该基因片断克隆到大肠杆菌(Eschenchia coli)中。从筛选到的阳性克隆中分离出肥胖基因，测定其序列，分析表明该片段为肥胖基因cDNA的部分序列，编码167个氨基酸组成的多肽，是成熟肽的主体部分。研究得到的基因片段与报道的猪脂肪组织中ob cDNA部分序列同源性达到99．41％。氨基酸序列同源性达到98．94％。以ob基因片段的克隆为基础，构建了优化的半定量RT-PCR法，以β-actin为内标，研究不同日龄的猪脂肪组织ob基因表达的差异，1-28日龄ob基因表达呈上升趋势，28日龄达到最高，随后，到56日龄ob基因表达又呈下降趋势。     

番茄ACO基因的克隆及其RNAi对乙烯释放的抑制

筛选番茄(Lycopersicon esculentum)幼苗cDNA文库获得与番茄抗性相关的克隆E11,设计引物,利用RT-PCR方法从受到病原侵染的番茄叶片中获得长1018bp的候选片段,同源性分析发现该片段与其它作物上发表的ACO基因序列高度同源,同源率83%～99%,推断该基因为番茄ACO基因家族的新成员。在此基础上,用BP克隆的方法构建该基因的RNA干涉(RNAi)载体pD311,对番茄进行遗传转化,获得卡那霉素抗性植株27棵,分子检测证实外源片段成功导入番茄基因组中。对获得的转基因植株的乙烯生成量测定结果表明,RNAi结构的导入大大抑制了内源ACO基因的表达,从而导致乙烯的生成大大降低。     

甜橙β-胡萝卜素羟化酶cDNA克隆及其瞬间反义表达*

根据已报道的温州蜜柑(Citrus unshiu)β-胡萝卜素羟化酶(BCH)cDNA序列(AF296158)，设计1对引物，以华盛顿脐橙(C.sinensis)成熟果实cDNA为模板，采用PCR扩增出1条长为1057bp的cDNA片段，将此片段克隆入pUCm—T载体，测序结果表明，所得序列与已报道序列同源99．62％，表明所获得的基因是BCH基因。将该基因反义插入到pBI121中构建成反义植物表达载体，其正确性得到测序的鉴定。含有该植物表达载体的根癌农杆菌(Agrobacterium tumefaciens)在脐橙白皮层中诱导了β-胡萝卜素积累。     

猪SOCS3基因克隆及其在不同生长阶段表达差异研究

从杜长大母猪的肠系膜脂肪中提取基因组RNA，用RT-PCR扩增SOCS3基因，获得1条约332 bp的片段，以pGEM-T Easy vector 为载体，将该基因片段克隆到大肠杆菌(Escherichia coli)DH5α中。从筛选的阳性克隆中分离出SOCS3基因，测定其序列，分析表明，该片段为 SOCS3 cDNA的部分序列，与GenBank中登录的猪SOCS3 cDNA部分核苷酸序列同源性达到100%。以SOCS3基因片段的克隆为基础，构建了优化的半定量RT-PCR法，以18s rRNA为内标，研究不同生长阶段猪肠系膜脂肪中SOCS3基因表达的差异。结果发现，从初生到90 kg，SOCS3基因在肠系膜脂肪中表达量呈逐渐增加的趋势，其中90 kg较初生时增加了141％（p<0.05）。     

乙烯受体基因LeFTR1和LeFTR4的克隆及在番茄果实中的表达

为研究野生型番茄(Lycopersicon esculentum Mill．cv．Lichun)和转反义ACS番茄果实中乙烯受体基因上eETR1和LeETR4的表达与乙烯的关系，实验用RT-PCR方法扩增了乙烯受体基因LeETR1和LeETR4片段，用两片段作探针进行Northern杂交。结果表明：两受体基因的表达在番茄果实成熟进程中变化不明显，LeETR4在同一时期的果实外果皮中表达水平低于其在辐射壁和中柱的表达。转反义ACS番茄果实中LeETR1和LeETR4的表达水平显著低于野生型番茄果实，外源乙烯处理转反义ACS番茄果实，促进两个受体基因的表达。可见果实中LeETR1和LeETR4的表达受乙烯调控的影响。     

乙烯受体基因LeETR1和LeETR4的克隆及在番茄果实中的表达

摘要：为研究野生型番茄（Lycopersicon esculentum Mill. cv . Lichun）和转反义ACS 番茄果实中乙烯受体基因LeETR1和LeETR4的表达与乙烯的关系，实验用RT-PCR方法扩增了乙烯受体基因LeETR1和LeETR4片段，用两片段作探针进行Northern 杂交。结果表明：两受体基因的表达在番茄果实成熟进程中变化不明显，LeETR4在同一时期的果实外果皮中表达水平低于其在辐射壁和中柱的表达。转反义ACS番茄果实中LeETR1和LeETR4的表达水平显著低于野生型番茄果实，外源乙烯处理转反义ACS番茄果实，促进两个受体基因的表达。可见果实中LeETR1和LeETR4的表达受乙烯调控的影响。     

Enzymatic synthesis of [3'-O-methyl-(3)H]malvidin-3-glucoside from petunidin-3-glucoside

Malvidin-3-glucoside has been labeled by enzymatic synthesis in a single-step experiment. Catechol-O-methyl transferase catalyzed the B-ring methylation of petunidin-3-glucoside, and S-Adenosyl-L-[methyl-(3)H] methionine was the methyl donor. Solid phase extraction and preparative high-performance liquid chromatography were necessary to separate [3'-O-methyl-(3)H]malvidin-3-glucoside from an isomer and the starting material. The specific activity was 2.2 Ci mmol(-1), and the yield of incorporation was 1.1%. A possible application of the labeled material is the study of anthocyanin reactions in complex mixtures such as red wine where products are difficult to isolate and analyze.     

Stereoselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol by lipase-catalyzed hydrolysis of 3-acetylthioesters

The enantioselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol, two potent sulfur-containing aroma compounds, by lipase-catalyzed hydrolysis of the corresponding 3-acetylthioesters was investigated. The stereochemical course of the kinetic resolutions was followed by capillary gas chromatography using modified cyclodextrins as chiral stationary phases. The enzyme preparations tested varied significantly in terms of activity and enantioselectivity (E). The highest E value (E = 36) was observed for the hydrolysis of 3-acetylthiohexanal catalyzed by lipase B from Candida antarctica resulting in (S)-configured thiol products. Immobilization of the enzyme (E = 85) and the use of tert-butyl alcohol as cosolvent (E = 49) improved the enantioselectivity. Modification of the acyl moiety of the substrate (3-benzoylthiohexanal) had no significant impact. The sulfur-containing compounds investigated possess attractive odor properties, and only one of the enantiomers exhibits the pleasant citrus type note.     

3YDX-3型烟草打顶抑芽机设计

为改变中国烟草手工打顶、打顶抑芽分步操作的落后局面,开发了3YDX-3型烟草打顶抑芽机。它主要有吹风系统、烟草高度识别系统、打顶系统、抑芽剂喷施系统、刀片消毒装置等组成,能够同时完成烟草打顶、抑芽剂喷施、刀片消毒3项作业。整体机架由液压油缸控制升降,滑动支架与整体机架滑动连接,使机组满足不同生长高度、不同种植行距的烟草使用。烟草高度识别系统、抑芽剂喷施系统采用机电一体化技术,实现了烟草高度的精确识别、准确打顶和对靶施药。试验结果表明,生产率为0.48 hm2/h时,该烟草打顶抑芽机工作安全稳定,打顶高度仿形,抑芽剂喷施准确,能够满足使用要求。     

The flavonol quercetin-3-glucoside inhibits cyanidin-3-glucoside absorption in vitro

At present, little is known about the mechanisms responsible for intestinal absorption of anthocyanins (ACNs). For example, it has not yet been established if ACNs are absorbed through an active transport mechanism, such as the sodium-dependent glucose transporter (SGLT1), or by passive diffusion. Previously, we found that the absorption of ACNs differs between regions of the digestive tract and is maximal in the jejunum, suggesting that an active transport mechanism is involved. In the present study, we examined the effect of d-glucose (main substrate of SGLT1), phloridzin (inhibitor of SGLT1), and quercetin-3-glucose (Q3G, a flavonol) on the absorption of cyanidin-3-glucoside (C3G; approximately 5 micromol/L) by mouse jejunum mounted in Ussing chambers. We found that the presence of either D-glucose (10, 20, and 40 mmol/L) or phloridzin (50, 100, and 200 micromol/L) resulted in a small but insignificant inhibition of C3G disappearance from the mucosal solution (decrease of disappearance with glucose, 33%; with phloridzin, 18%; NS). However, when the flavonol Q3G (50 micromol/L) was added to the mucosal solution together with the C3G, the disappearance of C3G was significantly decreased (74%; p < 0.001), and Q3G disappeared instead. In addition, we found phloretin and quercetin, the aglycones of phloridzin and Q3G, respectively, present in the mucosal solution and tissue extracts, indicating hydrolysis of these compounds by the enterocytes of the jejunum. In contrast, the aglycone cyanidin was not detected at all. Our results show that in the mouse small intestine, ACN absorption is not solely dependent on the activity of the SGLT1 transporter, as d-glucose and phloridzin had only a slight effect on uptake. Q3G, however, clearly inhibited C3G disappearance. These results suggest that there might be a competitive inhibition between C3G and Q3G absorption. It is possible that an absorption mechanism other than the SGLT1 is involved, which has a structural preference toward flavonols.     

改进人工神经网络降低NO3-选择电极的Cl-干扰

Fast detection of soil nitrate has an important significance for variable rate fertilization.As NO-     

Intracellular mediators of procyanidin-induced lipolysis in 3T3-L1 adipocytes

We have previously reported that grape seed procyanidins stimulate long-term lipolysis on 3T3-L1 fully differentiated adipocytes. To unravel the molecular mechanism by which procyanidins exert this effect, we checked the involvement of two main cellular targets in adipose cells: protein kinase A (PKA) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Procyanidin treatment increased intracellular cAMP levels in 3T3-L1 adipocytes, and their lipolytic effect was inhibited by simultaneous treatment with H89, a PKA specific inhibitor. BRL49653, a very highly specific ligand of PPAR-gamma, totally abolished the lipolytic effect of procyanidins. Simultaneous to this long-term lipolytic effect, the mRNA levels of some differentiation adipocyte markers decreased, although there were no changes in the triglyceride content of the cells. BRL49653 did not antagonize the decrements of differentiation markers. These results support a mediation of PPAR-gamma and PKA on the lipolytic effects of procyanidins on 3T3-L1 adipocytes.     

基于分形插值的三维路面重构与分析

原始精细的路面谱是研究车辆与路面耦合的重要基础,对车辆的通过性和噪声、振动及不平顺性NVH(noise,vibration and harshness)分析有重要的应用价值。为了构建与原始路面相同或相近的路面谱,利用非接触式激光路面不平度仪测量沥青路面、水泥路面、比利时路面和砂石路面的三维路面不平度;基于分形理论,采用迭代函数法重构这四种路面谱;结合路面不平度的统计特性评价指标和分形维数对四种路面的原始谱和重构谱进行评价。研究结果表明:重构前、后路面谱的平均值、标准差、峰度系数以及分形维数变化范围在±5%以内,除水泥路面的偏态系数变化范围均在±9%以内,重构路面谱与原始谱具有一致性;重构路面谱保持了原始路面的结构特性,并具有路面的细微结构。     

Two inhibition targets by [Cr(2gb)(3)](3+) and [Co(2gb)(3)](3+) on redox enzymes of spinach thylakoids.

[Cr(2gb)(3)]Cl(ZnCl(4)), [Cr(2gb)(3)]Cl(3), and [Co(2gb)(3)]Cl(3) were synthesized and characterized. Their chemical structures and the oxidation states of their metal centers remained unchanged in solution. The effects of these compounds, CrCl(3) and [Co(NH(3))(6)]Cl(3), on photosynthesis were investigated. The coordination compounds inhibit ATP synthesis and electron flow (basal, phosphorylating, and uncoupled), behaving as Hill reaction inhibitors. The target for [Cr(2gb)(3)]Cl(ZnCl(4)) is located at the Q(B) level. In contrast, the interaction sites of [Cr(2gb)(3)]Cl(3) and [Co(2gb)(3)]Cl(3) are located in the span from P(680) to Q(A) and at the b(6)f complex. Neither CrCl(3) nor [Co(NH(3))(6)]Cl(3) inhibited photosynthesis. The 100% inhibition on PS II of [Cr(2gb)(3)]Cl(ZnCl(4)) is explained in terms of a synergystic effect between the 2gb-chromium(III) coordination compound and the ZnCl(4)(2)(-) anion.     

巧用AutoCAD软件的“三维旋转”命令绘制实体课件

巧妙地应用AutoCAD软件的＂三维旋转＂命令,可以方便快捷地绘制带有倾斜结构及局部剖结构的实体课件,并用两个实例说明了具体的应用方法。     

基于Kinect V3的单株作物自动化三维重建与验证

为高效、精确地对单株作物进行三维重建,以点云方式无损测量作物表型信息,该研究提出一种基于Kinect V3深度相机的三维重建系统。使用步进电机搭建了一个旋转台,并将旋转台面设计成多颜色同心圆,利用同心圆计算平面法向量及圆心两特征信息,用于点云水平校准以及获取点云间的旋转平移矩阵;将Kinect V3采集的多视角点云变换到同一坐标系下,并结合裁剪迭代最近点（Trimmed Iterative Closest Point,TrICP）算法实现了多视角点云的粗配准与精配准,完成了作物三维重建。为检验该研究的三维重建效果,选取菜心、黄瓜苗为试验对象,与多视图立体视觉-运动恢复结构（Multi-View Stereo and Structure From Motion,MVS-SFM）算法重建点云进行对比,并提取叶面积参数与人工测量值进行比较。结果表明,两种方法下重建后的菜心点云间平均距离误差为0.59 cm,黄瓜苗点云间平均距离误差为0.67 cm,具有较高的相似度,而相较于MVS-SFM算法,该研究提出的方法的重建速度提高了约90%;该研究提出的方法所重建点云,菜心叶面积提取与标准参考值相对误差均值为5.88%,均方根误差为3.83 cm2,黄瓜苗叶面积提取与标准参考值相对误差均值别为6.50%,均方根误差为2.08 cm2,都显现出较高的准确性。该研究提出的方法能对单株作物进行快速三维重建,能有效提取叶面积参数,可为作物育种、栽培和农业生产提供高效技术手段和数据支持。