A functionalized Zr-based MOF, employing UiO-66 as the framework, polydiallyldimethylammonium (PDDA) as the interfacial modifier, and polydopamine (PDA) as the functionalizing agent, has been prepared by a simple one-pot hydrothermal reaction and characterized by TEM and XPS. The results indicate that UiO-66-PDDA-PDA exhibits excellent PDA dispersion and an abundance of functionalized hydroxyl and amino groups. Employing this novel functionalized MOF material, a reliable electrochemical sensor based on a UiO-66-PDDA-PDA-modified GCE was developed for the simultaneous determination of four Cd2+, Cu2+, Pb2+, and Hg2+. The prepared electrochemical sensor demonstrated effective selective detection of microgram levels of Cd2+, Cu2+, Pb2+, and Hg2+ in tea, water, and dairy products using cyclic voltammetry. This sensor exhibited exceptionally low detection limits and RSDs for these four heavy metal ions. This method could provide a quick screening method for inspectors to check the conformity of samples when goods in these categories enter or leave the country.

Tomato is one of the most popular horticultural crops, and many commercial tomato cultivars are particularly susceptible to Botrytis cinerea. Hydrogen sulfide (H2S) is an important gaseous molecule in various plant stress responses. In this study, it was found that endogenous H2S increases in tomato leaves in response to B. cinerea infection, along with a 3.8-fold increase in gene expression of DCD1 which encodes a H2S-generating enzyme D-cysteine desulfhydrase 1 in tomato at 3 DPI. Then we investigated the role of DCD1 in resistance of tomato leaves and fruits to B. cinerea. The mutation of DCD1 by CRIPSR/Cas9 greatly reduced the resistance of tomato leaves and breaker and red fruits to B. cinerea accompanied with increased reactive oxygen species (ROS) especially hydrogen peroxide (H2O2) and malondialdehyde (MDA) content increased by 1.2 and 1.4 times respectively at 5 DPI of leaves. Further investigation showed that DCD1 mutation caused decreased activity of antioxidative enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) in both leaves and fruits, in particular, CAT activity in dcd1 mutant was 25.0 % and 41.7 % of that in WT at leaves and red fruits at 5 DPI. DCD1 mutation also caused decreased expression of defense-related genes PAL (encoding phenylalanine ammonia-lyase) and PUB24, and their expression in the dcd1 red fruit is approximately 1.3 and 1.8 times higher than in wild-type red fruit at 5 DPI, respectively. Thus, the work emphasizes the positive role of DCD1 and H2S in plant responses to necrotrophic fungal pathogens. In addition, the work provides strong evidence that fruit at ripened stage is more susceptible to B. cinerea infection compared with green fruit, suggesting that senescence of plant tissues is more favorable to B. cinerea infection.

To clone and verify the cinnamate 4-hydroxylase（C4H） gene in tea plant and predict the biological function of C4H gene. Extracting total RNA from fresh tea leaves, and the C4H gene of tea was cloned by RT-PCR. The conserved domain, physical and chemical properties, subcellular localization, transmembrane domain and signal peptide, homology and phylogeny of the protein encoded by C4H gene were analyzed by bioinformatics software. The results showed that the C4H gene of tea contained a complete open reading frame（ORF） of 1518 bp, encoding 505 amino acids; conserved domain analysis showed that the protein encoded by tea C4H gene contained a domain of P450 superfamily protein（PLN02394）, with a molecular weight of 58.16 kDa and a theoretical isoelectric point of 9.29. It was a hydrophilic and unstable protein, localized in the endoplasmic reticulum, without signal peptide and transmembrane domain. The secondary structure of the protein was mainly a-helix and random coil. The C4H gene had the highest homology with oil tea at the amino acid level（92%）. In this study, the C4H gene of tea was successfully cloned and its biological function was analyzed, which laid a preliminary theoretical foundation for further study on the function of C4H gene and the molecular mechanism of tea synthesis.

对甘薯、番茄、拟南芥中63个SPL基因家族进行了系统进化树分析、保守蛋白基序(Motif)分析,筛选归纳出同源性较高的2个分支的12个SPL基因进行理化性质分析、核定位预测等,氨基酸序列比对结果表明这些基因的功能可能较为保守.通过对番茄中的SPL基因Solyc05g015510.2、Solyc10g078700.1进行表达量分析,发现这2个基因可能参与调控果实成熟衰老进程.另外,通过对非生物胁迫下的转录水平进行分析得知,拟南芥中的AT5G43270可能参与对盐胁迫、热胁迫条件下的响应,AT1G27360、AT1G27370可能参与热胁迫条件下的响应,AT2G42200可能参与冷胁迫条件下的响应,而AT3 G57920在非生物胁迫条件下表达量没有特别明显的变化,表明AT3 G57920可能不参与非生物胁迫下的响应.

真菌病害是制约水稻高产稳产的重要障碍因素之一.为了研究水稻种子中真菌发生情况,对分离自水稻种子中的一株真菌进行系统发育分析及功能评价.在形态学鉴定基础上,以通用引物ITS1/ITS4对目标真菌序列进行PCR扩增、序列对比分析、邻接法(NJ)构建系统发育树.通过菌落培养特征、孢子形态特征观察,分离菌株S-51的形态特征与文献中雪霉叶枯病菌(Microdochium nivale)的描述极为相似;序列比对结果表明,目标真菌与雪霉叶枯病菌(M.nivale)相近,相似率为100％;系统发育树显示,其与雪霉叶枯病菌株在同一条分支上,雪霉叶枯病菌M.nivale和M.musae的亲缘关系最近.利用ITS片段开展的序列分析和系统进化树构建,结合形态特征,能够确定分离自水稻种子中的S-51菌株为雪霉叶枯病菌(M.nivale).

对26份水稻材料进行抗病基因检测,包括8个白叶枯病抗性基因(Xa1、xa5、Xa7、xa13、Xa21、Xa23、Xa26、Xa27)和2个稻飞虱病抗性基因(Bph14、Bph15).其中23份水稻材料中含有Xa1基因材料20份,含xa5基因材料20份,含Xa7基因材料23份,含xa13基因材料12份,含Xa21基因材料9份,含Xa23基因材料23份,其中抗性4份,感性19份,含Xa26基因材料23份,含Xa27基因材料21份,其中抗性5份,感性16份.另有3份材料进行稻飞虱病抗性基因Bph14、Bph15检测,检测结果表明这3份材料均含有Bph14、Bph15基因.选取13个已报道的基因标记或连锁标记检测26份杂交稻材料,初步确定了其抗白叶枯病和抗稻飞虱病等位基因分布和利用情况,为水稻基础材料的筛选、亲本材料的选择及育种应用提供了良好的参考.

本文基于matlab语言和线性规划模型,建立了一种新的杂交水稻种子纯度预测的数学模型,并利用matlab GUI工具设计了杂交水稻种子纯度预测软件界面程序.在已知a/A拷贝数比的情况下,可以直接由软件计算出待测水稻种子纯度范围,适用于杂交水稻种子纯度快速初筛.

本试验从一批药品中分离到11株疑似芽孢杆菌菌株,采用VITEK 2 systems compact生化鉴定系统和16S rDNA基因测序技术对其进行鉴定.结果 显示,除2株株菌(6号菌株鉴定为Microbacterium oleivorans,8号菌株鉴定为Moraxella osloensis),其他9株株菌均属于芽孢杆菌属(Bacillus).但仅通过VITEK 2 systems compact鉴定和16S rDNA测序比对,均不能鉴定到种.因此,进一步选择gyrB基因以及其blast分析结果构建系统发育树,最终将1～5号菌株分别鉴定为B.oceanisediminis(99.08％),B.korlensis(90.71％),B.licheniformis(100％),B.marisflavi(97.26％),B.oceanisediminis(99.22％),9～12号菌株分别鉴定为B.megaterium(99.83％),B.boroniphilus(81.20％),B.jeotgali(90.70％),B.humi(81.39％).实验表明,利用16S rDNA与gyrB基因结合的方式能较好地将芽孢杆菌属鉴定到种,是一种快速准确的芽孢杆菌种间鉴定方法,适合于药品中分离菌株的种属及近缘种的鉴定与区分.

To improve the quantitative detection efficiency of transgenic rice,and promote the implementation of transgenic food quantitative identification system,the components of transgenic rice ‘ Bt shanyou 63 ’ were analyzed with droplet digital PCR quantitative detection method.Based on the exogenous inserting sequence of transgenic rice ‘Bt shanyou 63’ and the rice sucrose phosphate synthase (SPS) gene,high-efficiency primer probes were selected,designed and synthesized to amplify the endogenous gene and the strain specific sequence of ‘Bt shanyou 63’ with droplet digital PCR.The result of primer probes optimizing experiment demonstrated that SPS60-F/R/P and ‘Bt shanyou 63’-F/R/P was suitable for quantitatively detecting the components of ‘Bt shanyou 63’ with dual channel ddPCR method.The accuracy verification experiment showed that the measured value was similar to the standard value,and the biases were 0.03,0.15,0.15 and 0.03,respectively.SD of measured value was 0.08-0.48,RSD was 4.86％-15.10％,and they were less than the requirement of European Union,indicating that the accuracy was good.The established detection system of droplet ddPCR method for transgenic rice line ‘Bt 63’ quantification is simple,high efficient and accurate,and it can be used for quantitative analysis of transgenic rice line ‘ Bt 63’ in agricultural products and foods.

本实验用SSR分子标记技术鉴定两系杂交水稻“C两优513”和“深两优5814”的种子纯度.本研究从48对SSR引物中筛选到3组引物(RM277、RM17、RM7389),选取“C两优513”和“深两优5814”种子各5组,每组样品随机取192粒进行纯度检测.针对同一份样品,SSR标记与田间鉴定结果均接近,相差最大一组差异为3.3％,最小差异0.04％.此外,不同引物对同一样品的鉴定结果通常存在差异.结果表明,SSR标记技术适用于“C两优513”和“深两优5814”的种子纯度鉴定.为控制检测误差,宜用多对引物同时进行实验.

基于水稻细菌性条斑病菌的假定膜蛋白基因和水稻白叶枯病菌的rhs家族基因分别设计引物和探针,建立了这两种病菌的数字PCR检测技术.特异性测试结果显示,两种检测方法均可特异性检测到目标病菌的供试菌株,而其他对照菌和空白对照均为阴性.两种检测方法对目标菌的检测下限分别达到了9个和16个拷贝/反应,且成功检测到人工模拟带菌及自然带菌种子样品中的病菌.

[Objective] To establish and optimize the amplification system of methylation sensitive polymorphism ( MSAP) to study the methyl-ation level of Radix astragali.[Method] DNA of Radix astragali was extracted by magnetic bead method and digested with HpaII/MspI endo-nuclease.The digested products were connected with the linker sequence, and then pre-amplification and selectively amplification were carried out.Four pairs of primers were screened out from the 256 pairs of primers of MSAP, and were used to analyze the methylation levels for 12 dif-ferent habitat samples of R.astragali.[Result] For 12 samples, the rate of half methylation and full methylation were 12.5%-26.7% and 25.6%-51.1%, respectively.The total methylation rate was 48.7%-73.5%.The UPGMA clustering and principal component analyses (PCA) were obtained by analyzing the HpaII and MspI polymorphism data with NTSYSpc software.[Conclusion] MSAP technology can be used for the methylation analysis of R.astragali.The preliminary analysis of cluster analysis showed some regional characteristics of R.astra-gali from the UPGMA and PCA plots.

An amperometric glucose oxidase (GOx) biosensor based on gold nanoparticles (AuNPs) -reduced graphene oxide (rGO) was fabricated for the detection of glucose in beverages.AuNPs-rGO composite was synthesized by one-pot method with chitosan as reducing and stabilizing agent.It was used as the matrix for GOx immobilization via physical adsorption technique to fabricate the GOx-based biosensor.The biosensor was applied in the amperometric determination of glucose at-0.45 V(vs.Ag/AgCl) in a phosphate buffer (0.1 mol/L,pH 6.0).There was a linear response to glucose in the concentration range of 0.01-0.88 mmol/L,with a sensitivity of 22.54 μA · mmo1-1.L · cm-2 and a detection limit of 1.01 μmol/L.The apparent Michaelis-Menten constant was rather small(0.497 mmol/L).The constructed biosensor was successfully applied in the detection of glucose in beverages.

Callosobruchus maculatus are frequently intercepted in quarantine at port.In this paper,516 bp base sequence of COI gene of C.maculatus and allied species was detected and analyzed.The results showed that there are 353 conseved sites,163 mutation sites,134 parsimony information sites and 29 singletons sites in these genes.Model Kimura 2-parameter was used to analyse the genetic distance,and the results revealed that the genetic distance among the same species varied from 0.001 to 0.013 with the average value of 0.008,while that among different species ranged from 0.114 to 0.193 with the average value of 0.161.Neighbor-joining phylogenetic tree was built based on the COI gene sequences,and the result showed that the same species was gathered in the same branch,with the bootstrap value of 100％.The results indicated that the molecular identification of C.maculatus and its allied species is practicable based on COI gene sequence.

采用基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,简称MALDI-TOF-MS)技术对水稻作物的3种主要病原真菌进行分析,以获得稳定的指纹图谱.从菌物预处理方法、基质、点样方法等3个方面进行比较,对影响MALDI-TOF-MS分析结果的主要因素进行优化.结果表明,菌物预处理方法对检测结果的影响最大,热处理法在细胞壁较厚的真菌样品处理中可以获得较完整的生物信息;构建稻粒黑粉病病菌(Tilletia horrida)、稻曲病病菌(Ustilaginoidea virens)、恶苗病病菌(Fusarium moniliforme)的MALDI-TOF-MS鉴定规范化方法,扩充MALDI-TOF-MS指纹图谱数据库,可简便、快速、准确地对细胞壁加厚的真菌样品进行鉴定.

A method of real-time PCR is established for detecting Papaver somniferum ingredient in food and condiment.The TaqMan specific primers and probes are designed for Papaver somniferum berberine bridge enzyme bbe1 .The pre-processing and DNA extraction method is set up for detecting food and condiment samples.The limit of detection (LOD)of Papaversomniferum ingredient in food and condiment for this method is 0.01%.The absolute sensitivity of Papaver somniferum genomic DNA detection is 10 pg.Compared with the conventional PCR and SYBR Green fluorescent dye PCR such as SYBR Green,TaqMan probe method has better sensitivity and specificity,visible results and without pollution.This method could be applied for detecting Papaver somniferum in food and condiment.Meanwhile,it can provide references for other Papaver somniferum detection methods.

目的:建立Illumina Miseq深度测序筛查酸奶中微生物的方法,分析酸奶中微生物的多样性.方法:以9种酸奶样品为研究对象,提取酸奶中细菌基因组DNA,应用高通量测序技术测定细菌的16S rDNA v1-v3变异区序列,得到9种样品中微生物群体分布和丰度、以及不同样品间的菌种差异与进化关系.结果:酸奶中微生物主要为厚壁菌门(Firmicutes),占据99.6％之高,其中厚壁菌门主要以链球菌属(Streptococcus、87.1％)、乳杆菌属(Lactobacillus、10.3％)、乳球菌属(Lactococcus、0.3％)组成,9种酸奶中有3种同时含有链球菌属和乳杆菌属,其余6种样品中链球菌属几乎占据全部(＞97％).结论:Illumina Miseq深度测序技术可快速精确深入掌握酸奶中微生物多样性,结果对比显示不同酸奶菌种同质化程度高,其中链球菌属占绝对优势,同时发现部分酸奶标签标示不符.

Objective To investigate organochlorine pesticides residue in Radix Astragali collected from different medical institutions. Methods We collected samples and information of sources from different medical institutions.According to the "Chinese pharmaco-poeia"we used gas chromatography method.Samples were extracted with mixed solvents by ultrasonic,then purified with sulfuric acid. Splitless injection was adopted before the detection of ECD detector and determination of contents by external standard method.Results Organochlorine pesticides residue in Radix Astragali produced in Gansu and Inner Mongolia were much lower than the standard of Chinese pharmacopeia,but there were some differences among samples from different sources.Conclusions This method is quick and accurate,which can provide a guarantee for the safe use of drug.

[目的]对分离自不同产地和品种的蜂蜜中的芽孢杆菌属进行鉴定分型，探索利用蜂蜜中芽孢杆菌特征指纹图谱对蜂蜜产地和品种进行溯源的可行性。[方法] 从44种不同产地和品种蜂蜜中分离出44株芽孢杆菌，3株铜绿假单胞菌。采用基质辅助激光解析-电离飞行时间质谱（MALDITOFMS）条件对分离的芽孢杆菌进行鉴定分型和聚类分析。[结果] 44株芽孢杆菌中鉴定出蜡样芽孢杆菌（B.cereus）31株，短小芽孢杆菌（B.pumilus）5株，枯草芽孢杆菌（B.subtilis）3株，地衣芽孢杆菌（B.licheniformis）2株，炭疽芽孢杆菌（B.anthracis）1株，土壤短芽孢杆菌（Brevibacillus agri.）2株。通过多次重复试验，表明从同一份样品或同一品牌蜂蜜样品中能稳定分离到芽孢杆菌，获得的蛋白指纹图谱具有极好的稳定性。[结论]MALDITOFMS可根据获得的芽孢杆菌蛋白指纹图谱将芽孢杆菌划分为不同类型，可作为一种新的根据蜂蜜中芽孢杆菌的特征指纹图谱进行溯源的方法。

[目的]研究蜂蜜中蛋白质提取的有效方法,优化蜂蜜蛋白电泳条件.[方法]该试验使用丙酮、钨酸钠、硫酸铵、尿素-硫脲4种方法提取蜂蜜中的蛋白并进行比较,然后进行双向电泳分析,对影响双向蛋白电泳的IPG胶条的选择、蛋白质上样量、等电聚焦时间等主要因素进行优化.[结果]试验表明,采用硫脲法提取蜂蜜蛋白获得的蛋白含量最高,硫脲法和丙酮沉淀法获得的蛋白纯度最好,而硫脲法因干扰物质较少、易于裂解适于双向电泳技术,采用硫脲法提取的蜂蜜蛋白选择IPG胶条pH 5～8,蛋白上样量为150μg,等电聚焦时间达到32000 V·h的情况下能充分地聚焦,获得的双向电泳图谱最佳,优化后的洋槐蜜双向电泳图谱上可检测到326个蛋白点,重复性和分辨率均较好.[结论]蜂蜜类含糖样品中使用硫脲法提取蛋白能减少糖类物质干扰.