共查询到19条相似文献，搜索用时 93 毫秒
1.
黑籽南瓜(Cucurb ita ficifolia)具有很强的抗逆能力,对低温、干旱、贫瘠土壤以及瓜类枯萎病等逆境均具有较强的抗性,这些优良的抗性基因很难通过有性杂交转移到其它瓜类作物上,但可通过可转化人工染色体(TAC)以大片段的形式转化到其它瓜类作物中.本研究利用TAC载体构建了黑籽南瓜基因组DNA文库,该文库包含57 560个克隆,保存在150块384孔板中,其库容量相当于黑籽南瓜基因组的7倍.TAC文库克隆插入片段大小分布范围为20～100 kb,超过40 kb插入片段克隆占40％以上,插入片段平均大小为45kb.在挑取的15个TAC克隆中无空克隆;细菌继代培养30、60和100代后质粒的酶切鉴定表明,TAC克隆可以在大肠杆菌(Escherichia coli) DH 10B中稳定复制和保存,说明本研究构建的文库质量较高.该研究结果表明,TAC文库为黑籽南瓜抗性基因的克隆、功能验证、物理图谱的构建和基因组测序等有关研究提供了基础资料. 相似文献
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通过基因工程手段加强八氢番茄红素合成酶（Psy）基因的表达和通过RNAi抑制番茄红素β-环化酶（Lcy）基因（该基因是调控番茄红素转化为其他类胡萝卜素的主要的基因）的表达是培育高番茄红素品种的重要手段之一。根据GenBank中拟南芥的Psy基因序列（NM-121729），通过聚合酶链式反应（PCR）从拟南芥的cDNA中扩增出了长1296的Psy基因。根据GenBank中番茄的Lcy基因序列（X86452）和Pds启动子序列（U46919）分别设计特异引物从番茄中扩增出了Lcy基因的高度保守的长302bp的DNA片段和长1790的Pds启动子片段。根据RNAi的原理，将Lcy基因的长302bp的DNA片段以正反两个方向通过一段内含子序列连接在一起形成RNAi片段插入到抗Kan的pVCT2020的表达载体中，并通过具有果实特异性特点的启动子——八氢番茄红素去饱和酶基因（Pds）启动子控制该干扰片段的表达，同时将Psy基因在Pds启动子调控下插入到同一载体中，从而构建成了能同时加强Psy基因的表达和抑制Lcy基因表达的植物表达载体。 相似文献
3.
碱性土壤基因组DNA的分离纯化和基因文库的构建 总被引：4，自引：0，他引：4
直接从广西南宁市凤凰纸业排污沟碱性土壤样品中抽提和分离纯化混合基因组DNA，所获得DNA的产量为每克土壤样品10～30pg。采用限制性内切酶EcoR1酶处理后，构建了以pGEM-3Zf( )为载体的DNA部分文库。文库的容量为23650个转化子。外源片段DNA平均大小为3．2kb。建库效率为每克环境样品获得6000个左右的含1～15kb外源随机插入片段的克隆。通过DNA序列测定和同源性比较，对从文库随机词取的16个转化子序列进行分析，发现13个外源插入片段包含序列尚未确定的DNA片段。 相似文献
4.
为了研究盐生植物耐盐基因表达调控,本实验以海水浇灌的海马齿植株为供试材料,构建了盐胁迫下的全长cDNA文库。构建方法如下：采用改良的CTAB法提取总RNA,SMART法反转录合成cDNA,LD-PCR方法合成双链cDNA。LD-PCR产物经蛋白酶K消化和SfiⅠ酶切后,经CHROMA SPIN＋TE-1000分离柱子除去小片段DNA后,回收0.5kb以上的片段,按照适当的比例连接λTripIEX2载体。连接产物利用MaxPlaxTMLambda Packaging Extracts进行体外包装,得到海马齿初级cDNA文库。初始文库的独立克隆数为2.4×106pfu,初级文库滴度大于4.80×106pfu/mL,重组率为93.75%,插入片段为0.5～5kb,扩增文库的滴度为1.21×109pfu/mL,所得文库质量较高。本研究表明该cDNA文库适合于盐生植物海马齿相关基因的克隆和分析。 相似文献
5.
家蚕胞质肌动蛋白基因启动子的克隆及piggyBac转座子表达载体的构建 总被引：4，自引：0，他引：4
摘要: 利用PCR方法从家蚕（Bombyx mori）总DNA中克隆到细胞质肌动蛋白基因（cytoplasmic actin）启动子片段，序列分析表明，该片段中含有典型的组成型表达的细胞质肌动蛋白基因A3（BmA3）调控序列：SRE元件（serum response element）和ActE1元件(active element 1)。该序列的核苷酸序列与来自欧洲的一个家蚕品种的序列同源性为94%。通过多次重组，将A3启动子片段(不含信号肽序列)与转座子 piggyBac的转座酶编码区融合构建成辅助质粒；将其(含信号肽序列和部分编码区)与报告基因多管水母绿色荧光蛋白基因gfp融合，插入到人工合成的转座子piggyBac两反向重复末端序列之间，进而构建成基于转座子piggyBac的转基因载体。研究中构建的转座子转基因载体仅6.4 kb，并在两反向重复末端序列之间设计了1个多克隆位点区，便于目的基因的插入。 相似文献
6.
利用高盐法从鸡血中提取高分子量ＤＮＡ，以λＥＭＢＬ３为载体构建了鸡染色体文库，用鸡生长激素受体ｃＤＮＡ基因的部分序列作探针，从文库中筛选得到３个阳性克隆。通过对阳性克隆Ｉ插入片段的酶切和Ｓｏｕｔｈｅｒｎ杂交分析，表明它含有全长的鸡生长激素受体基因，建立了该基因的限制性酶切图谱。将插入片段亚克隆到ｐＧＥＭ－７Ｚｆ（＋）质粒上，对含有基因５’端序列的２．５ｋｂＥｃｏＲＩ酶切片段进行了序列分析。 相似文献
7.
由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50％以上。研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物。为了解水稻白叶枯病菌广西菌株GX1329中含有avrBs3／pthA家族基因的情况,本研究应用Alu I部分酶切其基因组DNA,构建了含有736个克隆的菌株GX1329的基因组文库。BamHI酶切分析随机挑取的15个文库克隆表明,克隆的外源DNA随机性良好,克隆的最小片段为27．7kb,最大为58．5kb,平均大小为39．9kb,文库克隆容量约为2．8×10^3Mb,该文库中包含基因组中任一个基因的概率为99．4％。利用来自水稻白叶枯病菌菲律宾菌株PX086的无毒基因avrXa10的第252位～第486位核苷酸序列作为探针,通过菌落原位杂交从GX1329基因组文库中筛选到37个含avrBs3／pthA家族基因的克隆。再通过Southern杂交分析,得到了17个独立克隆。这17个克隆中至少含有13个不同的avrBs3／pthA家族基因。这些基因在GX1329基因组中有的单独存在,有的两个或两个以上串联存在。本工作基本上明确了菌株GX1329基因组中avrBs3／pthA家族基因的数量,为进一步研究菌株GX1329中avrBs3／pthA家族基因的功能奠定了基础。 相似文献
8.
枯草芽孢杆菌菌株B 11对广泛的植物病原真菌和细菌都具有拮抗作用。以柯斯质粒pW EB∷TNC为载体构建了枯草芽孢杆菌菌株B 11的基因文库,文库含9 000个克隆。文库克隆中插入的DNA片段平均为42.1 kb,该文库含有菌株B 11基因组中任一基因的概率为99.99%。采用平板活性检测法筛选文库,筛选到1个对茄青枯假单胞菌菌株P 13具有拮抗活性的文库克隆GXN 9527,该克隆的重组质粒pGXN 9527含有50 kb的菌株B 11的DNA。文库克隆对革兰氏阴性植物病原细菌如水稻黄单胞菌水稻变种也具有拮抗活性,而对革兰氏阳性细菌如地衣芽孢杆菌和植物病原真菌如尖孢镰刀菌西瓜专化型、立枯丝核菌、水稻稻灰梨孢菌则没有拮抗活性。分别含有pGXN 9527的18、12、9、8 kb B amHⅠ片段的亚克隆对P 13均没有拮抗活性,说明编码该拮抗物质的生物合成基因很可能成簇存在。 相似文献
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10.
高盐极端环境土壤基因组DNA的分离纯化方法研究及基因文库的构建 总被引：5，自引：0，他引：5
以钦州市犀牛角镇晒盐池内(盐浓度>25%)的土壤样品为研究对象,对土壤混合基因组DNA的提取和纯化方法进行了深入研究,结果表明,冻融法、十二烷基磺酸钠(SDS)和蛋白酶K联合使用的提取法,能有效提高DNA得率,DNA的产量达到每克土壤样品10~15μg。交联聚乙烯吡咯烷酮(PVPP)、Sephadex G-200树脂以及大量胶回收的使用,可有效纯化DNA,纯化的DNA产量可达每克土壤样品4~6μg,DNA片段大小>30 kb。将纯化的DNA用B amHⅠ部分酶切后构建以pLAFR 3为载体的混合基因组文库,该文库包含15 600个克隆,外源片段DNA平均大小为18 kb。通过DNA序列测定和同源性比较,对从文库中随机挑取的10个克隆进行了序列分析,发现9个外源插入片段包含未知DNA序列。 相似文献
11.
Kochansky J 《Journal of agricultural and food chemistry》2000,48(7):2826-2828
Two deuterated insecticides were prepared for use as internal standards for gas-liquid chromatographic-mass spectrometric analyses. Diethyl chlorothiophosphate-d(10) was prepared by reaction of ethanol-d(6) with P(2)S(5) to give labeled diethyldithiophosphoric acid, followed by chlorination. Treatment of the acid chloride with 3-chloro-4-methyl-7-hydroxycoumarin and potassium carbonate in acetone at reflux gave labeled coumaphos. An analogous reaction with 4-methyl-7-hydroxycoumarin gave labeled potasan, and the technique should be usable for synthesis of labeled forms of other dialkyl thiophosphate insecticides. 相似文献
12.
Identification of oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin with H(2)O(2) 总被引：4，自引：0，他引：4
Zhu N Huang TC Yu Y LaVoie EJ Yang CS Ho CT 《Journal of agricultural and food chemistry》2000,48(4):979-981
(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are two important antioxidants in tea. They also display some antitumor activities, and these activities are believed to be mainly due to their antioxidative effects. However, the specific mechanisms of antioxidant action of tea catechins remain unclear. In this study are isolated and identified two novel reaction products of EGCG and one product of EGC when they were reacted separately with H(2)O(2). These products are formed by the oxidation and decarboxylation of the A ring in the catechin molecule. This study provides unequivocal proof that the A ring of EGCG and EGC may also be an antioxidant site. This study also indicates an additional reaction pathway for the oxidation chemistry of tea catechins. 相似文献
13.
This study evaluated the effect of lead (Pb(II)), zinc (Zn(II)) and copper (Cu(II)) on growth and sporulation of four Halophytophthora species (Halophytophthora vesicula, Halophytophthora elongata, Halophytophthora spinosa var. lobata, and an oogonia-producing Halophytophthora sp.) isolated from different mangrove sites in Taiwan. Results show that all isolates grew well or even better at 1 ppm concentration of the heavy metals tested. Growth of all test isolates was totally inhibited at 500 ppm, except for H. spinosa var. lobata exposed to Zn(II). For sporulation, all isolates produced moderate to abundant zoosporangia or oogonia at 1 ppm Pb(II) and Zn(II). Production of zoosporangia by H. vesicula, H. elongata and H. spinosa var. lobata was significantly affected or totally inhibited at 1 ppm Pb(II) and Zn(II) and all concentrations of Cu(II). Abnormal oogonia were produced by Halophytophthora sp. at 10 ppm Cu(II) and 100 ppm of the three heavy metals. In general, Cu(II) and Zn(II) were found to be the most toxic, and the least toxic was Pb(II). H. spinosa var. lobata was the most tolerant to all the heavy metals, while H. vesicula and H. elongata were the most sensitive. Results of this study shows that increased concentrations of Pb(II), Cu(II), and Zn(II) in the mangrove environment can significantly affect growth and impair normal reproduction of Halophytophthora species. 相似文献
14.
Nouri Hassan John D. Murimboh Chuni L. Chakrabarti 《Water, air, and soil pollution》2008,193(1-4):131-146
The competing ligand exchange method was used to investigate the competitive binding of Ni(II) by Al(III) and Fe(III) in model aqueous solutions and freshwaters. Graphite furnace atomic absorption spectrometry and adsorptive cathodic stripping voltammetry were used to monitor the rate of uptake of the Ni by Chelex 100 chelating resin and dimethylglyoxime as the competing ligands, respectively. The results have revealed that Ni(II)–humate complexes were more labile in presence of the mixture of Al(III) and Fe(III), compared to the lability of the Ni(II)–humate complexes when only one of the two, Al(III) or Fe(III), was present. The environmental significance of this work is that in model solutions simulating freshwater containing humic substances and the target trace metal Ni(II) and cations, Al(III) and Fe(III), the competitive binding of Ni(II), Al(III) and Fe(III) by humic substances makes Ni(II)–humate complexes labile, releasing free Ni2+–aqua complex, which reported to be toxic. 相似文献
15.
采用批处理法研究了锑/磷(Sb/P)在膨润土和高岭土上的竞争吸附等温线和动力学,并考察了pH的影响。结果表明:Sb和P吸附符合Langmuir或Freundlich方程(R~2=0.945 0~0.998 3,P0.000 1),存在极强的竞争吸附:P共存时Sb吸附容量(Q_(max))显著降低,分别从0.86和10.21 mmol/kg(先Sb后P)降低到0.64和2.61 mmol/kg(先P后Sb);Sb共存时P吸附亲合性(K)明显降低,分别从1.47和7.47 L/mmol(先P后Sb)降低到0.68和2.34 L/mmol(先Sb后P)。Sb和P的吸附总体随pH升高而降低,但相比高岭土,膨润土上的吸附受pH影响更大,意味着高岭土有更多配位吸附、膨润土有更多电性吸附。准二级动力学方程很好地拟合它们的竞争吸附(R~20.994,P0.000 1),并且在Sb/P共存下,膨润土对P吸附弱而慢(q_e=0.36 mmol/kg,,k_2=0.064 6 kg/(mmol·min))、对Sb吸附虽弱但快(q_e=0.19 mmol/kg,k_2=0.076 9 kg/(mmol·min));高岭土对P吸附强更快(q_e=0.66 mmol/kg,k_2=0.591 kg/(mmol·min))、对Sb吸附虽强但慢[q_e=0.39 mmol/kg,k_2=0.052 4 kg/(mmol·min)]。因此,Sb对P吸附的抑制在膨润土上更明显(主要是静电吸附竞争),P对Sb吸附的抑制则在高岭土上更突出(主要是配位吸附竞争)。 相似文献
16.
Photolysis, sonolysis, and photosonolysis of common groundwater contaminants, namely 1,1,1-trichloroethane, trichloroethylene, and tetrachloroethylene, were investigated using a flow-through photosono reactor system. Simulated groundwater containing the chlorinated volatile organic compounds (VOCs) was exposed to ultraviolet light (UV), ultrasonication (US), and UV and US concurrently (UVUS), without a photo catalyst. VOC removal efficiencies of the UV, US, and UVUS treatment processes were computed from the VOC concentrations in influent and effluent of the reactor. The process using UVUS exhibited larger degradation efficiencies than that with UV and US separately in most cases; however, statistical analysis showed that the UVUS treatment efficiency is likely to be additive of the UV and US treatment efficiencies. The results also showed that the increase of the detention time from 26 to 60 min had no significant effect on the VOC removal efficiencies in these processes. 相似文献
17.
Roy A 《Journal of agricultural and food chemistry》1999,47(12):5209-5210
Terpinolene oxide, a monoterpene belonging to the p-menthane group, is easily derived from naturally abundant (R)-limonene. It was isomerized with montmorillonite clay catalyst to karahanaenone (2,2, 5-trimethylcyclohept-4-en-1-one) by ring enlargement. The enantiomers of the corresponding alcohol, karahanaenol (2,2, 5-trimethylcyclohept-4-en-1- ol), known for their individual organoleptic properties, were resolved through Pseudomonas cepacia lipase mediated enantiospecific alcoholysis of its acetate derivative. 相似文献
18.
W R Dantzer J Hopper K Mullin S Hendrich P A Murphy 《Journal of agricultural and food chemistry》1999,47(10):4291-4296
14C-Fumonisin B(1) (FB(1)) was produced by Fusarium proliferatum M-5991 in modified Myro liquid medium and purified to >95% purity with a specific activity of 1.7 mCi/mmol. Nine male and nine female F344/N rats were each dosed by gavage with 0.69 micromol of (14)C-FB(1), (14)C-hydrolyzed FB(1), or (14)C-FB(1)-fructose/kg body weight. Urinary excretion of (14)C-FB(1) and (14)C-FB(1)-fructose was 0.5% and 4.4% of the total dose, respectively, and was similar between male and female rats. Urinary excretion of (14)C-hydrolyzed HFB(1) was significantly greater (P > 0.05) in female rats as compared with male rats (17.3% vs 12.8% of the total dose, respectively). There were no significant (P > 0.05) differences in biliary excretion of the three fumonisin compounds with a mean of 1. 4% of the dose excreted at 4 h after dosing. Lesser amounts continued to be excreted up to 9.25 h after dosing. Although biliary excretion of the (14)C-FB(1), (14)C-hydrolyzed FB(1), and (14)C-FB(1)-fructose was similar, increased urinary excretion of the (14)C-hydrolyzed FB(1) as compared to (14)C-FB(1) and (14)C-FB(1)-fructose indicated a greater absorption of the hydrolyzed form. 相似文献
19.
Prior RL Hoang H Gu L Wu X Bacchiocca M Howard L Hampsch-Woodill M Huang D Ou B Jacob R 《Journal of agricultural and food chemistry》2003,51(11):3273-3279
Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples. 相似文献