共查询到18条相似文献，搜索用时 531 毫秒
1.
脐橙基因组DNA文库的构建 总被引：1，自引：0，他引：1
研究构建了一个脐橙基因组DNA文库。以‘大山岛’脐橙幼叶为试材 ,CTAB法制备基因组DNA ,经CsCl密度梯度离心纯化 ,获得了大片段 ( >10 0kb)高纯度基因组DNA。以Sau3AI部分酶切 ,酶切片段 3’凹端不完全补平后与LambdaGEM 12XhoIHalf SiteArms连接 ,连接产物用Packagene Extract包装 ,所得噬菌体在KW2 51寄主上铺平板。文库经过扩增并保存。对该文库特性研究表明 ,该文库包含了 2 .15× 10 6个单个克隆 ,背景低于10 0pfu/ μgDNA ,插入片段平均大小约为 17kb ,证明是一个较为完整的脐橙基因组DNA文库。 相似文献
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平菇基因组文库的构建 总被引：3，自引：0，他引：3
本文采用原生质体裂解法制备高分子量平菇总DNA。通过Sau3A部分酶切和蔗糖密度梯度离心,获得15～25Kb平菇总DNA酶切片段。并以Lambda的衍生物EMBL3为载体,酶连形成重组DNA。然后将其包装,转染受体菌LE392,在TB平板上获得2.5×10~4个重纽子克隆。此重组子克隆群即构成中蔬10号平菇菌株基因组文库。 相似文献
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采用改良CTAB法提取美味猕猴桃(Actinidiadeliciosacv.Bruno)基因组DNA,经CsCl密度梯度离心纯化后用Sau3AI部分酶切,通过透析袋电泳的方法分级回收,剔除14kb以下的片段,回收长为14～23kb的酶切片段。部分酶切片段经dATP和dGTP部分补平后与LambdaFIXII载体连接,连接产物用GigapackⅢPackagingExtract进行包装,最后得到含有1.05×106pfu的基因组文库,扩增后文库滴度为2.5×109pfu/mL。利用6对根据植物基因保守区或猕猴桃基因序列设计的专一引物对基因组文库和基因组DNA进行PCR扩增,均得到与预计长度相符的目的基因片段。RR 相似文献
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结球甘蓝cDNA文库的构建和鉴定 总被引：4，自引：0，他引：4
构建了结球甘蓝cDNA 文库。文库的宿主菌为E. coli XL1-lue。对文库的部分特性进行研究,滴度为2 ×108～4 ×108 pfu/ mL , 插入片段的大小均在500 bp 以上, 以1kb 以上的片段占大多数, 重组体的重组率为95 % , 用抗病基因片段作探针进行噬菌体原位杂交, 杂交信号明显。对文库进行了扩增、保存。 相似文献
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利用PCR技术从马铃薯陇薯3号基因组DNA中扩增出长度约为600 bp的DNA片段,经与T载体连接,测序表明,克隆到的DNA片段大小为599 bp,该序列与Genebank中已公布的GBSS启动子序列同源性为99.67%;采用植物顺式调控元件数据库PLACE和PlantCare对其进行序列分析,结果表明,该片段含有启动子的保守序列TATA-box和CAAT-box。此外,还具有诸如TAACAAA、CTAACAC、CTCTT及CACT等序列,而这些特异序列可能是基因特异表达所必须的。 相似文献
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草莓镶脉病毒的PCR检测及特异片段的序列分析 总被引：5，自引：0，他引：5
用CTAB法从感病的草莓叶片中提取总DNA,以其为模板经PCR扩增获得与预期片段大小一致长约600bp的扩增产物,同时优化PCR反应程序,获得单一特异条带;通过总DNA浓度梯度稀释,进行PCR扩增,结果表明能检测到2.5μg叶组织中病毒的存在。回收PCR特异扩增产物,与pMD18-T载体连接,并进行转化、重组克隆的筛选、重组质粒的酶切鉴定和序列测定。扩增片段序列与已报道SVBVCP基因序列(序列号:Nc_001725)的核苷酸同源性为89.2%,氨基酸同源性为96.3%。该特异片段序列在GenBank中的登记号为AY862389。 相似文献
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牡丹ACC氧化酶基因的克隆与反义载体的构建 总被引：1，自引：0，他引：1
根据报道的牡丹ACC氧化酶基因(DQ337251)cDNA序列,设计一对特异引物,以牡丹品种"洛阳红"基因组DNA为模板,用PCR扩增方法克隆出牡丹ACC氧化酶基因的部分片段,并将其连接到pMD18-T载体上进行测序.结果表明,克隆的序列全长为467 bp,其中包括一个长度为157 bp的序列,推测它可能是一个内含子,其它序列与已报道序列同源性为98.2%;用SacⅠ和XbaⅠ对重组质粒和载体pBI 121酶切、连接,构建牡丹ACC氧化酶基因的反义表达载体. 相似文献
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甘蓝中硫氧还蛋白编码基因THL1的分子特性及表达研究 总被引：3，自引：0，他引：3
采用PCR和RT2PCR技术, 以‘E1’甘蓝基因组DNA和柱头cDNA为模板对THL1基因进行扩增克隆, 得到的片段长度分别为732 bp和455 bp。序列分析表明, 克隆的DNA和cDNA序列与甘蓝‘西园四号’THL1的DNA和cDNA同源性分别为97.9%和98.3%, 两条序列内含子的大小不同; 同时, 前者第2内含子不符合典型的GT2AG规则: 即第2个内含子3′端碱基为AT。将THL1基因cDNA序列定向克隆到原核表达载体pET-43.1a ( + ) , 构建融合表达质粒pET43.1a ( + ) -THL1, 在大肠杆菌BL21中表达出分子量为74kD的融合蛋白, 经胰岛素检测, THL1有氧化还原活性, 表明THL1在大肠杆菌中得到了正确表达。 相似文献
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随机扩增多态性DNA(Random AmplifiedPolymorphism DNA,RAPD),是Williams等于1990年发展起来的以PCR为基础的新型分子标记技术。基本原理是:利用一系列(通常数百个)不同碱基序列的随机引物(8～10bp)对基因组DNA进行PCR扩增,通过凝胶电泳分析扩增产物DNA片段的多态性。其中,每个扩增片段代表基因组上的一个位点,这些扩增片段多态性反映了基因组相应区域DNA多态性。RAPD所用一系列引物DNA序列各不相同,每一特定引物,在基因组DNA上都有其特定的结合位点;如果基因组在这些区域发生DNA片段插入、缺失或碱基突变,特定结合位点的… 相似文献
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AIM: To construct random eight-peptide library for the study on atherosclerosis and restenosis. METHODS and RESULTS: Random oligodeoxynucleotides encoded eight peptides were synthesized and amplified by polymerise chain reaction( PCR).The product was cloned into phage surface display vector fUSE5 in Sfi I site and electroporated into competent MC1061. The library was identified through PCR, hybridization, DNA sequencing and affinity biopanning of streptavidin. Because the upstream primer is complementary to part vector clone site sequences and part exogenous gene sequences, and the other one complementary to pⅢ gene of vector, thus only clones inserted exogenous gene could be amplified easily. Additionally we used the probe oligodeoxynucleotide complementary to vec for clone site sequences to identify clones which were not inserted exogeneous genes. Furthermore, two hybridizing positive clones were sequenced. Their sequences are consistent with two oligodeoxynucleotide probe sequences. As a result, 2.1×108 special clones were obtained. Affinity biopanning proved that the libraries could be amplified steadily.CONCLUSION: The eight-peptide library is reliable. 相似文献
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《园艺学报》2003,19(5):622-626
AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO 5. 0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0. 5% NP-40, and 0.05% Tween 40, at 60°C for 15 minutes, heated for 5 minutes at 100 °C, 10μL was used as template. The PCR react ion was performed in 50μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11) . PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was conf irmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen inmale control DNA are the presumpt ive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The ent ire procedure took <3. 5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue. 相似文献
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分别以琯溪蜜柚及其突变体红肉蜜柚（成熟期早20 ~ 25 d）的成熟果肉cDNA 作驱赶子和检测子,利用结合分子镜像选择技术的SSH 技术,构建了红肉蜜柚和琯溪蜜柚的正向差减cDNA 文库。通过PCR 检测cDNA 克隆外源插入片段,其大小介于100 ~ 1 000 bp。通过点杂交差异筛选文库,得到102 个表达增强2 倍或以上的克隆并测序,结果表明这102 个克隆代表44 个Unigenes,通过序列同源性比对分析,发现获得的Unigenes 在功能上主要涉及信号传导、蛋白质合成、应激反应、转运等代谢反应。 相似文献
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Miao Wang AnQun Tan LingYun Zhang JiaWei Yang ZhaoXin Wei ChunZhen Cheng ZhiQin Zhou GuangYan Zhong 《Scientia Horticulturae》2010
A deep-coverage bacterial artificial chromosome (BAC) library of Yanxiwanlu Ponkan, a late maturing cultivar, was constructed and evaluated. Results showed that the library contained a total of 61,000 clones. Restriction analysis indicated that the DNA insert sizes ranged from 30 to 170 kb with an average of ∼82 kb. No empty clone was found. Therefore, the library should cover ∼13.6-fold of the citrus genome. Continuous sub-cultivation of 5 randomly chosen clones for up to 100 generations showed no detectable change in their restriction profiles. To demonstrate the application of the library in discovery of genes, 7 ethylene-regulated genes were screened by PCR amplification from hierarchically pooled library clones with gene-specific primers. Five positive clones, each representing a different gene, were thus identified from 3840 clones. Sequencing of the clones showed that all of them matched the expected genes, indicating that the library was highly representative. 相似文献
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AIM:To screen the proteins interacting with human augmenter of liver regeneration (hALR) by yeast two-hybrid system and to study the mechanism of hALR action. METHODS:hALR bait plasmid was constructed by ligating the gene of hALR into pGBKT7, then transformed into yeast AH109. The yeast strain AH109 containing pGBKT7-hALR was mated with yeast Y187 containing human liver cDNA library plasmid. Diploid yeast was plated on SD/-trp-leu-his-ade (QDO) for screening and on QDO containing X-α-gal for further selection.The AD/library inserts were amplified by PCR and the PCR products were characterized by digesting with Sau3AⅠ and HaeⅢ restriction enzyme to eliminate the duplicates. After sequencing, the positive clones were analysed by bioinformatics. RESULTS:Several positive clones were obtaind. The sequencing and analysis shown that one of them is 669 bp DNA fragment encoding β subunit of Na+, K+-ATPase. The 224 bp 3'terminal DNA fragment is non-encoder region, and the 445 bp 5'terminal DNA encodes C-terminal 147 amino acid residues of Na+, K+-ATPase β subunit. CONCLUSION:The results of screening proteins using yeast two-hybrid system showed that hALR could interact directly with Na+, K+-ATPase in the yeast cell. 相似文献
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白菜亚硝酸还原酶基因BcNiR的克隆及表达分析 总被引：2，自引：0，他引：2
以白菜品种‘苏州青’自交系叶片cDNA为模板, 采用RT-PCR、3′RACE和5′RACE技术,
获得了编码亚硝酸还原酶基因(NiR) 的cDNA全序列1 852 bp, 包含有1 749 bp的开放阅读框, 编码583个氨基酸, 命名为BcNiR。所推导的氨基酸序列与拟南芥NiR1、烟草nii2编码的氨基酸序列具有较高同源性, 分别为83%和76%。生物信息学分析表明, BcNiR具有完整的NiR蛋白结构, 含血红素蛋白β - 化合物区域, 一个明显的西罗血红素siroheme结合位点和4Fe-4S区域, 可以在SWiSS-MODEL数据库中搜索到与之相近的三维结构。RT-PCR结果显示, 该基因在叶片中的表达量远远高于根系, 在以0、10、20、30、40 mmol·L - 1硝态氮各分别处理0、2、4、6、8、12 h的试验中, 30 mmol·L -1处理4 h可使BcNiR的表达量达到最大; 5和10 mmol·L - 1铵态氮处理试验表明, 高浓度的铵抑制该基因的表达。 相似文献
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一个与双孢蘑菇子实体品质相关的DNA片段的克隆 总被引：5，自引：0，他引：5
利用20个十碱基随机引物，在基因组水平上对双孢蘑菇（Agaricus bisporus）的优质低产型（G型）及低抽高产型（H型）菌析进行了RAPD差异显示，获得了一个与双孢蘑菇子实体品质相关的大小约为2000bp的差异DNA片段，将其克隆，并应用点杂交及RFLP技术对其区分两类菌株的有效性进行了检验。这为进一步建立和的特异性探针或PCR引物，从分子水平上提高双孢蘑菇菌种性状早期预测的准确性奠定了基础。 相似文献