从未引用的太平洋牡蛎生成虚拟序列的 cDNA 库的聚合策略
Summary
我们描述了如何使用未引用的太平洋牡蛎标本的RNA样本的策略,并通过与公开提供的基因组数据进行比较来评估遗传物质,以生成几乎序列化的cDNA库。
Abstract
以前在开发新型细胞系或基因组测序项目等关键实验中使用参考物种的生物材料,往往难以为进一步研究或第三方提供。样品的消耗性质。虽然现在广泛分布于亚洲、澳大利亚和北美的太平洋沿岸,但个别太平洋牡蛎标本在基因上相当多样化,因此不能直接作为基因库的起始材料。在本文中,我们演示了使用从区域海鲜市场获得的未引用的太平洋牡蛎标本来生成 cDNA 库。然后将这些库与公开提供的牡蛎基因组进行比较,并使用线粒体参考基因细胞色素C氧化酶亚单位I(COX1)和NADH脱氢酶(ND)选择最接近的相关库。生成的 cDNA 库的适用性也通过克隆和表达两个基因来证明编码酶 UDP-葡曲酸脱氢酶 (UGD) 和 UDP-xylose 合成酶 (UXS),它们负责 UDP-xylose 的生物合成。UDP-葡萄糖。
Introduction
由于交货时间长、创业推理或针对具体国家的海关法规,获取活的参考生物材料可能具有挑战性。作为替代方案,也可以从表象相同的标本中收集所需的生物材料。然而,这些样本在基因型水平上可能有很大差异,因此,由于新来源的材料与同一物种的数字化存储参考基因组的比较,往往变得困难,甚至徒劳。现有的DNA扩增方法。测序单个样本的高度保守的基因是识别物种1的广泛使用和强大的工具,例如常用的线粒体基因,这些基因经常用作cDNA库的质量评估的参考基因2 ,3,4,5,6.本文提出的方法的基本原理是,与参考基因组的相应序列相比,单个匿名牡蛎样本中的线粒体基因序列高度保存表明,其他基因也可能显示低发分,因为线粒体DNA进化速度比核DNA7要快,只需公开使用,即可对各种科学和工业相关的基因进行扩增和分离可作为参考的排序数据。
本文所述方法的总体目标是提供一个优化的工作流程,用于生成虚拟序列化的牡蛎 cDNA 库,该库可用作牡蛎基因克隆的模板 DNA。在虚拟测序中,绕过了新基因组测序;相反,一个已知的、数字存储的参考序列直接用于利用或设计用于生产cDNA的引物,最终构成一个库(或添加到预先存在的库)。其目的是生成收敛的 cDNA 库,这意味着生成的 cDNA 序列和参考序列之间的相似性可以从低到高背离排列。使用细胞色素 C 氧化酶亚单位 1 (COX1) 和 NADH 脱氢酶 (ND) 作为参考基因的一个关键优势是,由于这些线粒体基因的高度保护,即使高度地理分离的牡蛎标本也可以被分析。在证明了这些成熟的标记方法后,我们证明了其应用于两种酶候选体,它们涉及糖核苷酸生物合成,可能具有工业相关性8、9、10.太平洋牡蛎的生物技术潜力尚未开发。因此,我们认为,这种制备虚拟序列cDNA库的收敛方法也适合希望从这种相关生物材料中生成cDNA的非专家研究人员。
Protocol
注:图1显示了原理图概述。 1. 样本收集 获取牡蛎标本。在收获后、运输和实验室使用前,在购买后4-7天内将牡蛎留在冰上。注:根据本协议,牡蛎是从南京中彩批发市场(原产于中国福建宁德和中国江苏连云港)、青岛海杰水产品公司(原产于中国山东青岛)、居城购买牡蛎。烟台水产品公司(原产于中国山东烟台)和青岛金秀水产品公司(原产于中国山?…
Representative Results
图1显示了从太平洋牡蛎个体中提取的收敛cDNA库所述制备方法的原理图概述。图2显示了与参考材料的COX1和ND基因序列高度背离的远亲牡蛎标本的COX1和ND基因序列。图3显示了与参考材料的COX1和ND基因序列有低差异的密切相关的牡蛎标本的COX1和ND基因序列。图4显示了cDNA库成功克隆工业相关基因MgUGD和MgUXS。 <…
Discussion
通过将COX1和ND基因与公开的牡蛎DNA基因组数据库进行比较,允许对区域海鲜市场具有类似表型的无配表型牡蛎标本进行基因鉴定。该方法的意义在于其简单性,因为评估虚拟cDNA库只需要一个PCR反应。两个保守的线粒体COX1和ND基因从cDNA库被放大,该库由每个牡蛎的RNA提取物的逆转转录产生。通过直接在液氮中研磨牡蛎组织,简化了RNA分离方法(步骤2.1)。对每个试样的COX1和ND基因进行测序后,序列排列表明,…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了中国自然科学基金(赠款号31471703、A0201300537和31671854给J.V.和L.L.,授予G.Y.编号31470435)和100名外国人才计划(授予编号JSB2014012到J.V.)的支持。
Materials
| Chemicals: | |||
| 1% Triton X-100 | Solarbio | 9002-93-1 | *Alternative distributors possible |
| 2,5-Dihydroxybenzoic acid | Alfa Aesar | 490-79-9 | *Alternative distributors possible |
| Acetonitrile | Merck | 75-05-8 | *Alternative distributors possible |
| Agarose for molecular biology | Biowest Chemicals | 111860 | *Alternative distributors possible |
| Ampicilin | Solarbio | 69-52-3 | *Alternative distributors possible |
| Chloroform | Lingfeng, Shanghai | 67-66-3 | *Alternative distributors possible |
| DEPC water | Thermo Scientific | R0601 | |
| Ethanol | Jinhuada, Guangzhou | 64-17-5 | *Alternative distributors possible |
| Guanidinium thiocyanate-phenol reagent | Invitrogen | 15596018 | TRIzol reagent |
| Imidazole | Energy Chemical | 288-32-4 | *Alternative distributors possible |
| Isopropyl alcohol | Nanjing Chemical Reagent | 67-63-0 | *Alternative distributors possible |
| Isopropyl β-D-thiogalactopyranoside | Solarbio | 367-93-1 | *Alternative distributors possible |
| Kanamycin | Solarbio | 25389-94-0 | *Alternative distributors possible |
| LB Agar | Thermo Fisher | 22700025 | *Alternative distributors possible |
| LB Broth | Thermo Fisher | 10855021 | *Alternative distributors possible |
| Methanol | Jinhuada, Guangzhou | 67-56-1 | *Alternative distributors possible |
| MgCl2 hexahydrate | Xilong Huagong | 7791-18-6 | *Alternative distributors possible |
| NaCl | Xilong Huagong | 7647-14-5 | *Alternative distributors possible |
| NAD+ | Duly Biotech | 53-84-9 | *Alternative distributors possible |
| Phenyl-methylsulfonyl fluoride | Macklin | 329-98-6 | *Alternative distributors possible |
| Tris | Solarbio | 77-86-1 | *Alternative distributors possible |
| UDP-glucose | Wuhu Nuowei Chemicals | 28053-08-9 | *Alternative distributors possible |
| UDP-glucuronic acid | SIGMA | 63700-19-6 | *Alternative distributors possible |
| Tools/Instruments: | |||
| MALDI-TOF mass spectrometer | Bruker | Autoflex | *Alternative distributors possible |
| Metal block heater | Long Yang Scientific Instruments | Thermoshaker HB20 | *Alternative distributors possible |
| PCR thermocycler | Hema | 9600 | *Alternative distributors possible |
| Enzyme and Kits: | |||
| 10×Ligation buffer | Thermo Scientific | B69 | *Alternative distributors possible |
| 5×PrimeSTAR buffer | Takara | 9158A | |
| Alkaline phosphatase | ThermoFisher FastAP | EF0654 | *Alternative distributors possible |
| COX forward primer | Genscript | ATGTCAACAAATCATTTAGACATTG | |
| COX reverse primer | Genscript | ACTTGACCAAAAACATAAGACATG | |
| Cutsmart Buffer | NEB | B7204S | *Alternative distributors possible |
| dNTP mix | Invitrogen | 18427088 | |
| MgUGD forward primer | Genscript | ACATATGACCCTGTCCAAGATCTGTTGT | |
| MgUGD reverse primer | Genscript | ACTCGAGACTCTGTGAGGCGGTGGAG | |
| MgUXS forward primer | Genscript | CCATATGGCAGAATCCTCACAATCAC | |
| MgUXS reverse primer | Genscript | ACTCGAGCACATTTTTGAATTTGCAGACGT | |
| ND forward primer | Genscript | ATGAGATGGCAATTATTTTTTAAT | |
| ND reverse primer | Genscript | ATGTATTTTGGAAAAATCTCCAC | |
| PCR Cleanup Kit | AxyGen | AP-PCR-250 | *Alternative distributors possible |
| pET-30a(+) vector | Merck Millipore | 69909 |
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Citazione di questo articolo
Lyu, Y. M., Li, Y. Q., Song, H. B., Guo, J., Wang, T., Liu, L., Yedid, G., Voglmeir, J. A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters. J. Vis. Exp. (148), e59462, doi:10.3791/59462 (2019).