-
芋螺(Conus)是分布于热带海洋中的肉食性软体动物,全世界约有800种[1]。不同种类的芋螺都能通过分泌各自特有的肽类毒素(芋螺毒素、芋螺肽)捕食鱼类、虫类或其他软体动物。根据芋螺的食性,分为食鱼芋螺,食虫芋螺和食螺芋螺3大类[2]。芋螺的毒液是由长而卷曲的毒管上皮细胞分泌产生的。当芋螺捕食猎物时,毒腺的肌肉蠕动,推动毒液进入鱼叉状的舌状牙齿中,将毒液快速注射入猎物体内[3-6]。研究发现,每种芋螺毒液中含有约3 000种不同的芋螺毒素肽。在萼托芋螺(Conus episcopatus)的1个个体毒液中,竟发现了3 305个前体毒素基因序列[7]。由此预估全球芋螺可产生超过150万种的天然毒素肽,它们具有调节各种离子通道和受体等的特殊功能[8],已成为神经科学研究工具药和治疗新药研发的重要来源[9]。然而迄今为止,发现的芋螺毒素还不足其总量的1%,因而大量的新型芋螺毒素尚有待进一步开发。目前,获取芋螺毒素序列的方法主要有4种,包括粗毒液纯化[10]、芋螺毒素基因克隆[11]、毒管转录组测序[7, 12]、毒液蛋白质组学和多组学联用测序分析[13-14]。多组学是一种通过生物信息学将转录组学与蛋白质组学整合的综合策略[15],在芋螺毒素研究领域很受欢迎。芋螺毒素的研究需要持续不断地捕捞活体芋螺,但由于气候变暖,芋螺赖以生存的海洋环境也越来越恶化,再加上芋螺壳工艺品国际贸易的兴起,造成了芋螺资源的严重破坏,70%以上的芋螺遭遇生存危机。所以人们更应重视芋螺资源的保护和可持续利用,例如可建立不同芋螺种类的基因组DNA文库、毒管cDNA文库以及毒素肽库,长期保存并加以利用。粗毒纯化需要大量的芋螺,且只能发现一些表达量较高的毒素,为克服粗毒纯化的局限性,20世纪90年代,基因克隆为新型芋螺毒素的发现提供了极大的帮助[11]。由于芋螺毒素是由特定的前体基因表达的,所以可以用特定的引物通过PCR技术进行扩增[16-20]。因而,简单快速地获取高质量的芋螺基因组DNA是关键。有关海南产信号芋螺基因组DNA的提取方法优化的研究目前尚未见报道,为此,笔者拟从信号芋螺(Conus litteratus)的不同组织器官中,选取4种试剂盒,分别提取其基因组DNA,然后比较各种提法的效果,并对其中的优选试剂盒的提取条件进行优化,旨在建立简便快速提取信号芋螺基因组DNA的方法。
-
从表1可知,磁珠法DNA抽提试剂盒所提取的毒腺、毒管、腹足以及肝胰脏的A260/A280分别为1.69,2.01,1.73,1.61,其中,毒管和肝胰脏组织提取的纯度较低;改良的DNA快速提取试剂盒法的A260/A280分别为1.89,1.91,1.84,1.69,提取效果比磁珠法好,但肝胰脏有降解和蛋白污染等的问题;FastPure Tissue DNA提取试剂盒法的A260/A280分别为1.67,1.94,1.84,1.87,毒腺的A260/A280值偏低;海洋动物组织DNA提取试剂盒法的A260/A280分别为1.80,1.82,1.74,1.63,肝胰脏有明显的蛋白污染。比较4种方法的A260/A280可以看出,改良的DNA快速提取试剂盒提取的DNA纯度和海洋动物组织DNA提取试剂盒方法整体略优于其他2种方法,而且不同芋螺组织中,毒腺和腹足提取DNA的纯度更高。
表 1 4种方法提取信号芋螺不同组织基因组DNA的纯度及其产率
Table 1. The purity and yield of genome DNA of Conus litteratus extracted from the tissues of different organs
提取方法
Method样品名
Sample质量/mg
QualityA260/A280 浓度/(mg·L−1)
Concentration产率/(mg·kg−1)
Yield磁珠法试剂盒 毒腺 19 1.69 16.3±1.7 42.9±4.5 毒管 19 2.01 247.7±28.5 651.8±75 腹足 24 1.73 58.1±1.5 121.0±3.1 肝胰脏 11 1.61 350.2±9.3 1 590.9±42.3 改良DNA快速提取试剂盒 毒腺 25 1.89 68.9±10.2 137.8±20.4 毒管 12 1.91 109.9±21.1 457.9±87.9 腹足 20 1.84 80.3±9.8 200.8±24.5 肝胰脏 11 1.69 388.3±19.4 1 765±88.1 FastPure DNA提取试剂盒 毒腺 23 1.67 39.5±13.9 85.9±30.2 毒管 21 1.94 179.2±30.8 426.7±73.3 腹足 22 1.84 37.3±10.2 84.7±23.2 肝胰脏 12 1.87 357.7±20.7 1 490.4±86.3 海洋动物组织DNA提取试剂盒 毒腺 20 1.80 27.3±5.3 68.3±13.3 毒管 20 1.82 195.4±12.5 488.5±31.25 腹足 25 1.74 71.2±9.4 142.4±18.8 肝胰脏 10 1.63 162.1±28.9 810.5±144.5 注:质量和吸光度为均为3次测量的均值。
Note: Weight and A260/A280 are a mean of three measured values. -
从表1可知,改良的DNA快速提取试剂盒法整体优于其他试剂盒法,其中,毒管和肝胰脏组织的产率明显高于毒腺和腹足,腹足则略高于毒腺,结合A260/A280数值,4种方法提取腹足的基因组DNA纯度最高,A260/A280数值均接近1.80,因此,改良的DNA快速提取试剂盒法提取腹足组织能够获得较高纯度且较高产率的DNA(图3)。
-
将4种方法提取的不同组织的基因组DNA进行水平电泳检测(图4),从电泳条带看,条带均比较清晰。其中,改良的DNA快速提取试剂盒法电泳图中,毒腺,腹足提取的DNA条带明亮且清晰,大小均超过15 kb,DNA完整性较好且不存在明显污染情况;而毒管和肝胰脏DNA则完整性较差,条带弥散,且肝胰脏泳道最下方有明亮的条带,应是RNA污染,此结果在提取肝胰脏中较为常见,毒管泳道最下方也有模糊条带,可见毒管也有RNA污染的问题,毒腺和腹足组织DNA无明显的污染条带,此结果和NanoDrop 2000检测结果相符。这可能是由于毒腺和腹足密度较大,组织无法研磨充分,阻碍了蛋白质溶解,故而裂解过程比较困难,影响了DNA的产率。肝胰脏最易研磨,故而产率最高,但是由于其组织过于柔软,且富含DNA酶,在冷冻、解剖、以及提取过程中可能造成其DNA降解,故而电泳条带多呈现弥散状。
图 4 4种方法提取信号芋螺不同组织基因组DNA的琼脂糖凝胶的电泳图
Figure 4. Agarose gel electrophoresis of genomic DNA of Conus litteratus different tissues extracted with four different kits
通过4种试剂盒的电泳图(图4)可以发现,毒管和肝胰脏的DNA的降解严重,纯度低,对比毒腺与腹足的DNA产率,腹足略优于毒腺,况且毒腺组织材料相对少,不适用于大量提取DNA,所以腹足是更为合适的提取组织,且从图中(图3)可发现,提取腹足DNA时,改良的DNA快速试剂盒提取的DNA产率最高,因此,改良的DNA快速提取盒法提取腹足的DNA产率高、完整性好、纯度高。提取信号芋螺的腹足基因组DNA用该方法更合适。
-
以芋螺核基因特有的A超家族的α−芋螺毒素基因为引物,4种不同的方法提取的DNA为模板进行PCR,电泳发现1~16泳道均有明亮的条带,大小在100~250 bp之间(图5),与芋螺毒素的前体基因相符,测序后确定目的条带为α−芋螺毒素基因,大小为167~170 bp。因此,所提取的DNA可用于后续实验。
Optimization of Genomic DNA Extraction for Conus litteratus
-
摘要: 高质量的基因组DNA的获取是芋螺毒素基因克隆及基因组文库构建的关键。笔者采用4种方法提取信号芋螺(Conus litteratus)的不同组织器官的DNA,并综合比较了各组DNA的浓度和纯度、DNA片段的大小和完整性。结果表明,由改良的DNA快速提取试剂盒法提取的DNA纯度和浓度较好;4种组织器官中,腹足纯度较高,是更合适的提取组织;肝胰脏和毒管获取的DNA产率最高,但片段完整性差,污染严重;PCR反应获得了跟预期大小一致的特异扩增产物。因此,信号芋螺基因组DNA的提取最佳方法为改良的DNA快速提取试剂盒法,提取部位为腹足组织,可以获得高质量的基因组DNA,且符合后续实验要求。Abstract: The acquisition of high-quality genomic DNA is key to cloning of conotoxin genes and gene library construction. Four kits, Magnetic Bead Method Kit, Improved DNA Rapid Extraction Kit, FastPure DNA Isolation Kit, and TIANamp DNA Isolation Kit, were adopted to extract DNA from different tissues and organs of Conus litteratus, and the concentration, purity, size and integrity of the genomic DNA extracted from different tissues and organs by each kit were comprehensively compared. The results showed that the purity and concentration of the genomic DNA extracted with the Improved DNA Rapid Extraction Kit was better. Of the four different organs of C. litterantus, muscular foot had a higher purity in the genomic DNA extracted from its tissues and was the best organ used for extraction of the genomic DNA. The hepatopancreas and venom duct of C. litterantus gave the highest yield of the genomic DNA, but with poor fragment integrity and high contamination. Moreover, specific amplification products with the expected size were obtained through PCR. It is concluded that the best method to acquire the genomic DNA from C. litteratus is to extract the genomic DNA from the muscular foot with the Improved DNA Rapid Extraction Kit, with which high-quality genomic DNA can be produced and meet the requirements of subsequent research.
-
Key words:
- Conus litteratus /
- genomic DNA /
- extraction method /
- optimization
-
图 2 α−芋螺毒素基因克隆引物设计示意图
最左边黑线:5′端非翻译区;黑色方框:信号肽区;白色方框:前肽区;阴影框:成熟肽区;中间的黑色线:内含子;最右边黑线:3右端非翻译区。
Fig. 2 Schematic diagram of primer design for α-conotoxin gene cloning
Leftmost black line: 5′ untranslated region (5′ UTR); black box: Signal peptide region; white box: Propeptide region; shaded box: Mature peptide region; middle black line: Intron region; rightmost black line: 3′ untranslated region (3′ UTR).
图 4 4种方法提取信号芋螺不同组织基因组DNA的琼脂糖凝胶的电泳图
F1:磁珠法动物基因组DNA抽提试剂盒法;F2:改良的DNA快速提取试剂盒法;F3:FastPure Tissue DNA提取试剂盒法;F4:海洋动物组织DNA提取试剂盒法;M:DL15000 DNA Marker;1:毒腺;2:毒管;3:腹足;4:肝胰脏。
Fig. 4 Agarose gel electrophoresis of genomic DNA of Conus litteratus different tissues extracted with four different kits
F1: Magnetic Bead Method Kit; F2: Improved DNA Rapid Extraction Kit; F3: FastPure DNA Isolation Kit; F4: TIANamp DNA Isolation Kit; M: DL15000 DNA Marker: 1: Venom gland; 2: Venom duct; 3: Muscular foot; 4: Hepatopancreas.
图 5 α−芋螺毒素前体基因PCR扩增产物的电泳图
M:DL5000 DNAMarker;1~4:磁珠法动物基因组DNA抽提试剂盒法DNA;5~8:改良的DNA快速提取试剂盒DNA;9~12:FastPure Tissue DNA提取试剂盒法DNA;13~16:海洋动物组织DNA提取试剂盒法DNA;4种不同方法中从左到右依次为毒腺,毒管,腹足,肝胰脏。
Fig. 5 Electrophoresis of PCR amplified products of α-conotoxin precursor genes
M: DL5000 DNA Marker; 1−4: DNA extracted by the magnetic bead method kits; 5−8: DNA extracted by the Improved DNA Rapid Extraction Kit; 9−12: DNA extracted by the FastPure DNA Isolation Kit; 13−16: DNA extracted by the TIANamp DNA Isolation Kit; From left to right are venom gland, venom duct, muscular foot, and hepatopancreas.
表 1 4种方法提取信号芋螺不同组织基因组DNA的纯度及其产率
Table 1 The purity and yield of genome DNA of Conus litteratus extracted from the tissues of different organs
提取方法
Method样品名
Sample质量/mg
QualityA260/A280 浓度/(mg·L−1)
Concentration产率/(mg·kg−1)
Yield磁珠法试剂盒 毒腺 19 1.69 16.3±1.7 42.9±4.5 毒管 19 2.01 247.7±28.5 651.8±75 腹足 24 1.73 58.1±1.5 121.0±3.1 肝胰脏 11 1.61 350.2±9.3 1 590.9±42.3 改良DNA快速提取试剂盒 毒腺 25 1.89 68.9±10.2 137.8±20.4 毒管 12 1.91 109.9±21.1 457.9±87.9 腹足 20 1.84 80.3±9.8 200.8±24.5 肝胰脏 11 1.69 388.3±19.4 1 765±88.1 FastPure DNA提取试剂盒 毒腺 23 1.67 39.5±13.9 85.9±30.2 毒管 21 1.94 179.2±30.8 426.7±73.3 腹足 22 1.84 37.3±10.2 84.7±23.2 肝胰脏 12 1.87 357.7±20.7 1 490.4±86.3 海洋动物组织DNA提取试剂盒 毒腺 20 1.80 27.3±5.3 68.3±13.3 毒管 20 1.82 195.4±12.5 488.5±31.25 腹足 25 1.74 71.2±9.4 142.4±18.8 肝胰脏 10 1.63 162.1±28.9 810.5±144.5 注:质量和吸光度为均为3次测量的均值。
Note: Weight and A260/A280 are a mean of three measured values. -
[1] JIN A H, MUTTENTHALER M, DUTERTRE S, et al. Conotoxins: Chemistry and Biology [J]. Chemical Reviews, 2019, 119(21): 11510 − 11549. doi: 10.1021/acs.chemrev.9b00207 [2] PRASHANTH J R, DUTERTRE S, JIN A H, et al. The role of defensive ecological interactions in the evolution of conotoxins [J]. Molecular Ecology, 2016, 25(2): 598 − 615. doi: 10.1111/mec.13504 [3] ENDEAN R, DUCHEMIN C. The venom apparatus of Conus magus [J]. Toxicon: Official Journal of the International Society on Toxinology, 1967, 4(4): 275 − 284. doi: 10.1016/0041-0101(67)90056-6 [4] DUTERTRE S, JIN A H, VETTER I, et al. Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails [J]. Nature Communications, 2014, 5(1): 1 − 9. [5] MARSHALL J, KELLEY W P, RUBAKHIN S S, et al. Anatomical correlates of venom production in Conus californicus [J]. The Biological Bulletin, 2002, 203(1): 27 − 41. doi: 10.2307/1543455 [6] SALISBURY S M, MARTIN G G, KIER W M, et al. Venom kinematics during prey capture in Conus: the biomechanics of a rapid injection system [J]. The Journal of Experimental Biology, 2010, 213(5): 673 − 682. doi: 10.1242/jeb.035550 [7] LAVERGNE V, HARLIWONG I, JONES A, et al. Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks [J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(29): 3782 − 3791. doi: 10.1073/pnas.1501334112 [8] JONES R M, BULAJ G. Conotoxins - new vistas for peptide therapeutics [J]. Curr. Pharm. Des., 2000, 6(12): 1249 − 1285. doi: 10.2174/1381612003399653 [9] OLIVERA B M. E.E Just Lecture, 1996. Conus venom peptides, receptor and ion channel targets, and drug design: 50 million years of neuropharmacology [J]. Molecular Biology of the Cell, 1997, 8(11): 2101 − 2109. doi: 10.1091/mbc.8.11.2101 [10] CRUZ L J, GRAY W R, OLIVERA B M. Purification and properties of a myotoxin from Conus geographus venom [J]. Archives of Biochemistry and Biophysics, 1978, 190(2): 539 − 548. doi: 10.1016/0003-9861(78)90308-9 [11] SHON K J, GRILLEY M M, MARSH M, et al. Purification, characterization, synthesis, and cloning of the lockjaw peptide from Conus purpurascens venom [J]. Biochemistry, 1995, 34(15): 4913 − 4918. doi: 10.1021/bi00015a002 [12] PENG C, YAO G, GAO B M, et al. High-throughput identification of novel conotoxins from the Chinese tubular cone snail (Conus betulinus) by multi-transcriptome sequencing [J]. GigaScience, 2016, 5(1): 1 − 14. doi: 10.1186/s13742-015-0107-0 [13] HIMAYA S W A, LEWIS R J. Venomics-Accelerated Cone Snail Venom Peptide Discovery [J]. International Journal of Molecular Sciences, 2018, 19(3): 788. doi: 10.3390/ijms19030788 [14] UTKIN Y N. Modern trends in animal venom research-omics and nanomaterials [J]. World Journal of Biological Chemistry, 2017, 8(1): 4 − 12. doi: 10.4331/wjbc.v8.i1.4 [15] UTKIN Y N. Animal venom studies: Current benefits and future developments [J]. World Journal of Biological Chemistry, 2015, 6(2): 28 − 33. doi: 10.4331/wjbc.v6.i2.28 [16] SANTOS A D, MCINTOSH J M, HILLYARD D R, et al. The A-superfamily of conotoxins: structural and functional divergence [J]. The Journal of Biological Chemistry, 2004, 279(17): 17596 − 17606. doi: 10.1074/jbc.M309654200 [17] LU A, YANG L, XU S, et al. Various conotoxin diversifications revealed by a venomic study of Conus flavidus[J]. Molecular & Cellular Proteomics, 2014, 13(1): 105 − 118. [18] MCINTOSH J M, PLAZAS P V, WATKINS M, et al. A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha 9 alpha 10 and alpha 7 nicotinic cholinergic receptors[J]. 2005, 280(34): 30107 − 30112. [19] YUAN D D, HAN Y H, WANG C G, et al. From the identification of gene organization of alpha conotoxins to the cloning of novel toxins [J]. Toxicon: official journal of the International Society on Toxinology, 2007, 49(8): 1135 − 1149. doi: 10.1016/j.toxicon.2007.02.011 [20] WANG Q, JIANG H, HAN Y H, et al. Two different groups of signal sequence in M-superfamily conotoxins [J]. Toxicon: official journal of the International Society on Toxinology, 2008, 51(5): 813 − 822. doi: 10.1016/j.toxicon.2007.12.007 [21] 李雁达, 张永吉. DNA提取试剂盒的综述[J]. 全科口腔医学电子杂志, 2019(28): 14. [22] 丁青, 刘月鹏, 朱晓鹏, 等. 菖蒲芋螺不同大小基因组DNA片段的分离制备[J]. 热带生物学报, 2017, 8(3): 267 − 276. [23] 罗素兰, 张本, 长孙东亭. 芋螺基因组DNA提取方法的优化[J]. 中国海洋药物, 2004(1): 21 − 25. doi: 10.3969/j.issn.1002-3461.2004.01.006