论著

一种具厚几丁质外壳微小节肢动物无损伤凭证标本高质量DNA模板的获取方法

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  • 1 中山出入境检验检疫局检验检疫技术中心, 广东 中山 528403;
    2 中山大学生命科学学院, 广东 广州 510275
单振菊,女,硕士,农艺师,从事媒介生物鉴定工作,Email:173465531@qq.com

收稿日期: 2016-09-01

  网络出版日期: 2017-02-20

基金资助

国家质量基础项目(2016YFF0203205);国家质量监督检验检疫总局科技计划项目(2015IK067,2015IK069);广东省省级科技计划项目(2015A050502009);中山市科技计划项目(2014A2FC252)

A method of obtaining high quality DNA templates from tiny thick-chitin arthropods without damaging morphology of the voucher specimens

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  • 1 Zhongshan Entry-Exit Inspection and Quarantine Technical Center, Zhongshan 528403, Guangdong Province, China;
    2 College of Life Science Sun Yat-sen University

Received date: 2016-09-01

  Online published: 2017-02-20

Supported by

Supported by the National Quality Infrastructure Project(No. 2016YFF0203205), Administration of Quality Supervision, Inspection and Quarantine Research Projects (No. 2015IK067, 2015IK069), Guangdong Province International Cooperation Project(No. 2015A050502009), and the Zhongshan Research Project(No. 2014A2FC252)

摘要

目的 针对具厚几丁质外壳的微小节肢动物,研究一种无需研磨、不损坏凭证标本形态的高质量DNA模板的获取方法。方法 以具有厚几丁质外壳的土壤甲螨为材料,用几丁质酶消化其外壳,然后利用裂解液对整个个体进行裂解,以DNA条形码片段的扩增结果作为判断模板质量的依据,探讨几丁质酶的最佳工作浓度和工作时间;比较几丁质酶处理前后标本的外部形态。结果 该方法能获得高质量的DNA模板,在25℃,pH值6.0时,几丁质酶最佳浓度为1 mg/ml,最佳作用时间为24 h。经几丁质酶处理后,螨的外部形态保存完整,与处理前无明显差别,可以用于形态鉴定。结论 该方法首次将几丁质酶消化动物几丁质外壳与提取模板DNA相结合,不仅能保证微小节肢动物的形态特征不被破坏,而且获取的DNA模板质量高,作为凭证标本的微小节肢动物可以制成玻片标本保存,保证了凭证标本的形态完整性。

本文引用格式

单振菊, 邱德义, 岳巧云 . 一种具厚几丁质外壳微小节肢动物无损伤凭证标本高质量DNA模板的获取方法[J]. 中国媒介生物学及控制杂志, 2017 , 28(1) : 27 -30 . DOI: 10.11853/j.issn.1003.8280.2017.01.008

Abstract

Objective In order to establish a method of obtaining high quality DNA templates from tiny thick-chitin arthropods without grinding or damaging morphology of the voucher specimens. Methods Tiny soil oribatid mites with thick-chitin were used as research materials. We used chitinase to digest the external chitin skeleton first,after the digestion,the individuals were treated with lysate. Amplification efficiency of the DNA barcodes were used as the indicator for DNA template quality. Working concentration and condition of chitinase were optimized. Morphology of the samples treated with or without the chitinase were compared. Results High quality DNA template could be obtained with the method we established,working concentration of chitinase was optimized to 1 mg/ml, and working time was optimized to 24 h. The morphology of oribatid mites treated with the chitinase has no obvious changes compared to the untreated ones, and could be used in the morphology species identification. Conclusion Chitinase is first applied to the digestion of chitin in the extraction of template DNA, high quality DNA could be obtained without damaging the morphological characteristics, after the extraction, the tiny arthropods could be mounted on the slides and kept as voucher specimen.

参考文献

[1] 刘勇,宋毓,李晓宇. 基于线粒体COⅠ基因的DNA条形码技术在昆虫分子鉴定中的应用[J]. 植物检疫, 2010, 24(2):46-50.
[2] Stoeckle M. Taxonomy,DNA,and the bar code of life[J]. BioScience, 2003, 53(9):796-797.
[3] Hebert PDN, Cywinska A, Ball SL, et al. Biological identifications through DNA barcodes[J]. Proc Roy Soc B, 2003, 270(1512):313-321.
[4] 陈琪,姜玉新,郭伟,等. 3种常用封固剂制作螨标本的效果比较[J]. 中国媒介生物学及控制杂志, 2013, 24(5):409-411.
[5] 胡佳,岳巧云,邱德义,等. 医学媒介生物微量组织直接扩增DNA条形码序列方法的研究[J]. 中国媒介生物学及控制杂志, 2014, 25(4):297-300.
[6] 温硕洋,何晓芳. 一种适用于昆虫痕量DNA模板制备的方法[J]. 昆虫知识, 2003, 40(3):276-279.
[7] 高艳,卜云. 一种改进的小型节肢动物无形态损伤的DNA提取方法[J]. 四川动物, 2014, 33(2):216-220.
[8] 张志忠,吴菁华,吕柳新,等. 植物几丁质酶及其应用研究进展[J]. 福建农林大学学报:自然科学版, 2005, 34(4):494-499.
[9] 李瑶,范晓军. 昆虫几丁质酶及其在害虫防治中的应用[J]. 应用昆虫学, 2011, 48(5):1489-1494.
[10] 魏巍,贺淹才,刘爱花. 几丁质酶酶活性测定方法的研究进展[J]. 工业催化, 2006, 14增刊:11-14.
[11] 殷秀琴,马祝阳. Tullgren法对土壤动物的分离效率[J]. 东北师大学报:自然科学版, 2002, 34(2):84-91.
[12] 谢晓兰,曾炜,高平章,等. 黄粉虫几丁质酶的纯化及性质[J]. 泉州师范学院学报, 2012, 30(4):56-61.
[13] Mora C,Tittensor DP,Adl S,et al. How many species are there on earth and in the ocean[J]. PLoS Biol, 2011, 9(8):e1001127.
[14] 魏晓雅,邱德义,岳巧云,等. 应用DNA条形码技术鉴定口岸截获的国内未见分布蚊种[J]. 检验检疫学刊, 2014, 24(6):46-49, 67.

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