从白蛉提取内分泌物的方法<em> Bemisia tabaci</em
Summary
在这里，我们提出了通过解剖和过滤从粉虱烟粉虱中分离内分泌物的方案。扩增后，DNA样品适用于后续测序和研究内共生与粉虱之间的共生关系。
Abstract
细菌共生体与宿主形成亲密关系，在大多数情况下赋予宿主优势。基因组信息对于研究其宿主细菌共生体的功能和进化至关重要。由于大多数共生体不能在体外培养 ，所以分离足够量的细菌用于基因组测序的方法是非常重要的。在粉虱Bemisia tabaci中 ，已经确定了许多内生子，并且通过多种方法预测害虫的发育和繁殖是重要的。然而，支持协会的机制仍然很大程度上是未知的。障碍部分来自于粉虱中的内分泌物，主要是抑制细菌，难以与宿主细胞分离。在这里我们报告一个主要通过解剖的粉虱B.烟粉虱鉴定，提取和纯化内分泌物的分步骤方案开和过滤。通过该方法制备的内含子样品虽然仍然是不同内分泌物种的混合物，但适用于随后的基因组测序和分析烟粉虱内共生菌的可能作用。这种方法也可用于从其他昆虫中分离内分泌物。
Introduction
与节肢动物形成密切共生关系的细菌广泛存在于节肢动物中。在几乎每个发育阶段5中 ，内分泌已被证明影响宿主的方面，如营养代谢，繁殖，对环境胁迫2,3,4 等的反应。然而，支持协会的机制仍然很大程度上是未知的。研究细菌的潜在功能和作用时，基因组学是重中之重。一些基本信息， 即分类学状态，功能基因，代谢途径，分泌系统，可以从基因组序列推断出来，揭示了共生体在共生中的潜在作用。随着高通量测序的发展，大量的细菌基因组已被测序各种功能揭示6 。
内含子在半ter子中是至关重要的，如蚜虫7 ，臭虫8 ，木虱9 ，褐飞虱10和蝉11 。例如，作为专性共生体的蚜虫中的Buchnera已被证明参与必需氨基酸生物合成，以及来自蚜虫基因组12的基因。此外， Buchnera的转录调控也显示13 。在木麻黄中， 卡桑奈尔被测序，并且是发现的最小的细菌基因组14 。所有这些内含子的标志都是基于基因组序列推断的。由于这些内生子宫内膜不能在体外培养 ，因此已经采用了几种方法来分离足够的细菌uencing。在蚜虫中，通过离心和过滤提取内生子，并进行进一步的基因组和转录组学分析。在褐飞虱中，内共生连同整个昆虫基因组10进行测序。
烟粉虱是一种含有超过35种形态不可区分物种（隐蔽物种）的物种，其中两种入侵物种已经入侵到世界各地，对农业生产造成了巨大的危害15 。值得注意的是， 烟粉虱物种内的内生梭菌在害虫16的发育中已显示出重要性。到目前为止，在粉虱中已经鉴定出8个内含子，其中包括专性共生体， Candidatus Portiera aleyrodidarum和七个次生共生体Hamiltonella，立克次体 ， Arsenophonus ， Cardinium ，Wolbachia， Fritschea和Hemipteriphilus定义为17,18 。
与以前描述的半扁桃不同，粉虱烟粉虱是一只极小的昆虫，长度只有1毫米。大多数内生子体被限制在细菌19 （含有共生体的特殊细胞，其进一步在烟粉虱中形成细菌）。此外，这些内生子宫内膜不能在体外培养 。从烟粉虱获得内生菌的唯一方法是将细菌分解出来。然而，解剖有困难。首先，脆弱的细菌总是与粉虱的其他组织连接，这是很难分离的。其次，粉虱的微小尺寸限制了足够细菌的分离。第三，内生菌群聚集在细菌中，使获得单一种细菌非常复杂。
<p class ="“jove_content”">这里，我们报告一个简单而便宜的方案来隔离粉虱内分泌物用于随后的宏基因组测序。通过解剖，纯化和扩增，可以获得足够的内含子DNA，并可以确认细菌种类。所描述的方案可以在其他节肢动物中类似地使用。Protocol
Representative Results
Discussion
Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究的财政支持由国家重点研究发展计划（2016YFC1200601）和国家自然科学基金（31390421）提供。
Materials
| Taq DNA polymerase | Takara | R001A | including rTaq, 10×Buffer and dNTP |
| Gel DNA extraction kit | Qiagen | 28704 | |
| DNA sample sequencing system | ABI | ABI-3730XL | |
| Microtome | Leica | EM UC7 | |
| Transmission electron microscopy | Hitachi | H-7650 TEM | |
| Stereo microscope | Zeiss | Stemi 2000-C | |
| 20 μL microloader | Eppendorf | F2771951 | |
| Filter holder | Millipore | SX0001300 | |
| Filter membrane filter | Millipore | SMWP001300 | 5.0 μm SMWP |
| REPLI-g UltraFast Mini Kit | Qiagen | 150033 | DNA amlification kit |
| NanoDrop | Thermo Scientific | NanoDrop 2000 | |
| Qubit Fluorometer | Thermo Fisher Scientific | Q33216 | |
| Genome Sequencer | Illumina | Hiseq 2000 |
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