Crawford A M, Brauning R, Smolenski G, Ferguson C, Barton D, Wheeler T T, McCulloch A
AgResearch Invermay Agricultural Centre, Puddle Alley, Private Bag 50034, Mosgiel, New Zealand.
Insect Mol Biol. 2008 Jun;17(3):313-24. doi: 10.1111/j.1365-2583.2008.00802.x.
Purified RNA transcripts from venom glands dissected from the parasitoid wasp Microctonus hyperodae were copied, cloned and sequenced using traditional dideoxy sequencing methods. Using mass spectrometry analysis of the trypsinised PAGE gel protein bands we identified the RNA transcripts for the 3 most abundant proteins found in the venom and hence obtained their full protein sequence. Other abundant transcripts were also further sequenced. To reduce the effort required to obtain transcript information we dissected venom glands from a second parasitoid, Microctonus aethiopoides (Morocco biotype). The RNA transcripts were purified and reverse transcribed but instead of cloning the cDNA it was directly sequenced using Roche GS20 pyrosequencing. Results from a single GS20 sequencing run provided data similar to that obtained by the traditional methods used in analysing transcripts from M. hyperodae in a fraction of the time and cost. Comparing the transcripts between the two species showed that a similar range of genes are expressed with the putative orthologs of seven of the eight full length genes characterised from M. hyperodae being found in M. aethiopoides. Pyrosequencing should provide a valuable new method for rapidly sampling transcripts from a wide range of specialised insect tissues.
从寄生蜂超嗜微绒茧蜂解剖得到的毒腺中提取的纯化RNA转录本，使用传统的双脱氧测序方法进行复制、克隆和测序。通过对胰蛋白酶处理后的聚丙烯酰胺凝胶蛋白条带进行质谱分析，我们鉴定出了毒液中含量最高的3种蛋白质的RNA转录本，从而获得了它们完整的蛋白质序列。其他丰富的转录本也进一步进行了测序。为了减少获取转录本信息所需的工作量，我们从第二种寄生蜂埃塞俄比亚微绒茧蜂（摩洛哥生物型）解剖了毒腺。对RNA转录本进行纯化和逆转录，但不是克隆cDNA，而是使用罗氏GS20焦磷酸测序直接对其进行测序。单次GS20测序运行的结果提供的数据与分析超嗜微绒茧蜂转录本时使用传统方法获得的数据相似，且时间和成本仅为其一小部分。比较这两个物种的转录本表明，它们表达的基因范围相似，超嗜微绒茧蜂鉴定出的8个全长基因中的7个推定直系同源基因在埃塞俄比亚微绒茧蜂中也能找到。焦磷酸测序应为从广泛的特殊昆虫组织中快速采样转录本提供一种有价值的新方法。
