Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Reno, NV 89557, USA.
BMC Genomics. 2010 Mar 30;11:215. doi: 10.1186/1471-2164-11-215.
The mountain pine beetle (Dendroctonus ponderosae) is a significant coniferous forest pest in western North America. It relies on aggregation pheromones to colonize hosts. Its three major pheromone components, trans-verbenol, exo-brevicomin, and frontalin, are thought to arise via different metabolic pathways, but the enzymes involved have not been identified or characterized. We produced ESTs from male and female midguts and associated fat bodies and used custom oligonucleotide microarrays to study gene expression patterns and thereby made preliminary identification of pheromone-biosynthetic genes.
Clones from two un-normalized cDNA libraries were directionally sequenced from the 5' end to yield 11,775 ESTs following sequence cleansing. The average read length was 550 nt. The ESTs clustered into 1,201 contigs and 2,833 singlets (4,034 tentative unique genes). The ESTs are broadly distributed among GO functional groups, suggesting they reflect a broad spectrum of the transcriptome. Among the most represented genes are representatives of sugar-digesting enzymes and members of an apparently Scolytid-specific gene family of unknown function. Custom NimbleGen 4-plex arrays representing the 4,034 tentative unique genes were queried with RNA from eleven different biological states representing larvae, pupae, and midguts and associated fat bodies of unfed or fed adults. Quantitative (Real-Time) RT-PCR (qRT-PCR) experiments confirmed that the microarray data accurately reflect expression levels in the different samples. Candidate genes encoding enzymes involved in terminal steps of biosynthetic pathways for exo-brevicomin and frontalin were tentatively identified.
These EST and microarray data are the first publicly-available functional genomics resources for this devastating forestry pest.
山松甲虫（Dendroctonus ponderosae）是北美西部重要的针叶林害虫。它依靠聚集信息素来定植宿主。其三种主要的信息素成分，反式马鞭烯醇、外-法呢烯醇和法呢醇，被认为是通过不同的代谢途径产生的，但涉及的酶尚未被鉴定或表征。我们从雌雄中肠和相关的脂肪体中产生了 EST，并使用定制的寡核苷酸微阵列来研究基因表达模式，从而对信息素生物合成基因进行了初步鉴定。
从两个未归一化的 cDNA 文库中定向测序克隆，从 5' 端测序，得到 11775 个 EST 序列，经过序列清洗。平均读长为 550nt。EST 聚类为 1201 个 contigs 和 2833 个 singlets（4034 个暂定的独特基因）。EST 在 GO 功能组中广泛分布，表明它们反映了转录组的广泛谱。在最具代表性的基因中，有糖消化酶的代表和一个显然是特定于小蠹科的基因家族的成员，该基因家族的功能未知。代表 4034 个暂定独特基因的定制 NimbleGen 4-plex 微阵列与代表幼虫、蛹和未进食或进食成虫的中肠和相关脂肪体的 11 种不同生物学状态的 RNA 进行了查询。定量（实时）RT-PCR（qRT-PCR）实验证实，微阵列数据准确反映了不同样品中的表达水平。候选基因编码参与外-法呢烯醇和法呢醇生物合成途径末端步骤的酶，被暂时鉴定。
这些 EST 和微阵列数据是该毁灭性林业害虫的第一个公开的功能基因组学资源。
