2012 级制药工程 李晓梅 链霉菌次级代谢产物调控网络的研究思路与方法

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Introduction Streptomycetes are well known as their ability to produce a great variety of secondary metabolites. The control of secondary metabolite production is a complex process involving multiple levels of regulation (Lowest level the highest level). Pathway-specific-regulatory genes are usually found within their respective antibiotic biosynthesis gene cluster, but higher regulators can be located far away in the chromosome, thus making it difficult to infer their targets. LAL (Large ATP-binding regulators of the LuxR family) could play a role in higher steps of the regulatory cascade. But LAL regulators have been poorly studied.

Material and method To identify the genes affected by each mutation which will help us to establish regulatory networks. Construction of regulatory gene mutants DNA microarrays RT-PCR & quantitative RT-PCR EMSA

Gene expression analysis by RT-PCR Wild type strain internal control: cDNA of the hrdB three independent biological replicates Candidatets: SCO0877 SCO7173

Construction of △ SCO0877 and △ SCO7173 5’-ccggacgccggccgtccccctttagagtgggcttctgtgATTCCGGGGATCCGTCGACC-3’ 5’-gaactcttcactccagatggttacgtttcgcatgcgtcaTGTAGGCTGGAGCTGCTTC-3’ Apramycin resistance gene 0877 △ SCO0877

Growth pattern and actinorhodin production Reduced actinorhodin The same growth kinetics

DNA microarrays DNA 微阵列（ DNA microarray ）是由大量 DNA 或寡核苷酸 探针密集排列所形成的探针阵列，其工作的基本原理是通 过杂交检测信息，实质是核酸碱基的互补匹配。 把大量已知序列探针集成在同一个基片上，经过标记的若 干靶核酸序列通过与芯片特定位置上的探针杂交。 利用基因芯片杂交检测图像，可以对生物细胞或组织中大 量的基因信息进行分析。 基因芯片能够在同一时间内分析大量的基因，实现生物基 因信息的大规模检测。

DNA microarray Labeled Target Hybridized Array DetectionReagents DNA Microarray

Altered expression profile in LAL mutants 322 genes showed differential transcription 121 in mutant △ SCO in mutant △ SCO in common follow the same patter Regarding up- or down-regulation Including Genes involved in amino acid and carbohydrate metabolism nucleotide and coenzyme metabolism respiration and energy production

Phosphate starvation response genes 25 genes, including 15 whose transcription has been demonstrated to be directly controlled by PhoP ， are negatively regulated This profile indicates that the phosphate starvation response system is tightly controlled by both regulators LAL 0877 and LAL up-regulation of act genes by both regulators, especially significant in LAL 7173

Quntitative RT-PCR 可以根据 PCR 变化曲线计 算 出目标靶基因分子数。 用来检测某个基因的表达 主要采用荧光实时 RT-PCR 原理： 在实时荧光定量 PCR 反应中引入了一种荧光化 学物质，随着 PCR 反应的进行， PCR 反应产物不 断累计，荧光信号强度也等比例增加。每经过一个 循环，收集一个荧光强度信号，这样我们就可以通 过荧光强度变化监测产物量的变化，从而得到一条 荧光扩增曲线图。

Quntitative RT-PCR CT 值： 每个反应管内的 荧光信号到达设 定的阈值时所经 历的循环数。 Quantitative RT-PCR used on reversed transcribed RNA samples to confirm that ifferential expression indicated by the microarray data was supported by an independent method.

Validation of microarray results 11 wide ranged genes showing high Mc and a p-value (<0.0002) were chosen. Overall, the qRT-PCR data and microarray data showed a good concordance

Control of LAL regulators on phoP expression One of the most interesting outcomes of our transcriptomic studies was that both LAL regulators act as negative regulators of the phoRP system expression. So it was interesting to determine whether the level of expression of the LAL regulators studied varied with phosphate availability.

A.no variation of the LAL B.phoP expression varies C. a direct interaction between LAL 0877, or another regulator positively modulated by LAL 0877, and the phoP promoter. D. phoRP Control of LAL regulators on phoP expression

Conclusion Secondary metabolism is a complex playground where global and pathway specific regulators form a web of interactions that finally results in metabolite production. This study has introduced a method to Investigate other regulatory networks