2012级制药工程李晓梅

2012207347

链霉菌次级代谢产物调控网络的研究思路与方法

Main reference

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: SCO0877SCO7173

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 TargetLabeled Target

Hybridized ArrayHybridized Array

DetectionDetectionReagentsReagents

DNA MicroarrayDNA Microarray

Altered expression profile in LAL mutants

322 genes showed differential transcription

121 in mutant SCO0877 △263 in mutant SCO7173 △62 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 regulatedThis profile indicates that the phosphate starvation response system is tightly controlled by both regulators LAL 0877 and LAL 7173.

up-regulation of act genes byboth 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 rangedgenes showing high Mc and a p-value (<0.0002)were chosen.

Overall, the qRT-PCR data and microarray data showed a goodconcordance

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 LALB. 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