姓 名：逄爱萍

学 号： 2012207355

日 期： 2013.09.13

次级代谢产物是生物在特定条件下生成的具有重要生理功能和活性的化合物。

次级代谢产物的结构是由其编码的基因簇决定的，它的生成是基因协同表达的结果。

在链霉菌的次级代谢途径的合成基因簇中，功能基因往往十几个以上，多达几十个，而调控基因只有几个，甚至是一个。

背景

转录因子原核生物转录

转录因子对靶基因启动子区域 DNA序列的识别与结合是整个调控的核心。

发现次级代谢途径特异性转录调控因子，深入研究其转录调控机理，是微生物生理与代谢、生化与遗传的核心关键问题之一。

Isolation of RNA

RT-PCR

Expression and purification of GST fusion proteins

DNA-protein binding assays(EMSA)

Footprinting assays

Bioinformatic analysis

转录因子研究的一般方法

N C

Monocistronic ：单顺反子，一个启动子后仅具有一个编码序列。Polycistronic ：多顺反子，若干个基因由一个启动子控制，转录在一条 mRNA 上。

Organization of pimaricin gene cluster

a tetraene macrolide antibiotic produced by S.natalensis

Primers ： 400——600bp S2S3 IS2 CG FS0 S3S4 JI GF

Determining whether the neighboring genes could be co-transcribed by RT-PCR

If unbated transcription was observed ， the neighboring genes were co-transcribed.

93aa

192aa

143aa

Vector ： pGEX-2THost: BL21 （ DE3)

GST

Construction of expression plasmids

Heterologous expression of PimM and of its truncated versions

Expression ： 18℃ ， IPTG 0.1mM(OD=0.7) ， 14h

Purification ： affinity chromatography on Glutathione sepharose

EMSA原理：

based on the separation of free DNA from DNA-protein complexes

DIG Oligonucleotide 3’-End Labeling Kit, 2nd Generation (Roche Applied Science)

a.Digoxigenin labelled DNAb.DNA+protein incubationc.Electrophoresis:5% polyacrylamide native geld.chemiluminiscene

a

d

c

bDetermine the binding regions

DNA-protein binding assays ： EMSAOne retarded bands: pimKp, pimAp, pimEp, pimS2p,pimIp;

Two retarded bands: pimS1-Dp;

Four retarded bands: pimJp;

Two negative control reactions: absence of protein,and use of GST

Left lane: control without proteinRight lane: 60 uM of GST-PimM protein

B:a competition experiment between pimJp and pimCp. Addition of one to 1000-fold higher concentrations of pimCp competitor DNA failed to diminish the intensities of the pimJp retardation bands

C:control reactions made with pure GST protein were negative in all cases, excluding a possible binding of this protein to the promoters

B.PAS domain reduces binding affinity

Figure A demonstrates that binding ability relies on the DNA-binding domain, and is independent of the PAS domain.

Figure B demonstrates that truncated forms of the protein have significantly higher affinity.

A. binding ability relies on the DNA-binding domain

Dnase I 足迹实验（ footprinting assay ）

a.6-FAM labelled DNA +protein incubation

b.Dnase I digestions

c.Analyse with PEAK SCANNER program

Determine the binding sequence

The retarded band was observed upon the incubation of GST-PimM with all the promoters which are similar to PimM binding site.

A,B,C :homologous regulators from different polyene producers

The figure suggest the orthologous regulators of polyene biosynthesis share the same regulatory pattern

Genetic complementation of S.natalensis ΔpimM by orthologous regulators restores pimaricin production

DNA fragment: pimM,amphRIV, nysRIV, pteFVector: pSET152giving rise to pSETpimM pMamphRIV, pMnysRIV, pMpteF

Transfermation by conjugation

As expected,given its highest identity to PimM, pimaricin yield was the highest in the strain complemented with pteF (94%)Production of the strain complemented with amphRIV or nysRIV, which are more distantly related to pimM, was somewhat lower, (47%,61%)

When we introduce one copy of pimM into the genomes of S.nodosus and S.avermitilis, the polyene production boosted substantially, whereas no significant change in the growth curve was observed.

Expression of PAS-LuxR regulators is a bottleneck

Confirm the functional conservation among polyene biosynthetic gene clusters.