Molecular Identification Of Nktc1,A Temperature-conditional Mutant Cyanobacterium By Genetic Engineering Methods

Synechococcus elongatus PCC 7942?S.elongatus PCC 7942?is one of the simplest model organisms for circadian rhythms researches.The transcriptional activity of most genes in its genome is regulated by the endogenous circadian clock.Kai ABC genes encode the basic molecular components of the core clock oscillator of S.elongatus PCC 7942.Kai proteins play a pivotal role in the molecular regulation of the circadian rhythm in this cyanobacterium strain.The feature of temperature compensation is one of the three basic characters of all circadian clock models in known living organisms.It has been found that,the temperature compensation feature of circadian clock in cyanobacterium is generally controlled by the intact Kai genes.In previous works of our Lab,we have obtained a mutant nktc1?Non-Kai Thermo-regulatory Component 1?with a temperature conditional phenotype of circadian clock via a traditional method of chemical mutagenesis by using Ethylmethane sulfonate?EMS?as a mutagen.When a DNA fragment containing the whole Kai ABC gene loci was introduced back into the nktc1 genome DNA,the mutated temperature compensation feature of circadian clock in nktc1 mutants could not be rescued as that of wild-type cyanobacteria.This result meant that mutated genes in nktc1 were in charging of coding Non-Kai thermo-regulatory components of cyanobacterial circadian clock.In this dissertation,the mutant sites of nktc1 were molecular identified by De nove sequencing of genome DNA and other genetic technologies.Detail research contents of this thesis are listed as following.1.The mutated sites of nktc1 were identified and verified based on De nove sequencing of its genome DNA and gene cloning methods.The results showed that there were two mutation sites of nktc1 that were identified in the cyanobacteria genome DNA,one in the coding sequence of Synpcc 7942₀644?CikA gene?,and the other one located in Synpcc 7942₀095 ?RpaA gene?.The mutation site named nktc-cik A can lead to 559 Phe in wild-type CikA protein mutated to Ser;The mutation site named nktc-rpa A can lead to 118 Ala in wild-type RpaA protein mutated to Thr.2.Genetic engineering methods were used to construct 2 single-gene mutation-reconstructed cyanobacteria strains: nktc-cik A-m and nktc-rpa A-m;as well as 2 single-gene in situ complementary rescued cyanobacteria strains:19T-cik A-R and 19T-rpa A-R,also 2 single-gene ex situ complementary rescued cyanobacteria strains: NS-CikA-R and NS-RpaA-R,succeeding a double genes ex situ complementary rescued cyanobacteria strain:NS-CikA-RpaA-R.3.By testing the phenotypes of their circadian clock's temperature compensation feature in S149,nktc1 and all constructed cyanobacteria strains in abovementioned works,all the tested cyanobacteria strains were entrained by low-temperature signals under constant light conditions before their circadian rhythmic expression of KaiC gene was evaluated by RT-q PCR,as well as the phosphorylation rhythm of KaiC protein in these strains was detected by Western blot methods respectively.The results indicated that low-temperature entrainment of wild-type cyanobacteria S149,nktc-cik A-m,nktc-rpa A-m and NS-RpaA-R can effectively synchronize the phase of KaiC gene transcriptional rhythms,and It can also effectively synchronize the phase of protein phosphorylation rhythmicity of KaiC protein at the post-translational modification level.However,the period length of nktc-cik A-m became shorter while that of nktc-rpa A-m and NS-RpaA-R were elongated.In nktc1,NS-CikA-R and NS-CikA-RpaA-R strains neither their phases of KaiC gene transcriptional circadian rhythm at the transcription level,nor their phosphorylation rhythms of KaiC protein at the post-translational modification level can be entrained by the external low temperature time cues.To 19T-CikA-R and 19T-RpaA-R cyanobacteria strains,though low-temperature entrainment signals could effectively synchronize the phase of KaiC gene circadian rhythmical transcription at a transcriptional level,the phase of circadian rhythmical phosphorylation of KaiC protein could not be entrained by the same shifting temperature signals.However,the period length of 19T-CikA-R became significantly shorter than that of wild-type cells,while on the contrary,19T-RpaA-R showed a phenotype of elongated period.4.The possible functions of CikA and RpaA genes in the regulation of temperature compensation feature of circadian rhythm in the cyanobacteria were also studied.Based on the results of present work,this dissertation primarily concluded that CikA and RpaA genes,together with their coding proteins had involved in 2 distinct regulatory pathways that controlled the temperature responding features of circadian clock in S.elongatus PCC 7942 cyanobacterial strain.CikA gene and CikA protein were very likely to have taken part in a negative feedback regulatory pathway for temperature responding in cyanobacterial circadian clocks,nevertheless the RpaA gene and RpaA protein were possibly involved in the positive feedback regulatory process of which.